Superantigens, Endothelial Injury and Vasculitis in The Young.

University o f London
Superantigens, endothelial injury and

vasculitis in the young.
Paul Anthony Brogan BSc (Hon) MBChB (Hon), MRCP, MSc.
Submitted for the degree o f Doctor o f Philosophy.
Department o f Nephro-Urology, Institute o f Child Health,

University College London.
ProQuest Number: 10013879
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Acknowledgements
I would like to acknowledge my supervisors. Professor Michael Dillon and Dr Nigel
Klein for constant support, enthusiasm, ideas and discussion. It was truly a great privilege
to be Professor Dillon’s last research fellow before his retirement- Mike I hope this thesis
honours the fantastic contribution you have made to the study of the paediatric
vasculitides over the years. A special thanks must go to Mrs Vanita Shah for help and
guidance in the laboratory, and for much support and nurturing throughout the whole
duration of my PhD. Thanks also to Alka Patel for showing me how to turn on a flow
cytometer, and to Professor Nancy Hogg for the kind gifts o f the monoclonal antibodies,
and to Ms Carol Hutchinson for collecting control samples. Acknowledgement must also
go to the Charlotte Parkinson Research Fund and John Herring and Friends Trust, who
funded this research. Thanks also to the children and parents who participated in the
studies described in this thesis.
Lastly, I would like to say thankyou to my wife Gabi, for putting up \vith me throughout
all o f this.
Paul Brogan, October 2002.

Thesis Abstract
Superantigens, endothelial injury, and vasculitis of the young.
Limited data exist suggesting a role for superantigens (SAgs) in the aetiopathogenesis of
vasculitis syndromes in children and adults. The best evidence relates to Kawasaki
Disease (the second commonest vasculitic syndrome o f childhood), although this remains
a controversial issue. Even fewer data exist examining how SAgs could cause endothelial
and/or vascular injury. This thesis addressed these issues by examining peripheral blood
T cell activation and T cell Vp skewing in children with vasculitis. Secondly, an in vitro
model was established to examine the hypothesis that the endothelial cell may operate as
a competent SAg-presenting cell for T cells. Further evidence o f endothelial injury in
children with vasculitis was derived by examining circulating endothelial microparticles
(BMP) in children with vasculitis at various stages o f disease activity. Children with
active vasculitis had higher CD4 and CDS peripheral blood T cell CD69 expression than
those with inactive vasculitis and healthy controls, but not disease control children. No
difference in CD25 expression in either the CD4 or CDS T cell populations was observed
between active and inactive vasculitis groups, however. There was a significantly
increased variance of CD4 V pi2, and V pi7 and CDS V pi in the primary systemic
vasculitis group as compared to control and disease controls. Moreover, S0% o f the
primary systemic vasculitis children had one or more CD4 Vp expansions or deletions, as
compared with 30% of controls and 37% of the disease controls (p<0.002). In the KD
group, the mean % of CD4 VP2 T cells was higher than in controls or disease controls.
Co-culture of purified T Cells and MHC class HUVEC with SAg resulted in Vp-
restricted CD4 and CD8 CD69 upregulation. Additionally, there was CD4 and CD8 T
Cell VP-restricted adherence to the HUVEC monolayer at 4 hours, which was partially
abrogated by blockade of the integrin VLA-4, but not LFA-1. ICAM-1, E-selectin, and
VCAM-1 expression were upregulated on the MHC class 11^HUVEC following exposure
to SAg in the presence of T cells, and there was increased BMP release from the activated
HUVEC. Plasma from patients with active vasculitis contained increased numbers of E-
selectin and CD 105 positive BMP compared with patients in remission, controls and
disease controls. BMP correlated with clinical and laboratory indices o f vasculitic disease
activity, but there was overall a poor correlation between BMP and acute phase reactants
in the disease controls. In conclusion, these data provide indirect evidence of
superantigenic involvement in vasculitis syndromes o f the young, and suggest that one
possible mechanism of endothelial injury mediated by SAg could involve dual signaling
between the endothelial cell (EC) and T cell, with EC activation and injury. These data
also underscore the endothelium as both a target for injury in vasculitis, and as a potential
amplifier of abherrant inflammatory responses, and suggest that circulating BMP may
provide a window to the activated endothelium in vasculitis.
Figures
F ig u r e 1.1 S p e c t r u m o f v a s c u l it ic s k in r a s h in P A N ....................................................................................................2 3
F ig u r e 1.2 S y m m e t r ic a l p o l y a r t h r it is in a 12 y e a r o l d b o y w it h P A N .......................................................... 2 4
F ig u r e 1.3 R e n a l a n g io g r a p h y in P A N .....................................................................................................................................2 5
F ig u r e 1.4: G u id e l in e fo r t h e t r e a t m e n t o f c h il d h o o d P A N a n d r e l a t e d v a s c u l i t i d e s ....................2 8
F ig u r e 1.5: T h e t r e a t m e n t o f p a e d ia t r ic m ic r o s c o p ic p o l y a n g iit is a n d r e l a t e d c r e s c e n t ic
NEPHRITIDES.....................................................................................................................................................................................2 8
F ig u r e 1.6 D ia g n o s t ic c r it e r ia f o r K D ....................................................................................................................................3 2
F ig u r e 1.7 U K g u id e l in e f o r t h e t r e a t m e n t o f K aw asak i d i s e a s e ......................................................................3 8
F ig u r e 1.8 C o n v e n t io n a l v e r s u s s u p e r a n t ig e n p r e s e n t a t io n t o T c e l l s .................................................... 1 1 0
F i g u r e 1.9 F a t e o f a I c e l l f o l l o w i n g S A g s t i m u l a t i o n .........................................................................................113
F ig u r e 2.1 P u r it y o f C D 3 l y m p h o c y t e p o p u l a t io n a f t e r T c e l l n e g a t iv e is o l a t io n u s in g
MAGNETIC BEADS........................................................................................................................................................................ 1 4 0
F ig u r e 2 .2 P e r c e n t a g e o f c o n t a m in a t io n w it h M H C c l a s s II p o s it iv e c e l l s in t h e p u r if ie d C D 3 T
CELL POPULATION....................................................................................................................................................................... 141
F ig u r e 2.3 S t im u l a t io n o f h e a l t h y a d u l t P B M C w it h T S S T -1 ............................................................................ 145
F ig u r e 2 .4 S t im u l a t io n o f h e a l t h y a d u l t P B M C w it h S E B ...................................................................................1 4 6
F ig u r e 2 .5 T c e l l a c t iv a t io n in r e s p o n s e t o S A g s ........................................................................................................ 1 4 7
F ig u r e 2 .6 P h a s e c o n t r a s t l ig h t m ic r o s c o p y o f H U V E C .........................................................................................153
F ig u r e 2.7 F l o w c y t o m e t r ic c h a r a c t e r is a t io n o f H U V E C ....................................................................................1 5 4
F ig u r e 2 .8 C o n v e r s io n e q u a t io n fo r c a l c u l a t io n o f M P n u m b e r f r o m f l o w c y t o m e t e r c o u n t s
161
F ig u r e 2 .9 F l o w c y t o m e t r y g a t in g p r o t o c o l f o r m ic r o p a r t ic l e s ................................................................... 1 6 2
F ig u r e 2 .1 0 R e p r o d u c ib il it y o f M P a n a l y s i s ....................................................................................................................1 6 4
F ig u r e 2.11 E f f e c t o f d if f e r e n t c e n t r if u g a t io n p r o t o c o l s o n MP c o u n t ................................................. 1 6 6
F ig u r e 2 .1 2 E f f e c t of freeze-thaw o n MP n u m b e r ....................................................................................................... 1 6 8
F ig u r e 2 .1 3 E f f e c t o f d e l a y e d p l a s m a s e p a r a t io n f r o m w h o l e b l o o d o n MP p r o f i l e .......................1 7 0
F ig u r e 3.1 C D 4 T c e l l c d 6 9 e x p r e s s i o n ................................................................................................................................ 1 9 0
F ig u r e 3 .2 C D 8 T cell C D 69 e x p r e s s i o n ............................................................................................................................... 1 9 2
F ig u r e 3.3 CD4 T c e l l C D 6 9 e x p r e s s io n b y v a s c u l i t i s t y p e ...................................................................................193
F ig u r e 3 .4 CD8 T c e l l C D 6 9 e x p r e s s io n b y v a s c u l i t i s t y p e ...................................................................................1 9 4
F ig u r e 3.5 CD4 C D 2 5 e x p r e s s i o n ............................................................................................................................................... 1 9 6
F ig u r e 3 .6 CD8 C D 2 5 e x p r e s s i o n ............................................................................................................................................... 197
F ig u r e 3 .7 a and 3 .7 b : T c e l l V p r e pe r t o ir e s in s t u d y g r o u p s ...............................................................................1 9 8
F ig u r e 3 .8 a and 3 .8 b : ......................................................................................................................................................................... 2 0 0
F ig u r e 3 .9 a and 3 .9 b : v a r ia n c e r a t io s f o r e a c h vp f a m i l y .....................................................................................2 0 3
F ig u r e 4.1 P B M C in c u b a t io n w it h S A g (T S S T - 1 o r S E B ) r e s u l t s i n V p r e s t r ic t e d C D 6 9
UPREGULATION AT 4 HOURS...................................................................................................................................................2 2 4
F ig u r e 4 .2 P u r if ie d C D 3 ‘^l y m p h o c y t e p o p u l a t io n ( d e v o id o f c e l l s e x p r e s s i n g M H C c l a s s II)
INCUBATION WITH SA G AT 4 HOURS.................................................................................................................................. 2 2 6
F ig u r e 4 .3 U p r e g u l a t io n of MHC class II on HUVEC by IN F -y ...........................................................................2 2 9
F ig u r e 4 .4 v p spe c if ic C D 4 a n d C D 8 T l y m p h o c y t e a c t iv a t io n w it h in t h e a d h e r e n t p o p u l a t io n
OF T CELLS (MEAN, S E M OF 4 EXPERIMENTS)............................................................................................................... 2 3 0
F ig u r e 4 .5 v p spe c if ic C D 4 AND C D 8 T l y m p h o c y t e a c t iv a t io n w it h in t h e n o n - a d h e r e n t
POPULATION OF T CELLS (MEAN, S E M OF 4 EXPERIMENTS)....................................................................................2 3 2
F ig u r e 4 .6 V P - s pe c if ic T c e l l a d h e r e n c e to H U V E C ...................................................................................................2 3 5
F ig u r e 4 .7 : C D 4 a n d C D 8 V p r a t io s in r e s p o n s e t o T S S T -1 ( f ig u r e s 4 .7 a a n d 4 .7 b ) a n d S E B
( f ig u r e s 4 .7 c a n d 4 .7 d ) ( m e a n , S E M o f 3 e x p e r im e n t s ) .................................................................................. 2 3 6
F ig u r e 4 .8 F l o w c y t o m e t r y d o t -p l o t s o f c e l l a d h e s io n m o l e c u l e e x p r e s s i o n o n H U V E C c o
c u l t u r e d WITH pu r if ie d CD3'" T c e l l s in t h e p r e s e n c e o r a b s e n c e o f T S S T -1 ................................2 3 9
F ig u r e 4 .9 In t e g r in b l o c k a d e o f T S S T -1 m e d ia t e d T c e l l a d h e r e n c e t o H U V E C .................................2 4 3
F ig u r e 4 .1 0 A b s o l u t e n u m b e r o f a d h e r e n t T c e l l s : e f f e c t o f L F A -1 b l o c k a d e ................................. 2 4 4
F ig u r e 4.11 V p r e p e r t o ir e o f a d h e r e n t T c e l l s : e f f e c t o f L FA -1 b l o c k a d e .........................................2 4 6
F ig u r e 4 .1 2 V P - specdfic C D 6 9 e x p r e s s io n o f a d h e r e n t T c e l l s : e f f e c t o f L FA -1 b l o c k a d e ..........2 4 7
F IGURE 4.13 A b s o l u t e n u m b e r o f a d h e r e n t T c e l l s : e f f e c t o f V L A -4 b l o c k a d e ................................ 2 4 8
F ig u r e 4.1 4 v p r e p e r t o ir e o f a d h e r e n t T c e l l s : e f f e c t o f V L A -4 b l o c k a d e .........................................2 4 9
F ig u r e 4.1 5 V P - s p e c if ic C D 6 9 e x p r e s s io n o f a d h e r e n t T c e l l s : e f f e c t o f V L A -4 b l o c k a d e .........2 5 0
F ig u r e 5.1 E n d o t h e l ia l m ic r o p a r t ic l e f o r m a t io n (e x p r e s s e d a s m il l io n s M . o f su p e r n a t a n t )
FROM H U V E C IN RESPONSE TO T N F -a , S A g st im u l a t io n ( c o -c u l t u r e o f M H C c l a ss I f H U V E C
WITH C D 3 T LYMPHOCYTES IN THE PRESENCE OF T S S T -1 10 NG/ML) OR CONTROL (RESTING) H U V E C .
............................................................................................................................................................................................... 27 3
F ig u r e 5.2 In c u b a t io n o f p u r if ie d C D 3 ^ l y m p h o c y t e s w it h S A g a f t e r 24 h o u r s ................................. 2 7 4
F ig u r e 5.3 : T o t a l m ic r o p a r t ic l e n u m b e r s ................................................................................................................ 2 7 8
F ig u r e 5.4 E s e l e c t in a n d C D 105 m ic r o p a r t ic l e n u m b e r s ................................................................................2 8 0
F ig u r e 5.5 C o m p l e t e m ic r o p a r t ic l e p r o f il e .............................................................................................................2 8 2
F ig u r e 5 .6 C h a n g e s in M P p r o f il e b e f o r e a n d a f t e r in d u c t io n o f r e m is s io n o f v a s c u l i t i s .......... 2 8 5
F i g u r e 5.7 R e c e i v e r o p e r a t o r c h a r a c t e r i s t i c (R O C ) c u r v e s f o r E - s e l e c t i n a n d C D 105 E M P s f o r
THE DIAGNOSIS OF ACTIVE VASCULITIS .................................................................................................................... 2 9 2
F ig u r e 6.1 W h a t is T c e l l v p s k e w in g ? ...................................................................................................................... 3 0 4
F ig u r e 6 .2 v p s p e c if ic e x p r e s s io n o f C D 6 9 o n p e r ip h e r a l T c e l l s t a k e n f r o m a 3 - m o n t h o l d
INFANT W IT H K D .............................................................................................................................................................3 0 8
F ig u r e 6.3 S u r f a c e e x p r e s s io n o f E s e l e c t in o n T c e l l s f o l l o w in g c o - c u l t u r e o f T c e l l s w it h
H U V E C AND S A g ......................................................................................................................................................... 3 1 7
F ig u r e 6 .4 P r o p o s e d m o d e l f o r SA g -in d u c e d v a s c u l a r in j u r y ..................................................................... 3 1 9
Tables
T able 1.1: T he C la ssifica tio n o f P aed iatric V ascu litid es b a sed on F in k .............................................. 20
T able 1.2: K a w asaki D isea se : diagnostic c riteria (D a ja n i , T aubert and o t h e r s , 1993)................. 33
T able 1.3 C onventio nal a n tig en versus su pera n tig en presen ta tio n to T c e l l s .............................. 114
TABLE 1.4 S t u d i e s f o r a n d a g a i n s t t h e S A g t h e o r y i n KD ............................................................................122
TABLE 2.1 : F lu o ro c h r o m e -co n ju g a ted a ntibodies fo r flo w c y to m etr y ............................................... 136
T able 2.2: C entrifu g a tio n protocol fo r iso la tio n of M P fro m w h ole b l o o d ....................................165
T able 3.1 C linical features of pa tien ts w ith prim ary system ic v a sc u l it is ........................................ 181
T able 5.1 C orrelatio n b e t w e e n EM P and clinica l in d ices of v a sc u litic d ise a se a c t iv it y ........ 288
T a b le 5.2 T e s t c h a r a c t e r i s t i c s o f EMP s f o r t h e d ia g n o s is o f a c t i v e v a s c u l i t i s ............................ 290
Table of contents
FIGURES______________________________________________________________________________4
TABLE OF CONTENTS_________________________________________________________________ 7
ABBREVIATIONS_____________________________________________________________________ 12
CHAPTER 1; INTRODUCTION AND THESIS AIMS______________________________________ 15
1*1 ^^S^%UIjl 1 IS 'I'lHi

1.1.1 In tr o d u ctio n - defin itio n of v a s c u l it is ...................................................................................................16
T able 1.1: T h e Cla ssifica tion o f P a ed iatric V ascu litides b a sed o n F in k ......................................... 20
1.1.3 D escriptio n of in dividual v a scu litic s y n d r o m e s ........................................................................... 21
1.1.3.1 Polyarteritis nodosa (PAN) and cutaneous polyarteritis........................................................... 21
1.1.3.2 Microscopic polyangiitis...............................................................................................................26
1.1.3.3 Kawasaki disease (KD).................................................................................................................29
1.1.3.3 Kawasaki disease (KD).................................................................................................................30
T able 1.2: K aw asaki D isea se : d iagnostic c riter ia (D a ja n i , T aubert an d o t h e r s , 1993)............ 33
1.1.3.4 Wegener’s granulomatosis (WG).................................................................................................41
1.1.3.5 ChurgStrauss syndrome...............................................................................................................42
1.1.3.6 Primary angiitis o f the central nervous system........................................................................... 43
1.1.3.7 Henoch Schonlein purpura (MSP)...............................................................................................43
1.1.3.8 Hypersensitivity vasculitis............................................................................................................53
1.1.3.9 Hypocomplementaemic urticarial vasculitis...............................................................................54
1.1.3.10 Vasculitis associated with connective tissue disorders.............................................................54
1.1.3.11 Takayasu disease.........................................................................................................................55
1.1.3.12 Miscellaneous vasculitides o f the young....................................................................................56
1.1.4. C o n c l u s io n .........................................................................................................................................................57
L2 THE IMMUNO PATHOGENESIS OF VASCULITIS_______________________________ 58

1.2.1 In t r o d u c t io n ...................................................................................................................................................... 58
1.2.2 In fec tio n s and v a s c u l it is .............................................................................................................................59
1.2.2.1 Viruses causing vasculitis.............................................................................................................59
1.2.2.2 Bacteria causing vasculitis...........................................................................................................66
1.2.2.3 Parasites causing vasculitis..........................................................................................................69
1.2.2.4 Conclusion.....................................................................................................................................69
1.2.3 M alignancy and v a s c u l it is .........................................................................................................................70
1.2.3.1 Infectious causes o f vasculitis in malignancy.............................................................................. 70
1.2.3.4 Paraneoplastic vasculitis.............................................................................................................. 71
1.2.3.5 Angiocentric lymphomas............................................................................................................... 72
1.2.4 A u to antibodies and v a s c u l it is ..................................................................................................................73
1.2.4.1 Antineutrophil cytoplasmic antibodies (ANCA).......................................................................... 73
1.2.4.2 Anti endothelial cell antibodies....................................................................................................87
1.2.5 Im m u n e co m plex es and v a s c u l it is ............................................................................................................ 94
1.2.6 C ryog lo bu lin s and v a s c u l it is ................................................................................................................... 96
1.2.7 T he ENDOTHELIUM AS A TARGET AND AMPLIFIER OF INFLAMMATION IN VASCULITIS..........................97
1.2.7.1 The endothelial cell as a targetfo r injury in vasculitis.............................................................. 97
1.2.7.2 The endothelial cell amplifies inflammation...............................................................................97
1.2.8 T CELLS AND VASCULITIS..................................................................................................................................103
1.2.8.1 T cells and Giant cell arteritis................................................................................................. 104
1.2.8.2 T cells and small vessel vasculitides .....................................................................................................104
1.2.8.3 T cells a nd Kawasaki disease ................................................................................................................. 106
1.2.9 C o nclu d in g r em a rk s relatin g to th e pa th og enesis o f v a sc u l it is ...........................................107
C.TJI^n'IS •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••« 1OS

1.3.1 I n t r o d u c t io n .................................................................................................................................................... 108
1.3.2 W h a t is a su pe ra n tig en ? ............................................................................................................................. 108
1.3.2.1 Conventional antigen versus superantigen presentation to T cells .............................................. I l l
T able 1.3 C onv entional antig en versu s supera n tig en presen ta tio n to T c e l l s .........................114
1.3.2.2 Superantigens and vasculitis .................................................................................................................. 119
T able 1.4 S tudies fo r a nd against th e SA g th eo ry in K D .......................................................................122
1.3.2.3 Concluding remarks regarding S 4 g j and vasculitis ......................................................................... 129
1.4 AIMS OF THIS THESIS__________________________________________________________ 130
CHAPTER 2. MATERIALS AND
2.1 INTRODUCTION_________________________________________________________________ 132
2.2 SUBJECTS, CLASSIFICATION OF VASCULITIC SYNDROMES, AND ASSESSMENT OF

DISEASE ACTIVITY_________________________________________________________________ 132
2.2.1 S u bjects and cla ssifica tio n o f v a sculitic s y n d r o m e s .................................................................. 132
2.2.2 T h e assessm ent o f v a sculitic d isea se a c t iv it y .................................................................................133
2.3 MATERIALS_____________________________________________________________________ 135

2.3.1: F l u o r o c h r o m e -co n ju g a ted a ntibo d ies fo r flo w c y to m etr y ....................................................135
T able 2.1 : F lu o ro ch ro m e - con ju g a ted antibodies for fl o w c y t o m e t r y ..........................................136
2.3.2 C ytokines and pro tein r ea g en ts ..............................................................................................................137
2.3.3 T issue cu lture m e d ia ....................................................................................................................................137
2.4 MlETrH^)^)S .....M.........M..M.........MM..M.....................M...M..M.........M..M..M..M.M M . 137
2.4.1 : I so la tio n o f periph era l blood m o n o nuclear c ells and T cells f r o m w h ole b l o o d 137
2.4.1 P repa ra tio n o f pu rified CD3'" T ly m phocytes f r o m P B M C s ........................................................ 138
2.4.2 C a lcu la tio n of cell num ber and cell v ia b il it y ...............................................................................139
2.4.3 S to ra g e o f c e l l s .............................................................................................................................................139
2.4.4 F l o w cytom etry of PBM C or T c e l l s ....................................................................................................142
2.4.5 In v i t r o te c h n iq u e f o r v a l i d a t i o n o f e f f e c t o f S A gs o n T c e l l v p r e p e r t o i r e sk e w in g
AND T c e l l a c t i v a t i o n .............................................................................................................................................143
2.4.6 E n d o thelia l cell c u l t u r e ..........................................................................................................................148
2.4.7 H U V EC culture m e d ia ..................................................................................................................................148
2.4.8 F o eta l calf s e r u m ......................................................................................................................................... 149
2.4.9 P roto co l for isolation o f H U V E C ......................................................................................................... 149
2.4.10 H U V EC prim ary culture and sub -c u l t u r e .......................................................................................150
2.4.11 Id en tifica tio n o f H U V E C ..........................................................................................................................152
2.4.12 P ro tocol for d etectio n of cell ad h esion m o lecu le ex pr e ssio n b y flo w c y t o m e t r y .. 152
2.4.13 A ssay fo r en d o to x in c o n ta m in a tio n of H U V E C ........................................................................... 155
2.5 ISOLATION AND ANALYSIS OF WHOLE BLOOD CELLULAR AND PLATELET
2.5.1 P repa ra tio n o f platelet poor pla sm a fo r m ic r o pa rticl e a n a l y s is .........................................158

2.5.2 I so la tio n o f m icropa rticles fro m platelet po o r p l a s m a .............................................................158
2.5.3 : L a bellin g of m icro pa rticles w ith an n ex in V and m o n o clo n a l a n t ib o d ie s .......................158
2.5.4: F l o w cytom etric analysis of m ic r o pa rticl es .................................................................................. 159
2.5.5 D e term in a tio n o f a bsolute m icro pa rticle n u m b er per m l o f p l a s m a ....................................160
2.5.6 V a lidatio n of the technique fo r the ana lysis of m ic r o pa r t ic l e s ...........................................163
2.5.6.1: Reproducibility ofM P analysis.................................................................................................163
2.5.6.2: Effect o f different centrifugation protocols on microparticle analysis..................................163
Table 2.2 : Centrifugation protocol for isolation of MP from whole blood...........................165
2.5.6.3: Effect o f freeze-thawing on microparticle analysis.................................................................167
2.5.6.4: Effect o f delayed separation o f plasma from whole blood on microparticle analysis 169
2.5.6.5: Properties o f latex beads which affect microparticle analysis...............................................169
2.6 STATISTICS______________________________________________________________________172
CHAPTER 3; T CELL ACTIVATION AND V p REPERTOIRES IN CHILDHOOD

VASCULITIDES_____________________________________________________________________ 174
3.1 SUMMARY_______________________________________________________________________175
3.2 INTRODUCTION_________________________________________________________________ 177
3.3 lEN 1S IMETHÎDS .....m..m..m.........m..m.......m..m.....m..m......m.......m..m.....m..m..m.......................179

3.3.1 VASCULITIS PATIENTS................................................................................................................ 179
Table 3.1 Clinical features of patients with primary systemic vasculitis ...............................181
3.3.2 CONTROLS AND DISEASE CONTROLS.....................................................................................185
3.3.3 ANALYSIS OF PERIPHERAL BLOOD T CELL ACTIVATION MARKERS AND T CELL Vp
REPERTOIRES........................................................................................................................................... 186
3.3.4 IN VITRO TECHNIQUE FOR VALIDATION OF EFFECT OF SAgs ON T CELL Vp
REPERTOIRE SKEWING......................................................................................................................... 187
3.3.5 STATISTICS...................................................................................................................................... 187
3.4 RESULTS________________________________________________________________________ 188
3.4.1 PATIENT DEMOGRAPHICS...........................................................................................................188
3.4.2 T CELL ACTIVATION IN PATIENTS AND CONTROLS..........................................................189
3.4.4 T CELL Vp REPERTOIRES FROM PATIENTS AND CONTROLS: COMPARISON OF
MEANS........................................................................................................................................................ 195
3.4.4.1 PRIMARY SYSTEMIC VASCULITIS.........................................................................................195
3.4.4.2 KAWASAKI DISEASE................................................................................................................ 202
3.4.4.3 HENOCH SCHÔNLEIN PURPURA........................................................................................ 202
3.4.5 T CELL Vp REPERTOIRES: COMPARISON OF VARIANCE.................................................. 202
3.4.6 T CELL Vp EXPANSIONS AND DELETIONS............................................................................ 206
3.4.7 LONGITUDINAL STUDIES ON PRIMARY SYSTEMIC VASCULITIS CHILDREN........... 207
3.5 ^HS^Z-USSl^lN.........M..M.....M..M..M . . . . . . . . . .................... . M .. . M . .M . . .......M . . ....M .....M M ..... .M.....M..M......M...M..M..M..207
CHAPTER 4: Vp-RESTRICTED T CELL ADHERENCE TO ENDOTHELIAL CELLS: A

MECHANISM FOR SUPERANTIGEN DEPENDENT VASCULAR INJURY ___________ 214
4. 1 SU . M . . . . . . M . . M . . . . . M . . . . . . . . . . . . . . . M . . . . . . . . . . . . . M ..M .....M ....M ..M ..M .... . . . . . ....M ...M ......M .....M ..M ......M .M M ..M ..M ..... 215
4.2 lî 1R^)^)UC 1 IÎN ....... . . . . . . . . . M . . M . . . . . . . . . M . . . . . . . . . . . . M . . . . . M . . M . . . . . M . . . . . . . . M . .M .M ..........M ...M ..M .........M ......M ..M ...M .. 2 16
4.3 MATERIALS AND METHODS_____________________________________________________ 218

4.3.1 A ntibodies, cytokines, and protein reagents ......................................................................... 218
4.3.2 Isolation of human umbilical vein endothelial cells (HUVEC)....................................... 219
4.3.3 PBMC AND T CELL INCUBATION WITH SAG: DETERMINATION OF VP-SPECIFIC ACTIVATION........219
4.3.4 HUVEC-T cell co -culture experiments.................................................................................... 220
4.3.5 F low cytometry of T cells and HUVEC................................................................................... 220
4.3.6 Integrin BLOCKADE of SAg -mediated T cell adherence to HUVEC................................... 221
4.3.7 Statistics ..........................................................................................................................................222
4.4 RESULTS________________________________________________________________________ 223
4.4.1 SA gs activate T cells in a Vp-sPECiFic m a n n er , and req u ire M H C class II ex pr essin g
CELLS............................................................................................................................................................................... 223
4.4.2 H U V E C UPREGULATE M H C class II IN RESPONSE TO INF-y AND BECOME COMPETENT SAG-
PRESENTING CELLS FOR T CELLS RESULTING IN VP-SPECIFIC T CELL ACTIVATION AND ADHERENCE TO
H U V EC MONOLAYERS............................................................................................................................................... 228
4.4.3 SAG-DEPENDENT T CELL-MEDIATED ENDOTHELIAL CELL ACTIVATION: UPREGULATION OF CELL
ADHESION MOLECULES................................................................................................................................................ 238
4.4.4 In t e g r in blo ck a d e o f SA g - m ediated T cell a d h eren ce to H U V E C ........................................241
4.5 ^)ISC«%ISSI^)N.....w #.........M..w*. ......w......M.....w#..... . . . . . . . . . . . . . .............M.. 251
CHAPTER 5: ENDOTHELIAL AND PLATELET MICROPARTICLES IN CHILDHOOD

VASCULITIDES: A WINDOW TO THE ACTIVATED ENDOTHELIUM IN VASCULITIS OF
THE ^lUNCr. . M « . . . . M.....MM..M. . «. M. . . . ........ ....................M258
5.1 SUMMARY_______________________________________________________________________259
5.2 JLî l lt^)DU(.x 1 I^)N . .. . .M . .. . .. M . .. . .. . .. . .. . M . . .. . .. M .. . .. . M .. . . . . . . . . . . . M . . . M . . M . . M . . M . . . . . . . . . . . . . M . . . M . . . . . . . . . . M . M . . M . . M . . . . . M M . . 26 I

5.2.1 C ellu la r m em bra n e pho sph o lipid a sy m m etr y : pa th o ph y sio lo g ica l im p l ic a t io n s 261
5.2.2 E n d o th elia l m icro pa rticles (EM P)....................................................................................................... 263
5.2.3 EM P: relevan ce to the v a s c u l it id e s .................................................................................................... 265
5.2.4 S tud y a i m s ........................................................................................................................................................ 265
S 3 METHODS_______________________________________________________________________ 266
5.3.1 In VITRO stu d ies : analysis of en d othelia l m icropa rticles in H U V E C -T cell c o - culture
SUPERNATANTS..............................................................................................................................................................266
5.3.2 In VIVO stu d ie s : P a t ie n t s ............................................................................................................................ 266
5.3.2 P r epa ra tio n o f platelet poor p l a s m a ..................................................................................................268
5.3.3 I so la tio n o f m icropa rticles fro m pla telet po o r p l a s m a ............................................................ 269
5.3.4 S tatistica l a n a l y s is .................................................................................................................................... 269
5.4 RESULTS________________________ 270

5.4.1 SAG-DEPENDENT T CELL-MEDIATED ENDOTHELIAL CELL ACTIVATION AND INJURY: GENERATION OF
ENDOTHELIAL MICROPARTICLES (EM P).................................................................................................................. 270
5.4.2 C o m pa riso n o f m ea n m icro pa rticle num bers betw een pa tien t g r o u p s ................................. 276
5.4.2 L ongitudinal analysis of en d othelia l and platelet m icropa rticle pro files b efo r e and
AFTER INDUCTION OF REMISSION OF VASCULITIS..................................................................................................284
5.4.3 C orrela tio n of E M P s w ith BV A S and co nven tiona l acute ph a se r e a c t a n t s ...................284
5.4.4 T est ch a racteristics of E M P s fo r the diagnosis o f activ e v a s c u l it is ................................. 287
T able 5.1 C orrelatio n betw een EM P and clinica l in d ices of v a sc u l it ic d ise a se a ctiv ity ...288
T able 5.2 Test characteristics of E M P s fo r the dia g n o sis o f active v a s c u l it is ....................... 290
CHAPTER 6: GENERAL DISCUSSION AND FUTURE DIRECTIONS_____________________ 301
6.1 CrENFIj^l^Îj ^HSCfUSSIÎN.m*.m..................... m.m............... m. . . . . . . . . . . . . . 3 0 2

6.1.1 W h a t is true sk ew ing o f the v p r eperto ire ? .....................................................................................302
6.1.2 Vp-SPECIFIC ACTIVATION IN THE ABSENCE OF VP SKEWING.................................................................... 305
6.1.3 vp-RESTRICTED BINDING OF THE T CELLS TO THE H U V EC MONOLAYER: COMPARTMENTALISATION
OF THE IMMUNE RESPONSE IN VASCULITIS.............................................................................................................. 309
6.1.4 C l o n a l v ersu s poly clo n a l V p r estricted e x pa n sio n .................................................................... 310
10
6.1.5 F l o w cytom etry versus polym erase chain rea ctio n fo r the study of T cell Vp
REPERTOIRES.................................................................................................................................................................. 311
6.1.6 C olo n isa tio n of study subjects w ith SA g -pro d u cin g o r g a n ism s ............................................ 313
6.1.7 H L A - m ism a tch betw een T cells and H U V EC in c o -cu ltu re e x p e r im e n t s ........................... 314
6.1.8 E M P GENERATION FROM SA g -DEPENDENT T CELL MEDIATED ACTIVATION OF H U V E C ................ 315
6.1.9 A PROPOSED MODEL FOR SAG-INDUCED VASCULAR INJURY............................................................................. 318
6.1.10 F u tu re w o r k : SA g peptid e a n t a g o n ist s ............................................................................................ 320
6.1.11 C ellular m ic r o pa rticl es : P otential n ov el m ech a n ism s o f v a sc u l a r in ju r y ................. 320
6.1.12 O r g a n -specific end oth elia l m ic r o pa rticl es ? ................................................................................ 322
6.1.13 C on clud ing REMARKS..................................................................................................................................323
APPENDIX 1; CLASSIFICATION OF VASCULITIC SYNDROMES_______________________ 324
APPENDIX 2: THE BIRMINGHAM VASCULITIS ACTIVITY SCORE (BVAS) (LUQMANI,

BACON AND OTHERS, 1994)_________________________________________________________ 327
APPENDIX 3: 96-WELL ANTIBODY PLATE PLAN FOR DETERMINATION OF THE T CELL

v p REPERTOIRE____________________________________________________________________ 329
APPENDIX 4; 96-WELL ANTIBODY PLATE PLAN FOR DETERMINATION OF T CELL VP-

S^ÊC.IFIC^ C-^D69 ^lESSIÎN •••••«••••••••••••••••••••••••••••••••••••••••«•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••330
APPENDIX 5: FLOW CYTOMETRY INSTRUMENT SETTINGS FOR PBMC AND T CELLS,

HU\ ÊC. ^ H' I I CI ^ ^ ^ 1
APPENDIX 6: EQUATION USED TO DERIVE THE NUMBER OF 3 ^M LATEX BEADS ADDED

FOR THE ANALYSIS OF MICROPARTICLES (SIGMA PRODUCT INFORMATION)______ 332
APPENDIX 7; PLATE PLAN FOR T CELL AND HUVEC CO-CULTURE EXPERIMENTS 333
APPENDIX 8; PLATE PLAN FOR INTEGRIN BLOCKADE OF SAG-MEDIATED T CELL

ADHESION TO HUVEC..______________ 334
REFERENCE LIST__________ 335
]PUIfIjIC^^^lT^)^fS 'I'HlS 'H lESIS .............................M.. . . . . . . . . . . . . . . . . . . . . . . . M......M.M......M..M..... 402
11
Abbreviations
Abbreviation Definition
ACR American College of Rheumatology
AECA Anti endothelial cell antibody
AILD Angioimmunoblastic T cell lymphoma disease
ALCL Angiotropic large cell lymphoma
ANOVA Analysis of variance
APC Antigen presenting cell
BPSU British Paediatric Surveillance Unit
BSA Bovine serum albumin
BVAS Birmingham Vasculitis Activity Score
CAA Coronary artery abnormality
CAM Cell adhesion molecule
cANCA Cytoplasmic antinuclear cytoplasmic antibody
CDR3 Complementarity determining region
CINCA Chronic infantile neurological cutaneous articular syndrome
CLIP Class n associated invariant chain peptide
CNS Central nervous system
CRP C reactive protein
CSF Cerebro-spinal fluid
CSS Churg Strauss Syndrome
CTLA-4 CD152
EBV Epstein-Barr Virus
EC Endothelial cell
ECAF Endothelial cell attachment factor
ECG Electrocardiograph
ELISA Enzyme linked immunosorbant assay
EMP Endothelial microparticle
ESR Erythrocyte sedimentation rate
FACS Flow activated cell sorter
FCS Foetal calf serum
GBM Glomerular basement membrane
GCA Giant cell arteritis
HBV Hepatitis B virus
HCV Hepatitis C virus
mv Human Immunodeficiency virus
HMEC Human microvascular endothelial cell
HSP Henoch Schonlein Purpura
HUVEC Human umbilical endothelial cell
ICAM-1 Intercellular adhesion molecule-1
nF Indirect immunofluorescence
IL Interleukin
IMS Industrial methylated spirit
INF-y Interferon-y
12
ISKDC International Study of Kidney Diseases in Children
IVIG Intravenous immunoglobulin
JDM Juvenile dermatomyositis
JIA Juvenile idiopathic arthritis
KD Kawasaki disease
LFA-1 Lymphocyte functional antigen-1
LR Likelihood ratio
MAM Mycoplasma arthritides mitogen
MFI Median fluorescence index
MHC Major histocompatibility complex
MP Microparticle
MPA Microscopic polyangiitis
MPO Myeloperoxidase
MS Multiple sclerosis
NO Nitric oxide
NOS Nitric oxide synthetase
NPV Negative predictive value
PAN Polyarteritis nodosa
pANCA Perinuclear anti neutrophil cytoplasmic antibody
PBMC Peripheral blood mononuclear cells
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PMP Platelet microparticle
PPV Positive predictive value
PR3 Proteinase 3
PS Phosphatidylserine
PSV Primary systemic vasculitis
RAG Recombinase activating gene
RANTES Regulated upon activation, normal T cell expressed and secreted
RCT Randomised controlled trial
RFLP Restriction fragment length polymoprphism
ROC curve Receiver operator characteristic curve
SABE Subacute bacterial endocarditis
SAg Superantigen
SEB Staphylococcal enterotoxin B
SEM Standard error of the mean
SEE Systemic lupus erythematosus
SM Sphingomyelin
SV Systemic vasculitis
SW Small vessel vasculitis
TC T cell
TCR T cell receptor
TD Takayasu disease
TNF-a Tumour necrosis factor-a
TPA Tissue plasminogen activator
TSS Toxic shock syndrome
13
TSST-1 Toxic shock syndrome toxin type 1
TTP Thrombotic thrombocytopenic purpura
VCAM-1 Vascular cell adhesion molecule-1
VLA-4 Very late antigen-4
vzv Varicella zoster virus
WG Wegener’s granulomatosis
WHO World Health Organisation
14
Chapter 1: Introduction and thesis aims
1.1 Vasculitis of the young
1.2 The immuno-pathogenesis of vasculitis
1.3 Superantigens and vasculitis
1.4 Thesis aims
15
1.1 Vasculitis of the young
1.1.1 Introduction- definition o f vasculitis
The vasculitides are a group of disorders characterised by the presence o f inflammatory
infiltrates (which may be predominantly neutrophilic, eosinophilic, or mononuclear) in
the walls of blood vessels, with resultant tissue ischaemia and necrosis. The term
vasculitis refers to the presence of inflammation in a blood vessel wall (Petty and
Cassidy, 2001a). Perivasculitis describes inflammation around the blood-vessel wall but
without involvement of the mural structure itself (Petty and Cassidy, 2001a).
Vasculopathy is a broader term encompassing abnormalities o f blood vessels that may be
inflammatory, but may also be developmental or degenerative. More recently, the
distinction between certain degenerative vasculopathies and inflammatory vasculopathy
has become less clear with the emerging body of evidence that the pathogenesis of
atherosclerosis (previously considered to be a degenerative vasculopathy) involves an
important inflammatory component (Alexander and Dzau, 2000). Vasculitis may affect
blood vessels of all sizes and may be the major manifestation o f a disease (primary
systemic vasculitis e.g. microscopic polyangiitis), or one aspect of a more widespread
disease (secondary vasculitis e.g. systemic lupus erythematosus and the other connective
tissue diseases) (Flores-Suarez and Alarcon-Segovia, 2000).
16
1.1.2 Classification o f the paediatric vasculitides
Classification of the paediatric vasculitic disorders has proved difficult because o f the
absence of sensitive and specific diagnostic tests and an incomplete understanding of the
aetiology o f most vasculitis syndromes. Moreover, as in adult vasculitic syndromes,
many children have constellations of symptoms and findings that overlap the clinical
features o f the various individual diseases that will be discussed below.
The most widely used classification system for the purposes o f research is the Chapel Hill
Consensus on the classification of the primary systemic vasculitides (Jennette, Falk and
others, 1994) which is widely accepted in adult practice and is shown in Appendix 1.
This classification system is based upon the smallest blood vessel involved in the disease
process: Wegener’s granulomatosis, for instance, is classified as a small vessel disease
but vasculitis is detected in small and medium-sized arteries and larger vessels, including
the renal artery and aorta.
Importantly, the Chapel Hill criteria and the other main set of classification criteria for
the vasculitides- the American College of Rheumatology (ACR) criteria- both of which
are used for the description of patients in this thesis (see Chapter 2 and Appendix 1) have
never been evaluated in children (Petty and Cassidy, 2001a). Indeed there is much debate
regarding the most reliable classification system, with recent studies favouring the Chapel
Hill consensus. In this thesis, when considering aetio-pathogenesis (Chapter 3) both the
Chapel Hill and the ACR classification criteria were utilised because neither system on
its own classifies the paediatric vasculitides absolutely. For example, since the Chapel
17
Hill consensus defines a vasculitis based on involvement of the smallest artery
manifesting vasculitic changes and as such the diagnosis of classical polyarteritis nodosa
(PAN) would be precluded using this system if rash (such as purpura or livedo reticularis-
signs of small vessel vasculitis) was present. In contrast, the presence of livedo reticularis
is one o f the defining criteria for classical PAN using the ACR criteria and small vessel
vasculitic skin rash such as livedo reticularis is fi"equently observed in children with
classical PAN (Dillon, 1998; Petty and Cassidy, 2001 b; Ozen, Besbas and others, 1992).
For the purposes of this thesis, therefore, both the ACR and Chapel Hill criteria were
used, which classified the subjects adequately (but by no means absolutely), to allow
comparison with previously published studies. The limitations of such classification
systems must be borne in mind, however. Indeed, the sensitivity and specificity of the
ACR criteria for the classification of PAN in adults are relatively low at 82.2% and
86.6% respectively (Lightfoot, Michel and others, 1990), emphasising the limitations of
using such criteria for the classification vasculitis for research purposes.
In addition, an important concept worthy of emphasis is that classification criteria are not
the same as diagnostic criteria although the former are often confused and misused as the
latter (Hunder, 1998). Classification criteria work best in the study of groups o f patients
and work less well in the diagnostic evaluation o f individual patients. As an example, the
ACR criteria for PAN were designed to differentiate PAN from other types o f vasculitis
but not to diagnose vasculitis in the first instance (Hunder, 1998).
18
In clinical practice, one classification that relates specifically to the paediatric
vasculitides that has proved useful is based on that of Fink (Fink, 1986), and is outlined
in Table 1.1. This system, since it pertains to paediatric vasculitides, will be used as a
template for the consideration of individual vasculitic syndromes in this chapter.
19
Table 1.1: The Classification o f Paediatric Vasculitides based on Fink
T^ble 1:1 The g( cbWhood vasetilitliies hü&ed on Fisk
Polyarteritis
M acroscopic
M icroscopic
Cutaneous
Kawasaki disease
M ucocutaneous Lymph Node Syndrome
Granulomatous vasculitis
Wegener's Granulomatosis
Churg-Strauss Syndrome
Primary angiitis of the central nervous system
Leukocytoclastic vasculitis
Henoch-Schonlein purpura
Hypersensitivity angiitis
Hypocomplementemic urticarial vasculitis
Vasculitis associated with connective tissue disease
Systemic lupus erythematosus
Juvenile chronic arthritis
Mixed connective tissue disease
Dermatomyositis
Scleroderma
Giant cell arteritis
Takayasu disease
Miscellaneous vasculitides
20
1.1.3 Description o f individual vasculitic syndromes
1.1.3.1 Polyarteritis nodosa (PAN) and cutaneous polyarteritis
The classic form of polyarteritis, polyarteritis nodosa (PAN), is a necrotizing vasculitis
associated with aneurysmal nodules along the walls o f medium-sized muscular arteries,
and first described in detail in 1866 by Kussmaul and Maier (Kussmaul and Maier, 1866;
Dillon, 1998; Petty and Cassidy, 2001b). Although there is an overlap with smaller vessel
disease, it is distinct from microscopic polyangiopathy, and occurs more commonly in
childhood than this latter disorder (Brogan and Dillon, 2000a). The main clinical features
are malaise, fever, skin rash (figure 1.1), abdominal pain and arthropathy (figure 1.2)
(Besbas, Ozen and others, 2000; Brogan and Dillon, 2000a). Other features include
testicular pain, myalgia, hypertension, neuropathy, renal failure, organic psychosis, and
myocardial ischaemia (Besbas, Ozen and others, 2000; Brogan and Dillon, 2000a;
Lightfoot, Jr., Michel and others, 1990). Visceral angiography plays a key role in the
diagnosis (figure 1.3) (Bron, Strott, and Shapiro, 1965; McLain, Kelsch, and Bookstein,
1972; Brogan, Davies and others, 2002).
Cutaneous polyarteritis is a syndrome associated with crops o f painful skin nodules,
livedo reticularis, and often with a story of preceding upper respiratory tract infection
(David, Ansell, and Woo, 1993; Diaz-Perez and Winkelmann, 1974; Fink, 1991). The
condition responds to non-steroidal anti inflammatory drugs but can require steroids. If
streptococcus induced, prophylactic penicillin has a role. Cutaneous vasculitis usually
21
runs a benign course although in some patients the cutaneous features evolve into
systemic polyarteritis nodosa. Relapses, particularly in association with recurrent
22
Figure 1.1 Spectrum of vasculitic skin rash in PAN
Lie LID
lA : Severe skin necrosis and gangrene; IB: Ecchymotic and purpuric skin lesions;
1C: Livedo reticularis; ID : Patchy necrotic skin ulceration (“punched-out” vasculitic rash)
Figure 1.2: Symmetrical polyarthritis in a 12 year old boy with PAN afTecting knees and ankles.
Figure 1.3: Renal angiography in PAN
pqJA/CO
PD/X
A/CO
Figure 1.3A: Renal angiogram from Figure 1.3B: Renal angiogram

a 6 year old girl with PAN from an 8 year old boy with
demonstrating florid aneurysmal PAN demonstrating less florid
and non-aneurysmal changes. aneurysmal and non-
Large aneurysms(LA), small aneurysmal changes. Perfusion
aneurysms (SA), perfusion defect defect (PD), small aneurysm
(PD), arterial cut-off (CO), lack of (SA) in association with arterial
crossing of peripheral renal arteries cut-off (CO).
(X), collateral artery (Col).
streptococcal infection are seen in up to 25% of cases. Clinicians seeing this condition
usually advise continuing penicillin prophylaxis throughout childhood to prevent relapses
since it is amongst the relapsing group that systemic vasculitis tends to occur.
1.1.3.2 Microscopic polyangiitis
Microscopic polyangiitis (MPA, formerly microscopic polyarteritis) differs from classic
PAN by the presence of extensive glomerular involvement, and may be defined as small
vessel vasculitis with focal segmental glomerulonephritis but without granulomatous
disease of the respiratory tract (Savage, Winearls and others, 1985). Clinically, it can be
difficult to distinguish from Wegener’s granulomatosis, and often presents with rapidly
progressive pauci-immune glomerulonephritis (Jardim, Leake and others, 1992), in
association with perinuclear anti neutrophil cytoplasmic antibody (pANCA) positivity
(Jennette, Falk and others, 1994).
Treatment of PAN and MPA
Treatment for both PAN and MPA consists of steroids, anti-platelet agents, and an
additional cytotoxic agent, ordinarily cyclophosphamide (Adu, Pall and others, 1997;
Fauci, Katz and others, 1979). Cyclophosphamide is usually administered orally for 2-3
months at 2 mg/kg/day to induce remission (Brogan and Dillon, 2000b). Pulsed
intravenous cyclophosphamide may have advantages over the oral route in reducing the
total cumulative dose and hence side effects, but it may not be as effective as the daily
oral regimen in aggressive disease for the prevention o f relapses (Gaskin G and Pusey,
1998). Maintenance therapy is usually with oral azathioprine at a dose of 2 mg/kg/day,
with low dose alternate day prednisolone (0.2-0.5 mg/kg), and anti-platelet agents
26
(Brogan and Dillon, 2000b). If remission with this regimen is not maintained, then
cyclosporin or mycophenolate mofetil may prove useful, although the published evidence
for the use of these agents in this context is lacking. The guideline that is currently used
at Great Ormond St Hospital for Children, London (and hence the treatment that the
majority of the patients described in this thesis received) is outlined in figures 1.4 and
1.5. Currently, the mortality for PAN Great Ormond Street Hospital, London is about
10%, which compares favourably with many adult series, and other paediatric series of
PAN (Besbas, Ozen and others, 2000; Dillon, 1998).
27
F ig u re 1.4: G u id e lin e f o r t h e t r e a t m e n t o f c h ild h o o d PAN a n d r e l a t e d v a s c u litid e s
CONSIDER
• Single dose of IV
Cyclophosphamide 750
INDUCTION THERAPY (2/12)
mg/m2 if previously
• Prednisolone 60 mg/m^ OD for 4 /52, weaning over
next 6-8 weeks (depending on response to Rx) to 0.5 pven oral CYC for
mg/kg on alternate days, OR IV methyl prednisolone induction of remission
600 mg/ m^ (max 1g) for 3 consecutive days • Methylprednisolone 600
Failed mg/m2 (max Ig) IV X 3
followed by oral prednisolone as above remission
• Cyclophosphamide 2 mg/kg PO OD for 2/12 OR 750 if not previously given as
first line
mg/m2 IV once a month for 6 months (reduce dose if
renal failure) • 5 or 10 day course of
daily 2 volume plasma
• Aspirin 2 mg/kg OD (OR Dipyridamole 2.5 mg/kg
exchange with 4.5%
BD if aspirin contraindicated)
human albumin solution
• Second course of oral
cyclophosphamide
2mg/i:g OD for 2/12
Remission
MAINTENANCE THERAPY for 18/12 to 3 Major relapse

years for PAN; lifelong therapy for Wegeners whilst on
• Azathioprine 2-2.5 mg/kg PO OD (start 3-5 maintenance
days after stopping CYC) therapy
• Prednisolone 0.2-0.5 mg/kg alternate days
• Aspirin 2 mg/kg OD OR Dipyridamole 2.5
mg/kg BD NOTES:
• Consider ranitidine if abdo pain 1. Second line maintenance agents
• If stopping Rx, stop azathioprine first, then 1. MMF
wean prednisolone over next 12 months 2. Cyclosporine A
3. MTX
2. Consider sperm cryopreservation for
all post-pubertal males receiving CYC,
Minor relapse: increase oral prednisolone if and oocyte preservation for females
not steroid toxic 3. For monitoring of complications of
Recurrent minor relapses or "grumbling therapy refer to table 2 [4]
vasculitis": consider IV pulsed 4. Beware neutropaenia as
Methylprednisolone and/or switch to prednisolone dose is weaned during
second line maintenance therapy maintenance phase of therapy
5. Miscellaneous vasculitides such as
Behçet’s may require colchicine or
thahdomide
28
F ig ure 1.5: T h e treatm ent of paediatric m icroscopic poly a n g iitis and r ela ted crescentic
NEPHRITIDES
Crescentic GN on renal biopsy
Linear staining on Pauci-immune on Granular deposits on

immunofluorescence immunofluorescence- immunofluorescence-
consider microscopic indicative of immune complex
polyarteritis or "renal- disease
limited vasculitis"-
mav be ANCA +
Anti GBM + Electron microscopy
Plasma exchange for 10-14 days Treatment as per Treat specific disorders (refer to
(2 volume, 4.5% HAS)- or until vasculitis algorithm relevant protocols):
anti GBM Ab disappears above, but use plasma • HSP
Pulsed IV Methyl prednisolone exchange as first line • IgA nephropathy
600 mg/m2 (max 1g) X3, then oral induction therapy in • Post streptococcal
prednisolone 60 mg/m2 OD conjunction with steroid • SLE
(weaned over 2 months then stop) and cyclophosphamide • Membranoproliferative GN
Cyclophosphamide 2 mg/kg PO • Membranous GN
OD for 2/12. OR IV at 500
mg/M2 monthly (reduced dose
because of renal failure), possibly
increasing by 250 mg/m2 per
month (response dependent) to
maximum of 1000mg/m2 for 6
months
Consider prolonging therapy if

Anti GBM still detectable
29
1.1.3.3 Kawasaki disease (KD)
In 1967 Tomisaku Kawasaki described 50 Japanese children with an illness characterised
by fever, rash, conjunctival injection, erythema and swelling of hands and feet, and
cervical lymphadenopathy (Kawasaki, 1967). The mucocutaneous lymph node syndrome
that he described is now recognised as Kawasaki disease (KD), and is the second
commonest vasculitic illness o f childhood (Henoch-Schonlein Purpura being the
commonest). KD is associated with the development of systemic vasculitis complicated
by coronary and peripheral arterial aneurysms, and myocardial infarction in some patients
(Shulman, De Inocencio, and Hirsch, 1995). It is the commonest cause o f acquired heart
disease in children in the United Kingdom and the USA (Shulman, De Inocencio, and
Hirsch, 1995).
Since the early descriptions of KD there have been 21 randomised controlled trials
(RCTs), and at least 2 meta-analyses examining therapeutic interventions in KD. Despite
intensive research into the illness the cause remains unknown, and although there have
been significant improvements in diagnosis and treatment o f children with the disease
there are still a number of important unanswered questions regarding therapy.
Furthermore, there is still no diagnostic test available for KD.
Epidemiology of KD
KD is commonest in Japan where more than 125 000 cases have been reported (Tizard,
1999a) The disease is also commoner in Japanese and other Oriental children living
abroad (Shulman, De Inocencio, and Hirsch, 1995). Children aged 6 months to 5 years
30
are most susceptible, with peak incidence in children aged 9-11 months (Levin, Tizard,
and Dillon, 1991). Seasonal variation in the disease incidence has been reported, with
peak occurrence in the winter and spring months (Shulman, De Inocencio, and Hirsch,
1995). Outbreaks of KD have been linked to weather patterns with clusters o f KD cases
occurring in association with precipitation (Bronstein, Dille and others, 2000). Direct
person to person spread is not observed, although in Japan the disease occurs more
commonly in siblings of index cases with an estimated peak incidence of 8-9% in siblings
under the age of 2 years (Fujita, Nakamura and others, 1989). Currently, the estimated
incidence of KD in the United Kingdom is 8.1 per 100 000 children under the age of five-
an incidence which has doubled since 1991 (Hamden, Alves, and Sheikh, 2002).
Although KD is believed to be caused by an infectious agent in an immunologically
susceptible individual, the causative agent remains elusive.
Diagnosis of KD
There is no diagnostic test for KD, therefore diagnosis is based on clinical criteria (table
1.2, and figure 1.6) (Dajani, Taubert and others, 1993) and the exclusion o f other
diseases, particularly sepsis. The differential diagnosis includes toxic shock syndrome
(streptococcal and staphylococcal), staphylococcal scalded skin syndrome, scarlet fever,
and infection with enterovims, adenovirus, measles, parvovirus, Epstein-Barr virus,
cytomegalovirus, mycoplasma pneumoniae, rickettsiae, and leptospirosis (Brogan, Bose
and others, 2002). The differential diagnosis of IVIG (intravenous immunoglobulin)-
31
Figure 1.6: Diagnostic criteria for KD
Fever o f > 5 days duration + four o f five criteria:
Oropharyngeal Bilatéral non-purulent

Changes conjunctival injection
(90%+ of cases) (90%+ of cases)
Changes in peripheral
Polymorphous rash
extrem ities
(90%+ of cases) (95%+ of cases)
Cervical
lymphadenopathy
(75% of cases)
U)
K)
resistant KD includes PAN, systemic-onset juvenile idiopathic arthritis, and malignancy
(particularly lymphoma).
Table 1.2: Kawasaki Disease: diagnostic criteria (Dajani, Taubert and others, 1993)
:<urjterfon:
Fever Duration o f 5 days or more PLUS 4 o f 5 o f the following:
1. Conjunctivitis Bilateral, bulbar, non suppurative
2. Lymphadenopathy Cervical, >1.5 cm
3. Rash Polymorphous, no vesicles or crusts
4. Changes o f lips or oral mucosa R ed cracked lips; "strawberry" tongue; or difhrse erythema o f oropharynx
5. Changes o f extremities Initial stage: erythema and oedema o f palms and soles
_____________________________________ C oiiyal^cent stoge : P il in g skin from fingartips_____________________
KD may be diagnosed with fewer than 4 of these features if coronary artery aneurysms are detected.
Fever of 5 days duration plus 4 o f the 5 remaining criteria or the presence o f fever and
coronary arterial abnormalities (CAA) with 3 additional criteria are required for the
diagnosis o f "complete" cases (Tizard, 1999a). "Incomplete" cases comprise those with
less than the prerequisite number o f criteria. Irritability is an important sign, which is
nearly always present, although not included as one o f the diagnostic criteria (Brogan,
Bose and others, 2002). The exact mechanism o f the irritability is unclear, but it may be
related to the presence o f aseptic meningitis. Another clinical sign not incorporated into
the diagnostic criteria but which is relatively specific to KD is the developm ent of
erythema and induration at sites of BCG immunisations (Brogan, Bose and others, 2002).
The mechanism of this clinical sign is cross-reactivity o f T cells in KD patients between
specific epitopes o f mycobacterial and human heat shock proteins (Sireci, Dieli, and
Salerno, 2000). With an increasing number of infants receiving the BCG in the UK, it is
33
likely that this sign will become more common, and awareness o f it could result in earlier
diagnosis and treatment.
An important point worthy of emphasis is that the criteria may present sequentially such
that a so-called "incomplete" case can evolve with time into a "complete" case. Thus the
diagnosis o f KD must be considered in any child with a febrile exanthematous illness,
particularly if it persists longer than four to five days.
Other relatively common clinical findings in KD include arthritis, aseptic meningitis,
pneumonitis, uveitis, gastroenteritis, meatitis and dysuria, and otitis (Petty and Cassidy,
2001c). Relatively uncommon abnormalities include hydrops of the gallbladder,
gastrointestinal ischaemia, jaundice, petechial rash, febrile convulsions, nerve deafness,
and encephalopathy or ataxia (Petty and Cassidy, 2001c). Cardiac complications other
than coronary arterial abnormalities include cardiac tamponade, cardiac failure,
myocarditis, and pericarditis (Petty and Cassidy, 2001c).
Laboratory ilndings in KD
KD is associated with many non-specific laboratory findings. Acute phase proteins,
neutrophils, and erythrocyte sedimentation rate are usually elevated. Thrombocytosis
occurs towards the end of the second week of the illness and therefore may not be helpful
diagnostically (Petty and Cassidy, 2001 c). Liver function may be deranged. Sterile pyuria
is occasionally observed, and also CSF pleocytosis (predominantly lymphocytes)
representing aseptic meningitis (Shulman, De Inocencio, and Hirsch, 1995).
34
Treatment of KD
Treatment of KD is aimed at reducing inflammation, and preventing the occurrence of
coronary artery aneurysms (CAA) and arterial thrombosis. The optimal doses o f aspirin
and rVIG are discussed, and the treatment of refractory KD is considered.
Aspirin
It is worthy of note that treatment of KD with aspirin alone has never been subject to a
randomised controlled trial, although aspirin versus aspirin plus IVIG has been studied.
Aspirin is given in the acute phase of the illness at relatively high "anti-inflammatory"
doses (30-100 mg/Kg/day). Following defervescence of the disease aspirin is given as an
anti-platelet agent in a dose of 2-5 mg/Kg/OD, the duration being dependent on findings
on echocardiography but usually for a minimum of 6-8 weeks.
Recent reviews have recommended the higher anti-inflammatory dose o f 100 mg/Kg/day
in the acute phase of the illness (Tizard, 1999a) on the basis o f reducing duration o f fever
and length of hospitalisation compared with low dose aspirin (3-8 mg/kg/day) (Melish,
Takahashi, and Shulman, 1992). No such comparative data on fever and hospitalisation
exists regarding high dose aspirin versus moderate doses (30-50 mg/Kg/day).
Meta-analysis comparing moderate anti-inflammatory doses of aspirin (30-50 mg/kg/day)
with IVIG versus high dose aspirin (80-120 mg/Kg/day) with IVIG found no significant
difference in the incidence of CAA between the groups (Terai and Shulman, 1997).
35
Currently, it is our practice to administer aspirin at a dose o f 30 mg/Kg/day during the
acute phase of the illness, since this may be better tolerated in terms o f gastrointestinal
and other side effects (Matsubara, Mason and others, 1996). Some have advocated the
use of dipyridamole in addition to moderate dose aspirin as a synergistic anti-platelet
agent. The evidence for the effectiveness of this in this situation is, however, lacking.
Intravenous Immunoglobulin (IVIG)
Early recognition and treatment o f KD with aspirin and IVIG has been shown
unequivocally by meta-analysis to reduce the occurrence o f CAA (Durongpisitkul,
Gururaj and others, 1995; Terai and Shulman, 1997). Moreover, the prevalence of
coronary artery abnormalities in KD is highly dependent on total IVIG dose, but
independent of aspirin dose (Terai and Shulman, 1997). Treated with aspirin alone, 20-
40% of children develop CAA (Durongpisitkul, Gururaj and others, 1995; Terai and
Shulman, 1997). Combined therapy with aspirin and high dose IVIG given as a single
infusion reduces the occurrence of CAA to 9% at 30 days, and 4% at 60 days after the
onset of the illness (Durongpisitkul, Gururaj and others, 1995). The prevalence of CAA is
inversely related to the total dose of IVIG, 2 g/Kg of IVIG being the optimal dose,
usually given as a single infusion (Durongpisitkul, Gururaj and others, 1995; Terai and
Shulman, 1997). Meta-analysis of randomised controlled trials comparing divided lower
doses of rVIG (400 mg/Kg/day for 4 consecutive days) versus a single infusion o f high
dose IVIG (2g/Kg over 10 hours) has clearly demonstrated the therapeutic benefits in the
prevention of CAA with the latter regimen (Durongpisitkul, Gururaj and others, 1995).
One important practical point, however, is that infants who have cardiac compromise
36
may not be able to tolerate the fluid challenge associated with the high dose single
infusion, and consideration of divided doses given over several days may be appropriate
for this patient group.
rVIG treatment should be started early in the disease, preferably within the first 10 days
of the illness (Durongpisitkul, Gururaj and others, 1995; Terai and Shulman, 1997).
Importantly, however, clinicians should not hesitate to give IVIG to patients who present
after 10 days if there are signs of persisting inflammation.
Not all patients respond to a single dose of IVIG, and some require a second dose. It has
been observed that those children who received IVIG very early in the illness may require
a second infusion of IVIG for primary treatment failure or disease recrudescence (Han,
Silverman and others, 2000). Thus, the timing of IVIG administration appears to be
important, although this latter point should not dissuade clinicians from giving IVIG
before day 5 of fever if the diagnosis of KD is suspected.
Lastly, one question that remains unanswered is whether the type o f IVIG administered is
important, perhaps as a result of the presence of antibodies to epitopes derived from
different donor pools. There are currently no data to suggest that paricular preparations
derived from populations of different ethnicity or geography are superior.
The current UK guideline for the treatment of KD is given in figure 1.7 (Brogan, Bose
and others, 2002).
37
Figure 1.7: Establish diagnosis-1. ^Complete
UK guideline for
the treatm ent of KD (any age), or 2. Încomplete
Kawasaki disease
KD < lyear old
IVIG 2g/kg as a single infusion over 12 hours

(consider splitting the dose over 2-4 days in
infants with cardiac failure)
Aspirin 30-50 mg/kg /day in four divided doses
Perform echocardiography, and EC G
Aspirin 2-5 mg/kg/d^ when fever settled (disease Seek expert advice to
defervescence) continuing for a minimum of 6 consider:
No Disease
weeks. • Second dose of IVIG at
defervescence within 48
2g/kg over 12 hours
hours, or disease
• Consider pulsed methyl
recrudescence within 2
prednisolone at 600
Disease defervescence weeks
mg/m^ daily for 3 days,
or Prednisolone 2
‘Repeat echocardiography at 2 weeks and 6 weeks mg/kg/day OD weaning
over 6 weeks
No CAA CAA < 8mm, no stenoses ^CAA > 8mm, and/or stenoses
Stop aspirin at 6 • Continue aspirin • Lifelong aspirin 2-5
weeks • Repeat echocardiography mg/kg/day
Notes:
Lifelong follow & ECO at 6 monthly • Consider warfarin ^Treatment can be commenced before full 5
}jp at least every intervals • Consider coronary days offever i f sepsis excluded; treatment
2 years • Discontinue aspirin if angiography and exercise should also be given if the presentation is >
aneurysms resolve stress testing 10 days from fever onset
• Consider exercise stress • Repeat echocardiography **Incomplete cases >ly e a r old treated at
test if multiple aneurysms & ECG at 6 monthly discretion o f clinician- seek expert advice.
• Specific advice RE intervals *Refer to paediatric cardiologist
minimising atheroma risk • Specific advice RE ^ Other specific interventions such as PET
factors minimising atheroma risk scanning, addition o f calcium channel
w
00 • Lifelong follow up factors blocker therapy, and coronary angioplasty
• Lifelong follow up at discretion o f paediatric cardiologist.
Cardiac complications of KD
Echocardiographic and cardiac angiographic data indicate that 20-40% of untreated KD
patients develop coronary artery abnormalities. Approximately 50% of these lesions
regress within 5 years, and in most with mild CAA (3-4 mm) regression occurs within 2
years (Shulman, De Inocencio, and Hirsch, 1995). Giant aneurysms (>8 mm) are unlikely
to resolve, and some may develop stenosis with risk o f coronary thrombosis, myocardial
infarction, and death. In 1993, a report from the British Paediatric Surveillance Unit
(BPSU) indicated a mortality rate of 3.7% in the UK for KD (Dhillon, Newton and
others, 1993). Current mortality rates reported from Japan are much lower at 0.14%
(Tizard, 1999a). The reasons for this difference include improved therapy, and better case
recognition. Long-term sequelae may include the early development o f coronary
atherosclerosis (Bums, Shike and others, 1996).
All patients with KD should undergo echocardiography on diagnosis and six to eight
weeks after the onset of the disease (Brogan, Bose and others, 2002). Some also advocate
an intermediate echocardiograph at 10-14 days o f disease onset to pick up any missed
pathology (Brogan, Bose and others, 2002). Additionally, it is good practice to perform
echocardiography at least weekly (and occasionally 48 hourly in the acute stages with
ongoing active inflammation) for those who develop CAA to monitor aneurysm size
progression, or the development of thrombus formation. Long term aspirin at 2-5
mg/Kg/day is recommended for those with persisting aneurysms on echocardiography
(Tizard, 1999a). This can be discontinued if the aneurysms resolve. Depending on the
39
size of the aneurysms, ECG and echocardiography performed 6-12 monthly is
recommended.
Patients with aneurysms greater than 8 mm may require stress testing and possibly
coronary angiography to identify stenotic lesions. Most experts recommend the addition
of warfarin to aspirin therapy for those with giant (greater than 8 mm) coronary
aneurysms (Newburger, 2000), although randomised controlled trials supporting this
practice are lacking. If warfarin is to be commenced, it is imperative to cover the initial
warfarinization period with intravenous heparin to counteract the paradoxical
prothrombotic state which can occur soon after starting warfarin in some patients (Weiss,
SofiF and others, 1987). More recently, it has been suggested that platelet glycoprotein
nb/nia receptor blockade therapy may be a useftil addition to the therapeutic
armamentarium for those with giant aneurysms (Etheridge, Tani and others, 1998).
Limitation of strenuous activity is recommended in all patients with giant coronary
aneurysms and/or stenoses. Some patients with coronary artery stenoses may require
surgical revascularisation. In those patients who develop myocardial infarction, treatment
with streptokinase or tissue plasminogen activator (TPA) is indicated (Shulman, De
Inocencio, and Hirsch, 1995).
A rare but serious complication of KD is the development o f peripheral ischaemia and
gangrene. This particular complication is associated with peripheral arterial aneurysm
formation, particularly axillary aneurysms. Treatment with thrombolytic agents.
40
anticoagulants, and intravenous prostacyclin may be indicated in such patients (Shulman,
De Inocencio, and Hirsch, 1995; Brogan, Bose and others, 2002).
Prognosis of KD
The overall outlook for children with KD is good, although CAA are present in 4% of
treated cases 60 days after the acute illpess (Durongpisitkul, Gururaj and others, 1995).
A 1-2% acute mortality rate due to myocardial infarction has been reduced further in
many countries by alertness of clinicians to the diagnosis and early use of gamma
globulin with anti-platelet therapy- the mortality for the disease in Japan is currently
0.14% (Tizard, 1999a). Nonetheless, it has been postulated that adult atheromatous
coronary disease in some cases may have its origins in childhood due to covert or overt
KD (Brecker, Gray, and Oldershaw, 1988).
1.1.3.4 Wegener’s granulomatosis (WG)
WG is a necrotizing granulomatous vasculitis of the upper and lower respiratory tract,
associated with glomerulonephritis and variable small vessel vasculitis and first described
by Wegener in the 1930s (Stegmayr, Gothefors and others, 2000; Wegener, 1936).
Particular features that might lead clinicians to the diagnosis include subglottic stenosis
due to granulomatous involvement of the trachea, and other upper airway findings such
as sinus opacity, and nasal septum disease. Lower respiratory tract features frequently
masquerade as infection. Retro-orbital masses presenting as orbital pseudotumour are
also well-recognized (Moorthy, Chesney and others, 1977; Valmaggia and Neuweiler,
2001). The association with ANCA showing a cytoplasmic immunofluorescence
41
appearance has created another means of diagnosis as well as a method o f monitoring
disease activity, although this latter point is controversial (Moorthy, Chesney and others,
1977; van der Woude, Rasmussen and others, 1985; Moorthy, Chesney and others, 1977;
Rus and Handwerger, 2000).
Treatment is similar to polyarteritis and includes steroid, cyclophosphamide, anti-platelet
agents, and prophylactic antibiotics such as cotrimoxazole, with plasma exchange in life-
threatening situations (Besbas, Ozen and others, 2000; Brogan and Dillon, 2000b; Dillon,
1998; Rottem, Fauci and others, 1993; Stegmayr, Gothefors and others, 2000). Following
induction of remission with oral or pulsed intravenous cyclophosphamide, remission is
maintained with long term azathioprine or cyclosporin, with low-dose alternate day
prednisolone (Brogan and Dillon, 2000b; Dillon, 1998; Besbas, Ozen and others, 2000),
and further courses of cyclophosphamide (if necessary) to treat relapses. The mortality
for WG at Great Ormond Street Hospital is currently around 15% (Dillon, 1998),
although permanent morbidity is substantial.
1.1.3.5 Churg-Strauss syndrome
Churg-Strauss syndrome or allergic granulomatosis is extremely rare in childhood (Fink,
1977). The clinical picture consists of variable vasculitic features with asthma,
eosinophilia, infiltrates on chest X-ray and extra-vascular granulomata on biopsy
(Guillevin, Le Thi and others, 1988). Steroids are the mainstay o f therapy but cytotoxic
agents such as cyclophosphamide may need to be introduced to control disease activity
(Guillevin, Le Thi and others, 1988; Roberti, Reisman, and Churg, 1993).
42
1.1.3.6 Primary angiitis of the central nervous system
A primary angiitis of the central nervous system (CNS), sometimes referred to as primary
granulomatous angiitis is predominantly seen in adults but can occur in childhood. The
clinical presentation is usually of unexplained cerebral ischaemic episodes without
systemic features. Multifocal segmental arterial lesions of small or medium sized arteries
are usually present at angiography, and histologically there is evidence o f a lymphocytic
necrotizing or granulomatous vasculitis. Aetiology is unknown and in spite of treatment
with steroids and cyclophosphamide, prognosis is poor (Dillon, 1998; Moore, 2000).
1.1.3.7 Henoch Schonlein purpura (HSP)
Introduction
Henoch-Schonlein purpura (anaphylactoid purpura) is a multi-system small vessel
systemic vasculitis with a prominent cutaneous component. It is the commonest vasculitis
in the paediatric population, occurring with an incidence o f around 14 per 100 000-child
population (Stewart, Savage and others, 1988), and has the most favourable outcome,
with the large majority of children requiring no treatment. Younger children are most
frequently affected, the peak incidence occurring at around 4 to 5 years o f age and the
disease is more prevalent in boys. North American series report a higher incidence of
Henoch-Schonlein purpura in White compared with Black children (Allen, Diamond, and
Howell, 1960). The disease appears to follow a seasonal pattern, with a higher incidence
during winter and the early spring.
43
Presentation often follows an upper respiratory tract infection. A number o f early reports
implicated p-haemolytic Streptococcus as a significant specific cause, though subsequent
publications have failed to confirm this. Allergic reactions to both foods and drugs have
also previously been implicated, although again, definitive evidence to support these
theories is lacking.
Clinical features of HSP
Henoch-Schonlein purpura is a multi-system disease most frequently affecting the skin,
joints, gastrointestinal tract and the kidney (Tizard, 1999b). Other organs less frequently
involved include the central nervous system, gonads and the lungs. Many cases follow an
upper respiratory tract infection and the onset of the disorder may be accompanied by
systemic symptoms including malaise and mild pyrexia. Multiple organ involvement may
be present from the outset of the disease, or alternatively an evolving pattern may
develop, with different organs becoming involved at different time points over the course
of several days to several weeks (Tizard, 1999b).
Around one third of children have symptoms for less that 14 days, one third 2-4 weeks
and one third greater than 4 weeks (Allen, Diamond , and Howell, 1960). Recurrence of
symptoms occurs in around one third of cases, generally within four months o f resolution
of the original symptoms. Recurrences, which tend to be less severe than the initial
presenting episode, are more frequent in those with renal involvement.
44
Skin changes in Henoch-Schonlein purpura are a uniform finding. The skin lesion is
typically that of a purpuric rash which is generally symmetrical, affecting the lower limbs
and buttocks in the majority of cases, the upper extremities being involved less
frequently. The abdomen, chest and face are generally unaffected. The rash has a
predilection for the extensor sur&ces. The earliest skin changes may be those of urticaria
with associated oedema, which evolve into non-blanching purpuric lesions, with lesions
at different stages o f evolution often being present at the same time. New crops of
purpura may develop for several months after the disease onset, though generally fade
with time. Lesions can be induced by mild trauma.
Around two thirds of children have joint manifestations at presentation. The knees and
ankles are most frequently involved. Symptoms, which take the form o f pain, swelling
and decreased range of movement, tend to be fleeting and resolve without the
development of permanent damage.
Around three-quarters of children develop abdominal symptoms. The extent of
gastrointestinal involvement is highly variable, ranging from mild colicky abdominal
pain to severe pain with associated ileus and vomiting and gastrointestinal bleeding
manifest by haematemesis and melaena. Other complications include intestinal
perforation and intussusception. The latter may be difficult to distinguish fi-om abdominal
colic, though the incidence of intussusception is significant enough to warrant exclusion
by ultrasound where suspected.
45
Central nervous system involvement may present as headache, behavioural change,
seizure, hemiparesis or coma. Testicular swelling is well-recognised in males. Lung
involvement presents as pulmonary haemorrhage (Tizard, 1999b).
The reported incidence of renal involvement in Henoch-Schonlein purpura (Henoch-
Schonlein nephritis) varies according to the intensity with which evidence o f renal
involvement is sought. Overall, studies report an incidence o f between 20 and 100%,
though in studies where careful routine in-patient urinalysis was performed, the reported
incidence of renal involvement was 20-61% (Kobayashi, Wada and others, 1975;
Koskimies, Rapola and others, 1974; Stewart, Savage and others, 1988). Renal
involvement is normally manifest between a few days and a few weeks after clinical
presentation, but can occur up to 2 or months or (rarely) more from presentation. There
appears to be an increased risk of renal disease in those with bloody stools.
Renal involvement may present with isolated microscopic haematuria, proteinuria with
microscopic or macroscopic haematuria, acute nephritic syndrome (haematuria with at
least two of hypertension, raised plasma creatinine and oliguria), nephrotic syndrome
(usually with microscopic haematuria) or a mixed nephritic-nephrotic picture.
Investigation and Diagnosis of HSP
The diagnosis and classification of Henoch-Schonlein purpura is usually straightforward
and made clinically (see Appendix 1)- no single laboratory test has been shown to be
helpful. Immunological investigations including complement levels and anti-nuclear
46
antibodies are normal, though IgA is elevated in around one half o f children and a small
number exhibit ANCA positivity (Tizard, 1999b). Coagulation studies are normal and
platelet numbers are normal or occasionally increased. Where significant nephritis is
present at presentation, renal function and electrolytes may be correspondingly abnormal.
The differential diagnosis includes sepsis and other systemic vasculitides (SLE, PAN,
WG, and hypersensitivity vasculitis), all of which can present with similar clinical
features. Familial mediteranean fever can also mimick HSP.
Indications for renal biopsy in HSP are: presentation with nephritic and/or nephrotic
syndrome; impaired renal function (glomerular filtration rate (GFR) <80ml/min/1.73m2);
nephrotic proportion proteinuria (protein: creatinine ratio >250mg/mmol); significant
hypertension; plasma albumin <25 g/1 ; and persistently abnormal urinalysis after 1 year.
Pathological findings in HSP
The skin lesion of Henoch-Schonlein purpura is that of a leucocytoclastic vasculitis with
perivascular accumulation of neutrophils and mononuclear cells (Bagga and Dillon,
2001). Immunofluorescence studies reveal vascular deposition of IgA and C3 in affected
skin, though similar changes may be observed in skin unaffected by the rash (Bagga and
Dillon, 2001).
The renal lesion of Henoch-Schonlein nephritis is characteristically a focal and segmental
proliferative glomerulonephritis. The International Study of Kidney Diseases in Children
47
(ISKDC) classification of Henoch Schonlein nephritis was originally devised by Meadow
(Meadow, Glasgow and others, 1972) and adopted in a modified form by the ISKDC
(Counahan, Winterbom and others, 1977).
A broad correlation exists between the clinical presentation and the histological changes
on renal biopsy. Those with haematuria alone without significant proteinuria have
generally less severe histological changes which are highly likely to undergo spontaneous
resolution, whilst those with heavy proteinuria, a persisting nephritic syndrome or
nephrotic syndrome are likely to have more severe changes which are less likely to
resolve.
The renal lesion bears many similarities to that observed in IgA nephropathy, and the two
diseases are considered by many to share a common pathogenesis and perhaps represent
different spectra of one disease. Patients with IgA nephropathy do not, however, have
clinical evidence of extra-renal disease.
Treatment of HSP
The large majority of cases of Henoch-Schonlein purpura are mild and are associated
with a good prognosis, and immunosuppressive treatment is usually not justified-
children should receive symptomatic treatment only. The skin lesion usually requires no
treatment (except where there is severe haemorrhagic oedem affecting the face or
scrotum, where systemic corticosteroid therapy may be indicated) and the arthropathy
48
should be treated with rest and simple analgesia. Whilst never subjected to a controlled
clinical trial, there is some evidence to suggest that the more severe gastrointestinal
symptoms, particularly abdominal pain and gastrointestinal bleeding respond well to
corticosteroid therapy (Rosenblum and Winter, 1987), which has also been used for the
treatment of testicular involvement and pulmonary haemorrhage.
Few prospective randomised controlled studies have been completed relating to the
treatment of renal disease associated with Henoch-Schonlein purpura. Mollica et al have
reported the results of a prospective study where 168 children with no evidence o f renal
involvement at disease presentation were alternatively assigned to two weeks o f daily
prednisolone (1 mg/kg) or no therapy (Mollica, Li and others, 1992). No children treated
with steroids developed renal involvement, compared with 10 o f the 84 control patients.
To investigate this further, a randomised prospective study has recently commenced in
the UK to determine whether the administration of corticosteroids in all patients at
disease presentation results in an overall reduction in the subsequent incidence o f severe
renal involvement. Studies investigating anti-platelet therapy have failed to show any
benefit in terms of modifying the course of the disease or preventing the development of
nephropathy (Peratoner, Longo and others, 1990).
There is no evidence based on randomised controlled trials to support the use o f any of
the more potent immunosuppressive therapies in the treatment o f more severe grades of
Henoch-Schonlein nephritis. A number of uncontrolled studies have reported
improvement following therapy with azathioprine, cyclophosphamide, and chlorambucil,
49
anticoagulants and anti-platelet drugs, often in association with oral corticosteroid
therapy. Others, however, have failed to show any benefit. The presence o f a large
number of crescents may warrant more aggressive additional therapy with intravenous
methylprednisolone and plasma exchange, and there are a number o f uncontrolled studies
reporting good outcomes with these therapies (Gianviti, Trompeter and others, 1996;
Niaudet and Habib, 1998; Oner, Tinaztepe, and Erdogan, 1995). Where crescentic
changes are present, there are reports of good outcomes following 5-10 days o f plasma
exchange in addition to steroid and cyclophosphamide, as in other forms of crescentic
nephritis (figure 1.4). It is important to stress that there are no controlled trial data to
support these therapeutic interventions.
Long-term outcome of HSP
The overwhelming majority of the long-term morbidity associated with Henoch-
Schonlein purpura relates to the associated nephritis. All published data indicate that the
great majority of children with the disease make a full and uneventful recovery with no
evidence of ongoing significant renal disease. However, Henoch-Schonlein nephritis is
reported to be the cause of end stage renal failure in 1.6-3% of children in the UK (Lewis
MA, 1999) and Europe (Niaudet and Habib, 1998).
There is significant reported variability in the relative incidence of the various clinical
presentations o f Henoch-Schonlein nephritis and the long-term outlook associated with
these. Series from tertiary referral institutions report a significantly higher incidence of
long-term renal morbidity than unselected series.
50
The two largest unselected series of children with Henoch-Schonlein purpura are from
Finland and Northern Ireland (Koskimies, Mir and others, 1981; Stewart, Savage and
others, 1988). Koskimies et al found 39 of 141 (28%) children to have an abnormal
urinary sediment for more than one month after presentation. 29 o f these children were
followed up, and at an average of 7.2 years post diagnosis, only 1 child had developed
end stage renal failure and died, and two had developed chronic glomerular disease, the
overall incidence of chronic renal disease being 2.1%. Stewart er a/ reported 270 patients
presenting over a 13-year period. 55 (20%) were found to have initial evidence of renal
involvement. One child died during the acute phase of the illness. At an average o f 8.3
years follow-up, only 3 (1.1%) had evidence of persistent urinary abnormality with
normal renal function. In both of these studies, the large majority o f children had
relatively minor changes in urinary sediment.
In contrast, series o f highly selected patients from tertiary renal centres report
significantly higher rates of long-term renal impairment. A 23-year follow-up study from
Guys Hospital, London UK and Birmingham Children’s Hospitals, UK found the
incidence of long-term renal failure to be 19.2% in a cohort of 78 patients who underwent
renal biopsy early in the course of their illness (Goldstein, White and others, 1992). It
must be stressed that the fact that these children were referred into a tertiary centre and
underwent renal biopsy is indicative of the relative severity o f their renal disease at
presentation. A broad correlation was detected between clinical presentation and
outcome: 44% of children who presented with a nephritic, nephrotic or mixed
nephritic/nephrotic developed hypertension or renal failure on follow-up, whereas of
51
those with microscopic haematuria with or without proteinuria, 82% were entirely normal
at follow-up, with 7.7% developing hypertension or renal failure. A small number o f
patients showed clinical improvement at 5-year follow-up, only to deteriorate in the
longer term, and 16 of 44 successful subsequent pregnancies were complicated by
hypertension or proteinuria, including 12 where the mother had made an apparent
complete clinical recovery.
The severity of the histology on the initial biopsy correlated well with long-term
outcome: 42% of those with WHO grade IV or V changes developed renal failure or died,
compared with 7.4% of those with grade I or II changes.
From these studies and others, a number of conclusions can be drawn:
i] In unselected populations, the overall risk of significant long-term renal impairment is
around 2%
ii] Children with isolated haematuria with no proteinuria have a negligible incidence of
long-term renal morbidity
iii] Patients who present with isolated microscopic and or macroscopic haematuria may
have microscopic haematuria that persists for many months and years. Recurrence of
episodes of macroscopic haematuria may occur following upper respiratory tract
infections. The prognosis generally remains good here unless there is evidence of
significant proteinuria
iii] Where isolated haematuria is associated with proteinuria, the risk o f long term renal
dysfunction is around 5%
52
iv] Children presenting with the acute nephritic syndrome have a less fevourable
outcome, with a risk of long-term renal failure of 10-20%. Those with a mixed nephritic-
nephrotic presentation have the worst long-term outlook, with up to 33% developing
long-term renal failure .
v] Those with more aggressive renal biopsy changes are more likely to have a poorer
long-term outlook
vi] Children with significant renal impairment at presentation should remain under long
term follow-up.
vii] Some instances of hypertension have been reported many years after normalisation of
renal function and urinalysis: hence the need for long-term follow-up. Most would
advocate monitoring of blood pressure for 2 years after normalisation of urinary
sediment.
1.1.3.8 Hypersensitivity vasculitis
Hypersensitivity vasculitis is frequently a drug-induced condition but can be associated
with other antigens including infectious agents (Bagga and Dillon, 2001). The
predominant clinical manifestations involve the skin with palpable nodules or purpura
although other organs can be affected (Bagga and Dillon, 2001). Removal of the
precipitating agent is usually followed by resolution, but sometimes non-steroidal anti
inflammatory drugs and steroids are indicated. The ACR classification criteria for this
entity are given in Appendix 1 (Calabrese, Michel and others, 1990).
53
1.1.3.9 Hypocomplementaemic urticarial vasculitis
The clinical manifestations of this vasculitic disorder are recurrent urticarial lesions,
angioedema, arthritis and arthralgia, vomiting and abdominal pain. Severe systemic
disease with rapidly progressive glomerulonephritis and lung haemorrhage can occur
(Martini, Ravelli and others, 1994). There is classic complement pathway activation.
Therapy includes prednisolone and hydroxychloroquine but other immunosuppressive
agents may be required (Worm, Muche and others, 1998; Worm, Sterry, and Kolde,
2000 ).
1.1.3.10 Vasculitis associated with connective tissue disorders
Vasculitis is seen at times in systemic lupus erythematosus (SLE), juvenile idiopathic
arthritis (JIA), mixed connective tissue disease, juvenile dermatomyositis (JDM), and
scleroderma.
Juvenile dermatomyositis
Vasculitis is a major component o f JDM, and can pose a threat to life (Cassidy and Petty,
2001). The vasculitis affects striated muscle, skin, subcutaneous tissue and
gastrointestinal tract (Cassidy and Petty, 2001). Gastrointestinal perforation, bleeding and
acute pancreatitis can all result from mesenteric vasculitis (See, Martin and others, 1997).
Treatment of severe disease typically includes steroid, oral or intravenous
cyclophosphamide plus, in life threatening situations, plasma exchange (Dillon, 1998;
54
Brogan and Dillon, 2000b). IVIG and methotrexate also have a role in recalcitrant
disease. Recently cyclosporin has been shown to be effective in this disease (Heckmatt,
Hasson and others, 1989), and indeed many would now initially treat JDM with a
combination of prednisolone and cyclosporin, reserving more aggressive treatment
modalities for those in whom severe features emerge (Brogan and Dillon, 2000b).
For a recent comprehensive review of vasculitis in the connective tissue diseases, the
reader is referred to the work of Flores-Suarez (Flores-Suarez and Alarcon-Segovia,
2000 ).
1.1.3.11 Takayasu disease
Takayasu disease (TD) is a giant cell arteritis causing stenosis and aneurysmal dilatation
of large arteries, such as the aorta and its major branches (Lindsley CB, 2001). World
wide, it is the third commonest vasculitis of childhood, and may be related to infection
with tuberculosis (Lindsley CB, 2001; Wiggelinkhuizen and Cremin, 1978). Clinical
features include fever, anorexia, weight loss, arthritis, and later the development of
hypertension, heart failure and pulse defecits ( so-called “pulseless disease”) (Hall, Barr
and others, 1985; Lindsley CB, 2001). Diagnosis involves doppler ultrasonography,
magnetic resonance imaging, and conventional angiography (Lindsley CB, 2001;
South wood TR, 1988). Therapeutic regimens in the acute phase o f the disease include
steroids, cyclophosphamide and methotrexate (Shelhamer, Volkman and others, 1985).
More recently, there have been case reports o f the successful treatment of TD with
mycophenolate mofetil (Daina, Schieppati, and Remuzzi, 1999).
55
1.1.3.12 Miscellaneous vasculitides of the young
These include Behçet’s syndrome, familial Mediterranean fever and Cogan’s syndrome.
Behçet’s syndrome consists of the triad of aphthous stomatitis, genital ulceration and
iritis (Petty and Cassidy, 2001 d; Kari, Shah and others, 2001). Although relatively rare in
childhood, it is well recognised that a vasculitic component is an important feature (Petty
and Cassidy, 200Id).
Familial Mediterranean fever often manifests itself for the first time in childhood (Sohar,
Gafhi and others, 1967; Pras, Langevitz and others, 1996). Although not normally
considered to be associated with vasculitis there are now a number o f reports describing
both polyarteritis nodosa and Henoch-Schonlein purpura in affected patients (Glikson,
Galun and others, 1989). It is therefore important to consider the possibility o f an
associated vasculitis in patients with familial Mediterranean fever that may need treating
in its own right.
Non-syphilitic interstitial keratitis with vestibuloauditory dysfunction was first described
by Cogan (Cogan, 1945). Although rare and usually affecting young adults it has been
reported in children (Cogan, 1945; Kundell and Ochs, 1980) and in addition to the main
features there can be evidence of a widespread vasculitis that may require treatment with
corticosteroids and/or cyclophosphamide.
56
1.1.4. Conclusion
There is a wide spectrum of vasculitic disease affecting children. The distinction from
other conditions, especially those associated with infection, is often difficult, but it is
usually possible to categorise the disorders into clinico-pathological entities even though
there is a substantial degree of overlap. The current range o f investigative tools usually
allows the correct diagnosis to be established and modem therapeutic approaches have
made a major impact on the treatment o f these serious conditions. In spite o f this,
however, morbidity and mortality is not inconsequential.
57
1.2 The Immuno-pathogenesis of vasculitis
1.2.1 Introduction
The aetiology of vasculitis is unknown in most patients, although the immuno-
pathogenetic mechanisms which culminate in vascular injury are beginning to be
unravelled. Whilst an understanding o f the immuno-pathogenesis is critical for the design
of ftiture “magic-bullet” therapies based on strategies targeted to specific immunological
blockade (such as the success which has been achieved with monoclonal antibody
therapy against tumour necrosis factor-alpha in rheumatoid arthritis), it must be borne in
mind that many of the immunological processes that will be considered in this section
represent downstream events that arise in individuals with as yet undefined predisposing
factors, and triggered by some precipitating event. For example, whilst much is now
known regarding the immuno-pathogenesis of the vascular injury associated with ANCA,
comparatively little is known about the predisposing genetic or environmental factors
which result in a loss of immunological tolerance to neutrophil azurophilic enzymes in
the first instance.
This section will describe the current state of knowledge regarding the immuno-
pathogenesis of vasculitis, and will consider aetiology where it is clearly defined in the
context of particular vasculitic syndromes. The mechanisms involved in the pathogenetic
process can be considered under the headings of infections, malignancy, autoantibodies,
immune complexes, cryoglobulins, endothelial amplification o f inflammation (cell
adhesion molecules [CAMs] and cytokines), and T cells. It is proposed that a description
58
o f these mechanisms will set the scene and place in context the detailed discussion of
superantigens in vasculitis, which will be considered in section 1.3.
1.2.2 Infections and vasculitis
Many pathogens are known to cause vasculitis. For several o f these pathogens, vasculitis
may be the major manifestation of disease; others, however, typically present with
vasculitis as an occasional manifestation. The viruses, bacteria, and fungi that cause
vasculitis share a common target- blood vessels. Many infectious pathogens have tissue
tropism that includes endothelium; other agents may bind to the vessel wall because the
vascular endothelium expresses specific receptors for the pathogen (Naides, 2002). Even
when the agent does not enter the endothelial cell, the immune response to the agent may
be focused at the vessel wall because the pathogen is adherent to the endothelial cell
surface, resulting in bystander injury to the vessel. The advent o f sensitive molecular
techniques for the detection of infectious agents has encouraged searches for various
known pathogens in idiopathic vasculitis syndromes (Shingadia, Bose, and Booy, 2002).
1.2.2.1 Viruses causing vasculitis
Hepatitis C virus
Hepatitis C virus (HCV), discovered in 1989, has world-wide prevalence and is the cause
of most cases of the vasculitic syndrome previously known as “essential mixed
cryoglobulinaemic vasculitis”, and may also cause a vasculitic syndrome resembling
PAN (Cacoub, Lunel-Fabiani, and Du, 1992; Cacoub, Costedoat-Chalumeau and others.
59
2002). The 10 to 20-year latent period before hepatic or rheumatic manifestations of
disease explains the increasing number cases of hepatitis C virus-mediated vasculitis
currently being seen following the epidemic o f new cases in the USA in the 1980s
(Cacoub, Costedoat-Chalumeau and others, 2002).
Prior to the discovery of HCV in the late 1980s, the triad o f arthritis, palpable purpura,
and type II cryoglobulinemia was known as “essential mixed cryoglobulinaemia” and
considered an idiopathic vasculitis (Cacoub, Lunel-Fabiani, and Du, 1992; Cacoub,
Costedoat-Chalumeau and others, 2002). The advent of diagnostic testing for HCV
demonstrated that almost all of these cases were associated with HCV infection. Clinical
evidence of liver disease was found in 60 to 80% o f patients with essential mixed
cryoglobulinaemia, and liver biopsies showed chronic active hepatitis or cirrhosis
(Cacoub, Maisonobe and others, 2001; Levo, Gorevic and others, 1977a; Levo, Gorevic
and others, 1977b).
In mixed cryoglobulinemia patients, the hepatotropic antigen triggering the production of
antibodies that later can form immune complexes was sought for many years. Initially
hepatitis B virus (HBV) was implicated as the causative agent o f essential mixed
cryoglobulinaemia (Levo, Gorevic and others, 1977b). Numerous studies using more
recent tests for the detection of HBV surface markers and HBV DNA have failed to
validate these observations: HBV surface antigen (or antibodies to surfece antigen) was
found in only 17% of 33 patients with essential mixed cryoglobulinemia, and no patients
60
had detectable HBV DNA in their sera or cryoprecipitates (Cacoub, Fabiani and others,
1994).
Many studies of patients with mixed cryoglobulinemia have investigated the prevalence
o f anti-HCV antibody and HCV RNA. Studies on large populations found anti-HCV
antibodies in 43-85% o f highly selected patients with mixed cryoglobulinemia (Ferri,
Zignego and others, 2002). All patients were of Italian origin, and many also had
evidence o f HBV infection. In a prospective study (Cacoub, Fabiani and others, 1994),
anti-HCV antibody was demonstrated in 52% of patients with essential mixed
cryoglobulinemia. The anti-HCV antibody-positive patients had more cutaneous
involvement (Raynaud phenomenon, purpura, livedo reticularis, distal ulcers, or
gangrene), higher alanine aminotransferase, higher cryoglobulin levels, and lower serum
complement (CH50 and C4) levels.
Clinically, anti-HCV antibody-positive patients with mixed cryoglobulinemia have
cryoglobulin-related manifestations (see section 1.2.5) more often than anti-HCV
antibody-negative patients. There is a 2 to 3-fold increased frequency o f mixed
cryoglobulinaemia in HCV chronic hepatitis compared with HBV or non-A, non-B, non-
C chronic liver disease (Ferri, Zignego and others, 2002).
A possible explanation for the HCV-mixed cryoglobulinaemia association could be an
antigenic cross-reaction between HCV and the liver, particularly damaged liver (Cacoub,
Costedoat-Chalumeau and others, 2002; Ferri, Zignego and others, 2002). The production
o f antibody cross-reacting with liver and viral antigens is suggested by the finding that
HCV infection induces an antibody response to an endogenous antigen (Mishiro, Hoshi
and others, 1990). Hepatitis C virus RNA sequences have been detected in sera and the
cryoprecipitates from patients with mixed cryoglobulinemia and HCV antibody (Munoz-
Femandez, Barbado and others, 1994). Among 27 HCV-positive patients with mixed
cryoglobulinemia, 66% were HCV RNA-positive in the supernatant and 85% in the
cryoprecipitate. In fact, the presence of entire viral particles has been proposed to be
present in the cryoprecipitate. Furthermore HCV RNA sequences have been detected in
vasculitic lesions of patients with HCV-mixed cryoglobulinemia (Agnello and Abel,
1997; Casato, Agnello and others, 1997).
Thus it is clear that HCV infection can produce vasculitis, and this discovery has led to
effective anti-viral therapeutic regimens with interferon alpha and ribavirin (Zuckerman,
Keren and others, 2000a; Zuckerman, Keren and others, 2000b).
Hepatitis B virus
Hepatitis B virus (HBV) infection is the classic example o f virally mediated immune
complex disease (Dienstag, 1981a). Histologically there is lymphocytic venulitis and/or
neutrophilic vasculitis of small vessels with leukocytoclasis and fibrinoid changes
present. Clinical presentation is with an “urticaria-arthritis syndrome” (Berretty,
Neumann, and von Joost, 1981). Immune complexes o f HBV surfece antigen (HBsAg)
and antibodies to HBV surface antigen (HBsAb) circulate in the blood and are found
deposited in vessels in association with complement (Neumann, Berretty and others,
62
1981). The long latency period o f HBV allows time for an immune response to occur.
Viral replication increases HBsAg load, and is temporally associated with
jaundice(Hoofnagle and Di Bisceglie, 1991). The immune complexes eventually no
longer form in antigen excess and the serum sickness-like illness resolves.
HBV has also been associated with a vasculitic syndrome resembling PAN (Anon, 1983;
Avsar, Savas and others, 1998; Balkaran, Teelucksingh, and Singh, 2000; Chauveau and
Christophe, 1995; Darras-Joly, Lortholary and others, 1995). Onset o f this vasculitis is
early in the course of chronic HBV hepatitis. Again, immune complexes containing
HBsAg, HBsAb, and complement are found in the vessel wall (Dienstag, 1981b). The de
determinants o f small vessel versus larger vessel vasculitis in response to HBV infection
are unknown.
Human Immunodeficiency virus
Infection with Human Immunodeficiency virus (HTV) may present with a variety of
vasculitic syndromes. However, it is difficult to specifically attribute the various
vasculitides seen in HTV infection because of firequent co-infections with other pathogens
that may cause vasculitis in their own right. That said, HIV infection has been associated
with vasculitis affecting the central nervous system, skin, and retina (Berkefeld,
Enzensberger, and Lanfermann, 2000; Font, Miro and others, 1996; Gisselbrecht, Cohen
and others, 1998; Picard, Brunereau and others, 1997).
63
Herpes viruses
Herpes viruses are especially attractive as possible causes o f vasculitis (Shingadia, Bose,
and Booy, 2002). They are ubiquitous in the population, and many have been shown to
infect cells of a vascular lineage (Dal Canto and Virgin, 2000). All classes o f herpes virus
(a, p, and y herpes viruses) can cause panarteritis in animal models (Virgin, 2002). For
example, murine cytomegalovirus is widely used as a model for studying many aspects of
a human cytomegalovirus infection, particularly small vessel vasculitis (Dangler, Baker
and others, 1995). Another interesting model for analysis of mechanisms of vascular
injury and immune response in the large vessels is the rodent y herpes virus yHV68.
Analysis of the yHV68 genome demonstrates that this virus is closely related to primate
herpes viruses, including EBV and human herpes virus (Shingadia, Bose, and Booy,
2002). yHV68 infection causes a severe chronic vasculitis o f the great elastic arteries in
mice (Week, Dal Canto and others, 1997) and results in an expansion of splenic B cells.
This expansion is associated with hyp ergammaglobu linaemia and much o f the antibody is
not apparently directed to yHV68 antigens. Another unusual aspect o f the response of
CD8+ T cells to yHV68 infection is the dramatic expansion of Vp4 CD8+ T cells (Tripp,
Hamilton-Easton and others, 1997; Week, Dal Canto and others, 1997). The mechanism
underlying this selective Vp expansion and the physiologic importance o f this
phenomenon are currently unknown (Tripp, Hamilton-Easton and others, 1997; Week,
Dal Canto and others, 1997). Lack of either interferon-y (INF-y) or the INF-y receptor and
young age predisposes yHV68-infected mice to severe vasculitis of the great arteries.
INF-y receptor deficient mice died 1.5 to 14 weeks after infection with yHV68 and were
found to have severe large-vessel arteritis. It is important to be cautious about making
64
extrapolations from animal models to human disease, however, as there are species
differences in the pathogenesis of many diseases.
Newer molecular methods such as degenerate PCR are increasingly used to identify new
infectious agents that cause human disease. However, molecular methods have been used
only to a limited extent in human vasculitis syndromes. Kikuta et a l identified EBV DNA
in peripheral blood mononuclear cells and cardiac tissue o f KD patients by selective
DNA amplification techniques (Kikuta, Sakiyama and others, 1993). Rowley e t a l failed
to detect highly conserved viral and bacterial nucleic acid sequences in patients with KD
(Rowley, Wolinsky and others, 1994).
Herpes viruses including cytomegalovirus, varicella-zoster, herpes simplex viruses 1 and
2, and herpes hominis may be associated with retinal vasculitis in immunocompromised
patients (Kuo, Yip, and Chen, 2001; Savir, Grosswasser, and Mendelson, 1980).
Varicella-zoster may also cause a diffuse central nervous system small arterial
granulomatous vasculitis (Naides, 2002). Herpes simplex viruses 1 and 2 have been
associated with cutaneous vasculitis and necrotizing arteritis o f small and medium vessels
(Schmitt, Dietzmann and others, 1992). Epstein-Barr virus has been proposed as a cause
o f both small- and large-vessel disease in a number o f cases and short series (Naides,
2002). However, the ability to convincingly infer causality in many studies is made
problematic by the latency of herpes virus infection.
65
Other viral agents
Parvovirus B19 has been implicated in the aetio-pathogensis of PAN (Corman and
Dolson, 1992; Finkel, Torok and others, 1994; Martinelli, Azzi and others, 1997).
However, the feilure to eliminate B19 from pooled blood products is an important
confounding factor to consider when inferring causality (Prowse, Ludlam, and Yap,
1997). Vasculitis has also been reported following infection with rubella virus,
adenovirus, echovirus, coxsackie virus, parainfluenza virus, and hepatitis A virus
(Naides, 2002).
1.2.2.2 Bacteria causing vasculitis
Mechanisms of bacterial vasculitic injury
Bacteria may cause vasculitis by direct microbial invasion o f endothelial cells, by
immune-complex-mediated mechanisms, or by stimulation o f auto-reactive T or B-
lymphocytes by bacterial superantigens. This latter hypothesis will be discussed in detail
in section 1.3.
Bacterial seeding of vessels may lead to necrosis through direct bacterial infection.
Vessels may be seeded intraluminally at sites of endothelial injury or flow turbulence, or
seeding o f vasa vasorum may cause destruction of vessels from the outside in (“mycotic
aneurysm”). Spread from an infected site to a vessel may also occur. Immune responses
to bacteria or to bacterial components may also lead to or contribute to vascular injury,
usually by deposition of immune-complexes comprised of bacterial antigen, antibody to
bacterial antigen, and complement components.
66
In subacute bacterial endocarditis (SABE), dissemination o f septic emboli and immune
complex injury occurs resulting in the classic vasculitic stigmata o f SABE: myalgia,
arthralgia, Osier’s nodes, Janeway lesions, and septic infarcts. Bacteraemia may also
present as leukocytoclastic vasculitis.
Specific bacteria causing vasculitis
Neisseria species may be associated with small-vessel vasculitis. Neisseria gonorrhoea is
associated with necrotic cutaneous papules. In Neisseria meningi/ides infection, vasculitis
manifests in the skin and gastrointestinal tract, with the endothelial necrosis and
widespread thrombosis (Seaton, Nathwani and others, 2000).
Rickettsiae (causing Rocky Mountain and Mediterranean spotted fever) are obligate
intracellular bacteria with tropism for vascular endothelium (George, Brouqui and others,
1993). Infection results in widespread microvascular leak, focal thrombosis, and
ultimately multisystem failure.
Another example of microbial invasion of endothelial cells is Staphylococcus aureus {S.
aureus). It has been demonstrated that S. aureus binds more readily to endothelial cells
than most other bacteria (Cohen Tervaert, 2002). Following binding, bacteria are
internalised and can persist in phagosome-like vacuoles. The interaction between S.
aureus and endothelial cells may result in activation of the endothelial cells resulting in
enhanced expression o f cell adhesion molecules and in the production o f cytokines and
67
chemokines (Cohen Tervaert, 2002). Furthermore, endothelial cells may be damaged
following internalisation of alpha-toxin producing strains o f S. aureus (Cohen Tervaert,
2002 ).
Mycobacterial or fungal lung infections may mimic Wegener’s granulomatosis or Churg-
Strauss syndrome by causing a granulomatous reaction in vessel walls (Naides, 2002).
Spread of Mycobacterium tuberculosis to the aorta may causes tuberculous aortitis,
coronary arteritis and mycotic aortic aneurysm. Pseudomonas aeruginosa and other gram-
negative organisms can present as cutaneous vasculitis; vessel thrombosis results from
direct bacterial invasion of the vessels (Naides, 2002).
Syphilis, the “great imitator” (Witkowski and Parish, 2002), can present as large or
medium-sized vessel disease (aortitis or coronary arteritis), or as the small-vessel
vasculitic rash of secondary syphilis. Syphilitic aortic aneurysms were insidious in their
clinical presentation, and Treponema pallidum spirochaetes are rarely detected in the
fibrosed and scarred aortas(Aizawa, Hasegawa and others, 1998).
Borrelia burgdorferi (the cause of Lyme disease) produces vasculitic changes in the
central nervous system, retina, and temporal arteries (Naides, 2002).
68
1.2.2.3 Parasites causing vasculitis
Parasites rarely cause vasculitis. Toxocara canis can present as palpable purpura with
features suggesting Henoch-Schonlein purpura (Hamidou, Gueglio and others, 1999;
Magnaval and Morassin, 2000), or a more widespread vasculitis mimicking giant cell
vasculitis (GCA) (Hamidou, Fradet and others, 2002). Cysticercus has reportedly caused
vasculitis and arachnoiditis as it infects the central nervous system (Wolf, Allert and
others, 1966). Loa loa (a filarial parasite) can present with cutaneous leukocytoclastic
vasculitis and angioedema (Rakita, White, Jr., and Kielhofher, 1993). Angiostrongylus
nematodes may cause pulmonary vasculitis mimicking WG (Pirisi, Gutierrez and others,
1995).
1.2.2.4 Conclusion
Many infectious agents can cause vasculitis, but few have been convincingly
demonstrated as the cause of most idiopathic vasculitic syndromes. The advent of
molecular techniques for the detection of infectious pathogens has renewed efforts to
look for infectious causes for otherwise presumed autoimmune diseases, including the
systemic vasculitides. The predisposing factors which cause one individual to develop
vasculitis in response to infection, whereas another individual may escape this
manifestation remain elusive.
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1.2.3 Malignancy and vasculitis
Malignancy is a well-recognized association with systemic vasculitis, and is mainly
associated with cutaneous vasculitis, although systemic features are also described.
Vasculitis in this context can arise as a result o f an infectious complication operating on
the background of impaired immune function secondary to the presence o f malignancy,
as a paraneoplastic phenomenon (where the inflammatory cells infiltrating the blood
vessel wall are non-malignant), or as a result o f direct involvement o f the blood vessels
with the malignant process- usually associated with the so-called “angiocentric
lymphomas”. These mechanisms will be considered in turn.
1.2.3.1 Infectious causes of vasculitis in malignancy
Varicella zoster virus (VZV) has been reported to cause vasculitis in patients with
lymphoma (McKelvie, Collins and others, 2002). The presentation was that of a central
nervous system meningoencephalitis with vasculitis presenting in a patient with multiple
malignancies (non-Hodgkin’s lymphoma and cancers of breast and colon). Autopsy
showed necrotizing vasculitis of the leptomeningeal vessels, and polymerase chain
reaction detected VZV DNA in the cerebro-spinal fluid. Similarly, VZV vasculitis
affecting the skin was described in a patient with B cell lymphoma (Uhoda, Pierard-
Franchimont, and Pierard, 2000). Histology revealed a lymphocytic vasculitis, and
immunohistochemistry demonstrated VZV in endothelial cells.
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1.2.3.4 Paraneoplastic vasculitis
This entity is relatively common in adults, and usually takes the form of a cutaneous
vasculitis (Pavlidis, Klouvas and others, 1995). It has been estimated that 1-7% o f adults
with cutaneous vasculitis will have an underlying malignancy (Gran, 1997).
Paraneoplastic vasculitis has been described in association with several types of
malignancy. Erythema nodosum has been described with Hodgkin’s lymphoma (Blanco,
Neau and others, 1999). Henoch Schonlein purpura (HSP) is associated with non-
Hodgkin’s lymphoma (Day, Savage and others, 2001), carcinomas (lung, breast, prostate,
renal) (Pertuiset, Liote and others, 2000), adenocarcinoma o f the gastro-intestinal tract
(Pertuiset, Liote and others, 2000), and other haematological malignancies including
Hodgkin’s lymphoma, IgA multiple myeloma (Pertuiset, Liote and others, 2000), splenic
lymphoma (Farrell, Stem and others, 1999), and hairy cell leukaemia (Farrell, Stem and
others, 1999). In addition, in a series o f 14 adult patients with HSP, 4 cases were
associated with underlying malignancy (Pertuiset, Liote and others, 2000).
Proposed pathogenetic mechanisms for paraneoplastic vasculitis include formation of
immune complexes with tumour antigens (Pertuiset, Liote and others, 2000),
dysregulated IgA formation leading to immune complex formation (Pertuiset, Liote and
others, 2000), or the formation of cryoglobulins (Wooten and Jasin, 1996a; Wooten and
Jasin, 1996b).
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1.2.3.5 Angiocentric lymphomas
Angiocentric lymphomas are a heterogeneous spectrum of haematolymphoid
malignancies that share a particular histological characteristic, namely, an angiocentric or
perivascular growth pattern (Natkunam and Wamke, 2001). The consequence o f this is
that they present with lymphomatous vasculitis, which may mimic lymphocytic vasculitis
unless specific tumour markers are examined on the infiltrating tumour cells.
Angiocentric lymphoma should therefore be included in the differential diagnosis of
systemic vasculitis (al Chalabi and Abbott, 1995). They include a variety o f T-, B-, and
natural killer-cell derived lymphomas that have many different clinico-pathological
features, immunophenotype, and prognosis.
Vasculitic syndromes described in association with this group o f malignancies include
mononeuritis multiplex secondary to angiotropic large cell lymphoma (ALCL) (Roux,
Grossin and others, 1995), CNS vasculitis with ALCL, and cutaneous vasculitis with
angioimmunoblastic T cell lymphoma (Sugaya, Nakamura and others, 2001; Thomas,
Vuitch, and Lakhanpal, 1994).
72
1.2.4 Autoantibodies and vasculitis
Autoantibodies associated with vasculitis include antineutrophil cytoplasmic antibodies
(ANCA) and anti endothelial cell antibodies (AECA). Many other autoantibodies have
been described with propensity for vascular injury but for the purposes o f this thesis the
above two autoantibodies will be discussed in detail.
1.2.4.1 Antineutrophil cytoplasmic antibodies (ANCA)
Studies relating to ANCA and associated small vessel vasculitides have dominated the
vasculitis literature since these autoantibodies were first described by Davies in 1982
(Davies, Moran and others, 1982).
Vasculitic syndromes associated with ANCA
The small vessel vasculitides (S W ) associated with ANCA are Wegener’s
granulomatosis (WG), microscopic polyangiitis (MPA), and Churg-Strauss syndrome
(CSS) (Jennette and Falk, 1997; Pirisi, Gutierrez and others, 1995; Pirisi, Gutierrez and
others, 1995; Savage, Harper and others, 2000). ANC A target autoantigens are contained
primarily within neutrophil azurophil granules, although it is becoming increasingly
apparent that the tissue distribution of autoantigens targeted by ANCA is wider than first
appreciated (Pirisi, Gutierrez and others, 1995; Savage, Harper, and Holland, 2002). In
WG, the ANCA recognise proteinase-3 in more than 90% cases, whereas in MPA either
proteinase-3 or myeloperoxidase may be recognized. In CSS myeloperoxidase is
recognized most often (Pirisi, Gutierrez and others, 1995; Savage, Harper, and Holland,
2002 ).
73
The aetiological agents that initiate the autoimmune response resulting in loss of
tolerance to neutrophil enzymes and the development o f ANCA are unknown, although
certain drugs (such as propylthiouracil (Casis and Perez, 2000; Chastain, Russo and
others, 1999; Colakovski and Lorber, 2001; Dolman, Cans and others, 1993),
minocycline (Schaffer, Davidson and others, 2001; Schrodt, Kulp-Shorten, and Callen,
1999; Schrodt and Callen, 1999)), as well as infections (superantigen-producing strains of
S. aureus (Stegeman, Tervaert and others, 1994; Popa ER, van der Meer B and others,
2002; Popa, Stegeman and others, 1998)), and silica (Hogan, Satterly and others, 2001;
Koeger, Rozenberg, and Bourgeois, 1994) have been implicated.
Do ANCA cause small vessel vasculitis?
There is strong circumstantial evidence that the vasculitis in ANCA-associated S W is
the result of the presence of ANCA, but until very recently direct evidence was lacking.
Recently, however, 4 studies have demonstrated in animal models that ANCA are the
cause of the vascular injury. At the time of writing o f this thesis, these have only been
published in abstract and were presented at the 10* International Vasculitis and ANCA
Workshop, Cleveland USA, 2002.
Jennette et al were able to induce pauci-immune necrotizing crescentic
glomerulonephritis by the intravenous administration o f anti-MPO antibodies to a mouse
model deficient in recombinase activating gene-2 (RAG-2 -/-) (Jennette JC, Xiao H and
others, 2002). These mice effectively lack B and T lymphocytes and therefore serve as a
74
pure model to study the passive transfer of potentially pathogenetic autoantibodies.
Passive transfer of anti-MPO antibody resulted in the onset o f haematuria and
proteinuria. Mice sacrificed at day 6 that had received anti-MPO (n=5) all had focal
necrotizing glomerulonephritis (mean 18% glomeruli with necrosis) and crescents (mean
11% crescents), whereas mice that received anti-BSA (bovine serum albumin) antibody
(control, n=3) had no symptoms or histological lesions. The authors concluded that in
mice with no T or B-lymphocytes, circulating anti-MPO (MPO-ANCA) causes pauci-
immune necrotizing and crescentic glomerulonephritis.
Again using the RAG-2 -/- model, the same group were able to induce necrotizing and
crescentic glomerulonephritis and S W by transfer of anti-MPO producing splenocytes
(Xiao H, Heeringa P and others, 2002). Mice that received anti-MPO splenocytes
developed circulating anti-MPO within 3 days, and the titer rose until sacrifice at 13 days.
Mice that received anti-BSA splenocytes developed circulating anti-BSA. Mice that
received MPO-ANCA splenocytes developed renal failure, haematuria and proteinuria,
whereas mice that received anti-BSA (control splenocytes) did not. All mice that received
anti-MPO splenocytes (n=4) developed severe crescentic nephritis; 3 developed
pulmonary capillaritis; 1 had necrotizing vasculitis in spleen and lymph nodes; and 1 had
necrotizing granulomatous inflammation in spleen. Mice that received anti-BSA (n=14)
splenocytes did not develop vasculitis.
Using another mouse model which spontaneously develops crescentic nephritis fi'om
early life (the SCG/Kj mouse) Ishida-Okawara et a l correlated the development of
75
vasculitis with neutrophil MPO release, neutrophil superoxide generation, and MPO-
ANCA titer in the serum of these mice (Ishida-Okawara, Ito-Ihara and others, 2002).
They found positive correlation between neutrophil MPO release, MPO-ANCA, and
histological scores of vasculitis severity, and suggested on the basis o f these data that
neutrophil activation, in association with MPO-ANCA contributed to the vascular injury
observed in the kidneys of these mice.
Lastly, Smyth et al used a rat model to study MPO-ANCA associated pauci-immune
focal segmental glomerulonephritis (Smyth CL, Smith J and others, 2002). In that study,
Wistar Kyoto rats were immunised with purified human MPO (50 micrograms
intramuscularly). Over 2 to 4 weeks all rats developed anti-myeloperoxidase antibodies
as confirmed by ANCA indirect immunofluorescence (on rat and human neutrophils) and
enzyme-linked immunosorbant assay (ELISA). Haematuria was detected in 95% by week
5, accompanied by mild proteinuria. Kidney sections taken from rats killed at 8 weeks
showed glomeruli with segmental inflammation and occasional fibrinoid deposits, tubular
red cell casts, and tubulo-interstitial inflammation. Lung sections showed evidence of
fresh haemorrhage and haemosiderin deposition. Immunofluorescence microscopy
revealed no deposits of IgG and only scanty tubular deposits of C3 (i.e. pauci-immune
glomemlonephritis).
Such studies therefore support the view that ANCA are themselves pathogenic, although
as mentioned in previously, care must be taken when extrapolating animal studies to
human disease states.
76
How do ANCA cause vasculitis?
The most accepted model of pathogenesis proposes that ANCA activate cytokine-primed
neutrophils, leading to bystander damage of endothelial cells and escalation of
inflammation with recruitment of mononuclear cells (Harper and Savage, 2000). ANCA
bind with their target antigen on the surface o f neutrophils after the cells have been
primed. This priming involves neutrophil stimulation with an agent such as
1ipopolysaccharide (LPS) or the cytokine tumour necrosis factor alpha (TNF-a) at a
concentration that does not of itself cause full functional activation, but rather results in
the translocation of antigen from intracellular granules to the cell surface (Csemok, Ernst
and others, 1994). It has been demonstrated that patients with active S W have
circulating neutrophils in the primed state, with enhanced expression of surface
proteinase-3 (Muller Kobold, Mesander and others, 1998; Muller Kobold, Kallenberg,
and Tervaert, 1998) and increased expression o f the activation markers CD66b, CD63,
and CD64 (Muller Kobold, Mesander and others, 1998; Muller Kobold, Kallenberg, and
Tervaert, 1998). Subsequent studies have confirmed these data and show that resting
neutrophils do not express proteinase-3 or myeloperoxidase (Yang, Tuttle and others,
2000). Furthermore primed circulating neutrophils not only have enhanced proteinase-3
expression but also have increased basal superoxide production (Harper, Cockwell and
others, 2001).
In addition to neutrophil priming by LPS or proinflammatory cytokines, in vitro studies
have suggested that low concentrations of PR3-ANCA can themselves prime neutrophils
77
for activation (Hattar, Sibelius and others, 2001). The priming effect appears to be
dependent on the intact ANCA IgG molecule. TNF-a priming and ANCA activation of
neutrophils lead to a respiratory burst and degranulation, generating potential for
endothelial injury.
The binding of ANCA to antigens expressed on the sur&ce o f primed neutrophils is not
enough in itself to cause full neutrophil activation: binding o f Fc receptors is also
necessary (Mulder, Heeringa and others, 1994). The intracellular signaling events that
lead to the functional response of typical of activated neutrophils are currently
incompletely defined, but are likely to involve tyrosine kinases and protein kinase C, and
G-proteins (Savage, Harper, and Holland, 2002).
After neutrophil stimulation by ANCA, numerous cytotoxic mediators are released,
including reactive oxygen species, chemokines, cytokines, proteolytic enzymes, and
nitric oxide (NO)(Savage, Harper, and Holland, 2002). The release of NO is independent
of nitric oxide synthase (NOS) (Tse, Williams and others, 2001), but whether this source
of nitric oxide is protective or damaging to endothelium is unclear.
If neutrophils are the cause of endothelial injury, they must be brought into close
proximity with endothelial cells. Studies in a flow model using a platelet monolayer as a
surrogate vessel wall have demonstrated that neutrophil “rolling adhesion” on the platelet
monolayer can be converted to firm stationary adhesion by either PR-3-ANCA or MPO-
78
ANCA (Savage, Harper, and Holland, 2002). This conversion from rolling adhesion to
firm adhesion is blocked by antibodies against neutrophil GDI lb or FcyRIIa receptors.
Firm adhesion of activated neutrophils to endothelial cells would be predicted to result in
endothelial cell damage, and perhaps recruitment o f other inflammatory cells including
monocytes and T cells. Interestingly, endothelial cell damage by PR3 is mediated (in
part) by endothelial cell apoptosis via a mechanism involving PR3 internalization into the
endothelial cell independent of its proteolytic enzyme function (Yang, Kettritz and
others, 1996; Yang, Preston and others, 2001).
Following neutrophil activation by ANCA, neutrophils are driven down an accelerated
but abherrant apoptotic pathway (Harper, Ren and others, 2000). Thus, the neutrophils
will develop the morphologic features of apoptosis, but there is failure of cell surface
changes such as bi-lipid cellular membrane phosphatidylserine (PS) externalisation
(Harper, Ren and others, 2000; Zwaal and Schroit, 1997), which is normally an early
feature of all apoptotic cells. The externalization o f PS allows apoptotic cells to be
recognized and safely removed by phagocytes so that inflammation is not provoked by
release o f damaging cell contents (in other words, non-phlogistic cell death). After
ANCA stimulation, as a result of the lack of PS extemalization, phagocytes are less able
to process the apoptotic neutrophils in a non-phlogistic fashion, explaining the well-
characterised finding of leucocytoclasis often seen in the vasculitic lesions o f certain
vasculitic syndromes.
79
T cells and monocytes may also contribute to vascular injury in ANCA associated
vasculitides
ANCA associated vasculitic damage is accompanied by T cell and monocytic recruitment
(Jennette, 2002). It has been demonstrated that peripheral blood T-cells from patients
with either active or quiescent SSV proliferate in response to PR3 or MPO (King, Brooks
and others, 1998). The observation that T cell activation continues after disease remission
was confirmed by Christensson et al (Christensson, Pettersson and others, 2000), who
showed reduced CD28 and increased CD69 (an early T-cell activation marker, also
known as the activation induction molecule) on CD3 T cells, and CD38 on CD8-positive
T-cells (CD38 is present on T cells stimulated with mitogen, and furthermore CD38^
populations of T cells in the mouse are capable o f producing high levels of interleukin-4).
Increased levels of soluble interleukin-2 receptor during clinical remission were also
found. These studies suggest that T cells may contribute to the remitting and relapsing
nature of S W .
Lastly, it has been proposed that stimulation of T and/or B cells by superantigens may
result in the proliferation of autoreactive B cells capable o f ANCA production (Cohen
Tervaert, Popa, and Bos, 1999). This speculative theory will be discussed in more detail
in section 1.3.
Tissue expression of antigen targeted by ANCA
Although the antigens targeted by ANCA are mainly distributed on the neutrophil, there
is also evidence that monocytes express PR3 and MPO (Preston, Yang and others, 2002).
80
Moreover, ANCA-induced monocyte activation can result in the production of
interleukin-8 (Ralston, Marsh and others, 1997), contributing to the potentially injurious
pathways involved in ANCA associated S W . ANCA-mediated monocyte activation is
accompanied by increased expression o f CD 14, a lipopolysaccharide receptor, and CD 18
(one of the members of the p2 family of integrin receptors) (Nowack, Schwalbe and
others, 2000). Thus ANCA-activated monocytes may contribute to tissue injury (Preston,
Yang and others, 2002). Interestingly glomerular epithelial cells also are reported to
express proteinase-3 mRNA, raising the possibility o f direct glomerular epithelial injury
by ANCA (Schwarting, Hagen and others, 2000).
Genetic predisposition to ANCA associated vasculitides
Several investigators have examined potential genetic factors predisposing to vasculitis,
including vasculitic syndrome associated with ANCA (Kallenberg, Rarok, and Stegeman,
2002). Since no clear association with HLA alleles has been found attention has focused
on genes that control the neutrophil inflammatory response. These will be considered in
turn.
81
a-1-antitrypsin deficiency and ANCA associated vasculitides
An association between PR3-ANCA and the deficient a - 1-antitrypsin PiZZ phenotype
has been described (Esnault, Testa and others, 1993; Esnault, 1997). Analysis of the
incidence o f antibodies against neutrophil components in PiZZ-deficient sera has shown
that there is an increased incidence of antibodies against a granules and human leukocyte
elastase, but not against other classical ANCA antigens such as PR3, MPO, lactoferrin, or
bactericidal permeability-increasing protein (BPI protein). It thus appears that a-1-
antitrypsin deficiency is not sufficient in itself to cause ANCA-positive vasculitides, but
may act as an amplifying factor (Audrain, Sesboue and others, 2001). To support this
theory, PiZ heterozygosity in patients with PR3 ANCA vasculitis is associated with a
poor prognosis, disseminated disease, and increased mortality compared with non
carriers (Segelmark, Elzouki and others, 1995).
Proteinase-3 polymorphisms and ANCA associated vasculitides
Any genetic predisposition to altered PR3 processing, potentially resulting in abnormal
surface expression of PR3 on neutrophils, would be an attractive mechanism explaining
the observation of persistent PR3 expression on neutrophils in patients with ANCA
associated vasculitides, and the increased risk o f vasculitis in individuals who
constitutively express higher levels of surface PR3 (Witko-Sarsat, Lesavre and others,
1999). Such observations have provoked analyses o f PR3 polymorphisms. Of 10
identified polymorphisms, an association with WG was demonstrated for the A-564G
polymorphism in the proteinase-3 promoter (Gencik, Meller and others, 2000a). This site
82
contains a possible transcription factor-binding site, which could affect the level of
surface proteinase-3 expression.
p2-integrins and ANCA associated vasculitides
There may be a role for p2 neutrophil integrins in ANCA-mediated activation (Reumaux,
Vossebeld and others, 1995; Radford, Savage, and Nash, 2000). A restriction Aagment-
length polymorphism (RFLP) in exon 11 of the GDIS gene was associated with MPO-
ANCA SSV, although no relevant polymorphisms were identified in the vicinity of genes
for other cell adhesion molecules including intercellular adhesion molecule-1 (ICAM-1),
E-selectin, GDI lb, or human urokinase plasminogen activator receptor gene (Gencik,
Meller and others, 2000b).
F ey receptors and ANCA associated vasculitides
The Fey receptors FcyRIIa and FcyRIHb are constitutively expressed on neutrophils, and
polymorphisms have been studied in the context of ANCA associated vasculitides. No
association has been found between FcyRIIa receptor polymorphism and S W (Tse,
Abadeh and others, 1999), although there may be an association between the functionally
more active NAl allele of FcyRQIb in patients with WG and renal involvement (Tse,
Abadeh and others, 2000). Patients with WG homozygous for the FcyRII-R131 and
FcyRHIa-F158 may however be more susceptible to disease relapse (Dijstelbloem,
Scheepers and others, 1999).
83
Other genetic polymorphisms and ANCA associated vasculitides
Proinflammatory cytokines such as TNF-a and interleukin-1 may be involved in the
development o f vasculitic lesions. One study has failed to identify associations among
TNFa and interleukin-Ip restriction fragment length polymorphisms, and WG (Huang,
Giscombe and others, 2000). However, the same investigators did show an association
with a polymorphism of CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) and
WG CTLA-4 is a co-stimulatory molecule, which suppresses antigen-specific immune
responses by suppressing the CD28 pathway. The investigators found that the prevalence
of the shortest CTLA-4 allele was decreased in patients with WG compared with healthy
individuals, and proposed that genetic polymorphism o f this molecule could contribute to
the pathogenesis of WG by allowing an increased T cell activation by antigen.
ANCA from the paediatric perspective
In comparison with the extensive work relating to ANCA and vasculitis in adults, work in
children has been limited. Undoubtedly, the ANCA associated vasculitides occur in
children, as previously discussed, and a number o f reports have described typical C-
ANCA and P-ANCA patterns in children with vasculitis, predominantly Wegener's
granulomatosis, microscopic polyangiitis and renal crescentic glomerulonephritis (Nash
and Dillon, 1997; Ellis, Wood, and Beriy, 1995; Vogt, Kim and others, 1994). Generally
speaking clinical experience suggests that findings in adults with these conditions can be
extrapolated to children.
84
ANCA have also been reported in other chronic inflammatory disorders in children,
including inflammatory bowel disease, SLE, and cystic fibrosis (Nash and Dillon, 1997;
Schultz, Csemok and others, 2000; Sediva, Bartunkova and others, 1998). However, in
these disorders the antibodies are directed against a variety o f epitopes, and there is little
correlation with organ involvement or disease activity (Wong, Shah, and Dillon, 1995).
The significance of ANCA in these chronic inflammatory conditions in childhood is
unclear. ANCA are not present in haemolytic uraemic syndrome (Fitzpatrick, Shah and
others, 1992); IgG and IgM ANCA are not found in children with HSP, a relatively
common systemic vasculitis of childhood (Nash and Dillon, 1997). The prevalence and
significance of IgA isotype ANCA in Henoch-Schonlein purpura is a matter of debate
(ODonoghue, Nusbaum and others, 1992).
Some studies have suggested ANCA are found in acute Kawasaki disease, with an
elevated titre and atypical pattern on UF (Savage, Tizard and others, 1989). Later studies
using only samples taken before administration of IVIG therapy did not confirm this, and
the same IIF pattern was seen in children with fever resulting from acute infection as in
those with Kawasaki disease (Nash, Shah and others, 1995). This is consistent with the
experience in adults in which an atypical pattern can be seen in a range of acute and
chronic inflammatory conditions, and suggests that this pattern may be due to non
specific binding of antibody secondary to generalised immune activation and polyclonal
B cell activation.
85
Thus although the ANCA associated vasculitides do occur in children, these forms of
vasculitis are less common than ANCA negative vasculitides such as HSP and KD. Most
of the paediatric practice in dealing with the ANCA associated vasculitides is based on
data derived from adult studies, and many assumptions are made (rightly or wrongly)
regarding similarities in the clinical management and pathogenesis o f ANCA associated
vasculitides in children and adults.
Conclusion
ANCA-associated systemic vasculitis may develop as a result of a “two-hit” process by
the interplay of exogenous and endogenous frctors. As regards the genetic contribution,
immune response genes are likely to be involved (as in other autoimmune diseases). For
example, in PR3-ANCA-associated vasculitis, genetic polymorphism in the PR3
molecule altering its expression on the neutrophil may influence induction and clinical
course of WG.
Whilst much is now known about the downstream events mediating ANCA associated
vascular injury, relatively little is known about the factors which predispose to the loss of
immunological tolerance to neutrophil enzymes. Significant advances in the
understanding of the genetic influences that may predispose to ANCA associated
vasculitides are anticipated.
86
1.2.4.2 Anti endothelial cell antibodies
Anti endothelial cell antibodies (AECA) are antibodies that react with endothelial cell
(EC) structures (Praprotnik, Blank and others, 2001). They were first reported in the early
1970s in sera from patients with various rheumatic diseases, including systemic lupus
erythematosus (SLE) and scleroderma (Lindqvist and Osterland, 1971; Tan and Pearson,
1972). Since then AECA have been demonstrated in a wide variety of inflammatory
diseases, sometimes inconsistently. AECA bind to different structures on endothelial
membranes, mainly through the F(ab)2 portion o f the immunoglobulin, and IgG, IgM,
and IgA isotypes of those antibodies have been reported. The endothelial epitopes for
AECA have not yet been defined, but it is clear that they are likely to be more than one
(Praprotnik, Blank and others, 2001). It is now becoming apparent that AECA may have
preferential specificity for blood vessels of different sizes, presumably as a result of
differential antigen expression on endothelial cells derived firom vessels of different size
(Praprotnik, Blank and others, 2001). Thus, sera positive for AECA have been shown to
display a broad reactivity against endothelial cells obtained from different human
anatomic sources: from large arterial (aorta) or venous (umbilical cord vein, saphenous
vein) vessels as well as from small vessels such as renal, skin, omental, and brain
microvasculature (Shoenfeld, 2002). Interestingly, AECA are not species specific, since
they cross-react with human, bovine, and murine endothelial cells. In this respect, AECA
may be non-specific antibodies that are directed against ubiquitously expressed
endothelial antigens (Shoenfeld, 2002).
87
Based on studies showing that AECA derived from patients with large vessel diseases
such as Takayasu disease (TD) bind to and activate macrovascular human umbilical vein
endothelial cells (HUVEC) in vitro, and not microvascular endothelial cells (Blank,
Krause and others, 1999; Shoenfeld, 2002), Shoenfeld et al proposed that AECA from
different sources recognise different types of endothelial cell target molecules, which
may correlate with the predominant vessel size involved in individual diseases. They
therefore have suggested a classification system for AECA based on microvascular or
macro vascular endothelial cell binding propensity (Praprotnik, Blank and others, 2001;
Shoenfeld, 2002).
A Pitfall in the detection of AECA: methods of detection have variable sensitivity
and specificity
Non-standardised methods of detection at least in part explain some o f the inconsistencies
in the literature relating to the prevalence of AECA in different disease states (Meroni PL
and Youinou P, 1996). The use of indirect immunofluorescence permitted the original
description of AECA in rodent tissue. Despite its high specificity, this method is limited
by low sensitivity (Meroni PL and Youinou P, 1996). Subsequent reports documented a
similar reaction using whole human endothelial cells maintained in culture or membrane-
coated plates. Enzyme-linked immunosorbent assay (ELISA) is the method most widely
employed for the detection of AECA. Whole endothelial cells from different sources
(mainly HUVEC) or enriched endothelial cell membranes are used as substrate (Meroni
PL and Youinou P, 1996). Although ELISA is easy to perform, false-positive results are
possible, probably due to autoantibodies reacting with cytoplasmic or nuclear
88
components that contaminate the membrane preparation (Meroni PL and Youinou P,
1996). Additionally, few reports have compared the detection o f AECA on fixed and
unfixed endothelial cells, possibly leading to considerable discrepancies in the results.
Recently, it has been suggested that endogenous antibodies reacting to fetal calf serum
protein fi'om cell culture medium may give rise to false positivity in a cyto-ELISA for
AECA (Revelen, Bordron and others, 2000).
Flow cytometry can be used to detect AECA, but this technique requires a large number
of endothelial cells in suspension (Praprotnik, Blank and others, 2001). Moreover,
detachment of EC grown in vitro by enzymatic treatment in order to create such
endothelial cells suspensions may result in contamination o f endothelial cells surface
proteins with nuclear and cytoplasmic components exposed upon disruption of the of
endothelial cells (Praprotnik, Blank and others, 2001).
Therefore, it is clear that different methodologies can produce different results making
the interpretation of studies that employ different techniques problematic. That said, it is
now generally accepted that if appropriate cells are used as substrate, ELISA may display
the highest specificity and sensitivity for the detection o f AECA.
AECA disease associations
Many autoimmune diseases have been associated with AECA. These will be considered
in turn.
89
Systemic vasculitides and AECA
The largest disease group in which AECA are detected includes patients with systemic
vasculitides. Several investigative groups have reported the occurrence o f AECA in both
WG and MPA sera (Meroni PL and Youinou P, 1996). Some authors found that the
presence o f AECA in WG reflected the activity o f the disease and may represent an early
marker predictive o f disease relapse (Gobel, Eichhorn and others, 1996). Prospective
analysis showed that the rise in AECA titers preceded the development o f relapses in
ANCA-negative WG patients, and the persistence o f AECA after remission was
associated with a highly increased risk o f relapses (Gobel, Eichhorn and others, 1996).
In vitro studies support a pathogenetic role for AECA in systemic vasculitis. AECA from
the sera o f patients with WG are able to activate endothelial cells (Del Papa, G uidaii and
others, 1996): HUVEC incubated with AECA IgG from WG patients can up-regulate the
expression o f cell adhesion molecules, and can support leukocyte adhesion to HUVEC. In
addition, once bound to HUVEC AECA also induce an increase in production of
proinflammatory cytokines by the HUVEC including IE-1, IL-6, IL-8, and monocyte
chemotactic protein. These observations could provide pathogenic mechanisms for
vascular injury mediated by AECA.
Further evidence for the pathogenicity o f AECA comes from animal studies. Vasculitic
lesions are associated with murine AECA induced by immunizing naive mice with the
IgG fraction from the sera o f WG patients (Blank, Tomer and others, 1995). Following
the injection o f the human WG IgG fraction (displaying both PR3-ANCA and AECA
90
activity), naive mice produced both murine ANCA and AECA accompanied by
histopathological signs of renal and pulmonary vasculitis. The investigators went on to
immunize mice with IgG fraction containing only AECA (i.e. deplete o f PR3-ANCA).
Again, the mice developed murine AECA followed by glomerular vasculitis (Blank,
Tomer and others, 1995). This experimental model, therefore, suggests that AECA
potentially are involved in the pathogenesis of vasculitic lesions.
Conflicting data have been reported relating to the prevalence of AECA in KD. Kaneko
et al studied the sera from patients with respect to binding activity to HUVEC using a
cellular based ELISA technique (Kaneko, Savage and others, 1994). They detected IgM
AECA in 16/22 sera, and IgG AECA in 19/22 sera. This result was significantly different
when compared to sera obtained from healthy control children. In addition, in that study
8/16 sera showed complement-dependent cytotoxicity against HUVEC, which was
further enhanced by pre-treatment of the HUVEC with TNF-a. IgM AECA titres
measured by ELISA were positively correlated with cytotoxicity. The authors concluded
that AECA worked synergistically with TNF-a to mediate complement-dependent
endothelial damage in some patients with KD.
In contrast, Nash et al from the same group repeated the study of AECA in KD patients
using the same ELISA technique, but this time compared the results with sera obtained
from febrile control children (Nash, Shah and others, 1995). They found no difference
between KD patients and febrile controls in terms o f levels o f IgM AECA when total
IgM had been taken into account. The authors concluded that the previously observed
91
difference in IgM AECA between KD and afebrile controls was attributable to
differences in total IgM levels, and suggested that AECA were an epiphenomenon related
to a non-specific acute phase response.
Other vasculitic diseases where AECA have been detected include Behçet’s disease
where the prevalence of AECA was 50% in one study (Cervera, Navarro and others,
1994). Moreover, the prevalence was higher in patients with active disease. Importantly,
however, no febrile control group was included in this study and hence this observation
may again have represented a non-specific epiphenomenon related to increased total
circulating immunoglobin.
Other AECA disease associations
AECA have also been described in association with SLE and antiphospholipid syndrome,
rheumatoid arthritis, systemic sclerosis, inflammatory bowel disease, organ
transplantation, hyperprolactinaemia, thrombotic thrombocytopenic purpura (TTP),
heparin induced thrombocytopenia, and in preparations of pooled human
immunoglobulin (Meroni PL and Youinou P, 1996).
Conclusion
AECA would appear to be a logical and obvious pathogenetic mechanism for systemic
vasculitis. Definitive proof that AECA are involved in the pathogenesis of vasculitis are
lacking, however. Moreover many studies relating to AECA have been hampered by
92
methodological artefacts, and lack of appropriate disease control groups. Moreover,
potential AECA epitopes are currently poorly characterised.
93
1.2.5 Immune complexes and vasculitis
Immune complexes have already been considered in the context of the association
between HCV and cryoglobulinaemia, but some expansion on this pathogenetic
mechanism is required. For many years immune complexes have been a recognised
association with vasculitis (Dixon FJ, VasquezJJ, and Weigle WO, 1958). Acute serum
sickness, which is the prototypic model of immune complex-mediated disease, was one
of the first animal models of vasculitis to be described. Our current understanding o f the
cellular and molecular mechanisms responsible for the inflammation and tissue damage
that occurs in immune complex-mediated diseases, such as in SLE or hypersensitivity
vasculitis, is based to some extent on knowledge gained from the elucidation of the
immunopathogenesis of acute serum sickness in animals.
In this model, which is essentially an example of a type m hypersensitivity reaction,
following the injection of a large dose of bovine serum albumin (BSA), rabbits develop
antibodies to the foreign protein, with the subsequent formation o f circulating BSA-anti-
BSA immune complexes. Deposition of those immune complexes in various tissues,
including the glomerulus, synovium, and the walls of blood vessels then occurs. The first
component of complement, Clq, binds to the Fc portion of IgG or IgM antibodies in the
deposited immune complexes and activates the classical pathway o f complement
activation. Activated complement components (C5a) with chemotactic properties are
generated, which attract neutrophils to the area and activate them. The activated
neutrophils bind to the deposited immune complexes through their Fc and complement
receptors, and attempt to phagocytose the complexes, but are unable to do so. As a
94
consequence o f this “frustrated” phagocytosis, the neutrophils degranulate and release
lytic enzymes. When this sequence of events occurs in blood vessels it causes an acute,
necrotizing, leukocytoclastic vasculitis (Cochrane and Koffler, 1973). Left untreated, the
involved blood vessels in animals with acute serum sickness develop fibrinoid necrosis of
the media, and prominent mononuclear cell infiltrates around all layers of the blood
vessel walls (Neild, Ivory, and Williams, 1984a; Neild, Ivory, and Williams, 1984b).
Treatment of rabbits with acute serum sickness with cyclosporin inhibits the development
of mononuclear infiltrates in or around vessels, but does not prevent necrosis. These data
suggest that the formation and maintenance of mononuclear cell infiltrates in acute serum
sickness model is a T cell- dependent phenomenon (Neild, Ivory, and Williams, 1984a;
Neild, Ivory, and Williams, 1984b).
The deposition of virus-anti-virus immune complexes in vessel walls appears to play a
central role in the pathogenesis of many virus-associated vasculitides, including the
vasculitis that occurs in patients infected with HBV and HCV (see section 1.2.2.1).
Immune complex deposition is also central to the pathogenesis o f most types of
leukocytoclastic vasculitis. The post-capillary venule is an important site o f initiation of
damage. Immune complexes, formed in a state o f antigen excess, circulate in the vascular
compartment and precipitate in the walls of blood vessels. The trapping o f immune
complexes in the microvasculature initiates the cascade o f events described above,
causing small vessel vasculitis (Bagga and Dillon, 2001).
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1.2.6 Cryoglobulins and vasculitis
Cryoglobulins have been mentioned previously in the context o f HCV infection (section
1.2.2.1). Cryoglobulins are immunoglobulins that persist in the serum, precipitate with
cold temperature, and resolubilize when rewarmed (Cacoub, Costedoat-Chalumeau and
others, 2002). Cryoglobulins are classified according to Brouet et al. (Brouet, Clauvel and
others, 1974): type I cryoglobulins are single monoclonal immunoglobulins; types II and
in are mixed cryoglobulins, composed of different immunoglobulins, with a monoclonal

component in type II and only polyclonal immunoglobulins in type m. Type I
cryoglobulins are always associated with haematological malignancy. Mixed
cryoglobulins (type II or HI) are associated with connective tissue disease, malignant
haematological disorders, or infections (including HCV, Epstein-Barr virus, HIV,
cytomegalovirus. Treponema, and Leismaniasis) (Cacoub, Costedoat-Chalumeau and
others, 2002). When mixed cryoglobulins are found in the absence of well-defined
disease, the syndrome has been designated “essential mixed cryoglobulinemia”.
The syndrome of mixed cryoglobulinemia is characterised by the clinical triad of
purpura, arthralgia, and asthenia associated with type II or type m mixed cryoglobulins.
The disorder represents the consequence of an immune complex-type vasculitis (see
section 1.2.5) with depressed levels of complement and deposition of immunoglobulins
and complement in lesions. Cryoglobulinémie vasculitis may involve many organs,
particularly the peripheral nervous system and the kidneys. Cryoglobulinaemic vasculitis
is rare in childhood, but should be considered in any child with small vessel vasculitis,
and/or membranoproliferative glomerulonephritis (Bagga and Dillon, 2001).
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1.2.7 The endothelium as a target and amplifier o f inflammation in vasculitis
An emerging concept is that the endothelium is not only the prime target for many of the
pathogenetic mechanisms described above, but is also a protagonist o f the inflammatory
cascade associated with vasculitis, and may play an active role in focusing the
inflammatory process at vascular sites by mechanisms which will include antigen
presentation, cell adhesion molecules, and cytokine production. In addition to
amplification of the inflammatory response, the vascular response to inflammation results
in vascular remodelling and repair- ultimately leading to vessel occlusion and end organ
damage.
1.2.7.1 The endothelial cell as a target for injury in vasculitis
As will now be appreciated from the preceding description o f the various pathogenetic
mechanisms involved in vasculitis, the endothelial cell is a target for injury by infectious
agents, malignant processes, ANCA and AECA mediated vascular injury, and immune
complex-mediated injury. But it now appears that endothelial cells are not just “innocent
bystanders”, and as will be discussed, actively participate in the inflammatory process.
1.2.7.2 The endothelial cell amplifîes inflammation
Endothelial cells can amplify inflammation by 4 main mechanisms: adhesion molecule
expression, cytokine production, angiogenesis, and as will be discussed in considerable
97
detail in subsequent sections, may play a role in antigen or superantigen presentation to T
cells.
Endothelial cell adhesion molecules
Numerous studies have described upregulated soluble adhesion molecule expression in
serum, on circulating leukocytes, and in lesional tissue o f vasculitis syndromes (Sundy
and Haynes, 2000; Cid, 2002). Adhesion molecules influence the binding o f neutrophils
and mononuclear cells to endothelial cells. Adhesion molecule expression in lesional
tissue o f vasculitis syndromes may reflect the type o f cellular infiltrate. For example,
vascular cell adhesion molecule-1 (V CAM -I), the ligand for lymphocytic VLA-4, is
expressed on vessels in lymphocytic cutaneous vasculitic lesions, whereas E selectin
(CD62E) expression is upregulated in leukocytoclastic vasculitic lesions characterised by
neutrophil infiltration (Bradley, Lockwood, and Thiru, 1994; Burrows, Molina and
others, 1994).
Passage o f inflammatory cells from the vascular space into inflamed vascular tissue
requires adhesion molecule-mediated, leukocyte-leukocyte, and leukocyte-endothelial
cell interactions. Adhesion molecule expression on leukocytes and endothelial cells is
influenced by local cytokine production and adhesion molecule-ligand interactions o f
leukocytes and endothelial cells in turn influence cytokine production from both
leukocytes and endothelial cells (Bradley, Lockwood, and Thiru, 1994; Sundy and
Haynes, 2000).
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Constitutive adhesion molecule expression on endothelial cells is modulated by regional
factors such as vessel location and sheer forces (Cines, Poliak and others, 1998). Thus,
adhesion molecule expression in the healthy state varies from organ to organ, and by
vessel size. For example, lung endothelium uniquely expresses Lu-ECAM-1 in humans,
whereas endothelial cell venules in Peyer’s patches constitutively express Mad-CAM-1.
This heterogeneous pattern of adhesion molecule expression may, in part, explain the
propensity of different vasculitis syndromes to affect vessels o f a particular size or
location (Sundy and Haynes, 2000).
Studies on cell adhesion molecule expression in vasculitis
Lesional endothelial adhesion molecule expression to date has been studied in patients
with cutaneous leukocytoclastic vasculitis, KD, PAN, and GCA (Leung, Cotran and
others, 1989; Sais, Vidaller and others, 1997; Coll-Vinent, Cebrian and others, 1998; Cid,
Cebrian and others, 2000). Common findings in these syndromes were upregulation of E-
selectin, VCAM-1 and ICAM-1 by endothelial cells. In glomerular lesions of WG and
MPA VCAM-1 and ICAM-1 expression can be observed in the glomerulus, tubular
epithelial cells, and peritubular capillaries (Cid, 2002). In GCA adhesion molecule
expression occurs in neovessels at the adventitia and within the inflammatory lesions
suggesting that the infiltrating leukocytes come from the vasa vasorum (Cid, Cebrian and
others, 2000).
It is likely that interactions mediated by adhesion molecules are pathogenetically
important in vasculitis. In the study by Chakravorty and others (Chakravorty, Howie and
99
others, 1999) peripheral blood T cells adhered to intraglomerular, periglomerular, and
tubulo-interstitial regions of the cortex of kidney sections taken from patients with renal
vasculitis. In that study, blocking monoclonal antibodies against tissue-expressed ICAM-
1, VCAM-1, and the CS-1 domain of fibronectin (CS-lFn) differentially attenuated T cell
adhesion. Additionally, in a mouse model of vasculitis induced by immunisation against
mycobacterium butyricum, the administration of blocking monoclonal antibodies
demonstrated the important participation of interactions mediated by selectins and by a4
integrins in leukocyte adhesion and transmigration (Johnston, Issekutz, and Kubes, 1996).
Endothelial cells, cytokines and vasculitis
Endothelial cells may contribute to the inflammatory response in vasculitis by the
production of a variety of cytokines and growth factors. Notably, they can produce IL-la
and IL-6 which may contribute to the acute phase response observed in vasculitis (Cid,
2002). Through the production of colony-stimulating factors, they may prolong the
survival and promote the proliferation of leukocytes in the perivascular compartment,
thus perpetuating the chronic vascular inflammation (Schliesser, Pralle, and Lohmeyer,
1992). Several chemokines including IL-8, and RANTBS (regulated upon activation,
normal T cell expressed and secreted) can be produced by endothelial cells (MacKay,
2001). Chemokines selectively attract leukocytes, thus endothelial cells may actively
contribute to tissue targeting in systemic vasculitis, and by attracting additional
leukocytes may perpetuate and amplify vessel inflammation.
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Thus, there is now a considerable body o f evidence that would support the notion that
cytokine and adhesion molecule-mediated pathways may contribute to the inflammatory
vascular response to injury by focusing and perpetuating leukocyte infiltration into the
vascular and perivascular compartments.
Therapy of vasculitis aimed at modulation of cytokine and adhesion molecule
function
Therapies designed to modify abherrant cytokine production and/or cell adhesion
molecule expression may be a step towards the therapeutic “Holy Grail” or “magic
bullet” therapy for vasculitis. As discussed in section 1.1, treatment o f vasculitis requires
non-specific and aggressive cytotoxic immunosuppressive regimens. This has
implications in terms o f toxicity, which is o f particular concern for paediatric patients
(Brogan and Dillon, 2000b). Evidence relating to the role o f cytokines and adhesion
molecules in vasculitis offers the prospect o f therapies that block key pathogenic cytokine
or adhesion molecule pathways. This is an approach that has been used with considerable
success in rheumatoid arthritis, for example (Emery and Buch, 2002).
The use o f anti-TN F-a monoclonal antibody and soluble TNF receptor for the treatment
o f rheumatoid arthritis has validated the rationale for disrupting cytokine pathways in
treating inflammatory disease. Since TN F-a is ubiquitously upregulated in vasculitis,
T N F-a blockade may be an obvious and logical therapeutic choice. Anecdotal evidence
suggests efficacy in treating rheumatoid vasculitis, temporal arteritis, and WG (Sundy
and Haynes, 2000). Recently, a randomised, placebo-controlled multi-centre trial in WG
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using etanercept (a TNF-a receptor p75 fusion protein) has started in the USA. The
principal aim of the trial is to test the efficacy o f etanercept as add-on therapy to standard
maintenance treatment to maintain disease remissions (Stone JH, 2000).
Caution must be the order of the day, however, since such a “logical approach” resulted
in deterioration in multiple sclerosis in response to TNF-a blockade (Wiendl and
Hohlfeld, 2002). The experience in MS demonstrates that theoretically promising agents
may paradoxically increase disease activity.
Other potential cytokine therapy targets in the vasculitides include inhibition o f signaling
by IL-1 and IL-6 by administration ofIL-1 receptor antagonist and soluble IL-6 receptor;
and IL-10 (which downregulates both Thl and Th2 immune responses) (Sundy and
Haynes, 2000).
Finally, drugs that inhibit the adhesion of leukocytes to endothelial cells including
antibodies to CD 18, intercellular adhesion molecule type 1 (ICAM-1) antisense, and anti-
ICAM-1 antibodies may prove useful in the treatment of vasculitis in the future (Cohen
Tervaert, Stegeman, and Kallenberg, 2001; Griffin, Chapman and others, 1999).
Lockwood et al. recently reported the use of a humanized monoclonal anti-CD 18
antibody in patients with vasculitic tissue injury that resulted in ulceration or limb/digital
infarction with incipient gangrene (Griffin, Chapman and others, 1999; Lockwood, Elliott
and others, 1999). Remarkable prompt clinical healing of ulceration and the restoration of
limb function was noted in four out o f five patients.
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Angiogenesis and inflammatory amplification
Angiogenesis is part of the vascular response to inflammation in vasculitis, and is
particularly relevant to GCA (Cid, 2002). Angiogenesis plays a dual role; on the one hand
it presumably constitutes a protective response to organ ischaemia as a result of vascular
injury, but on the other hand may contribute to the amplification of the inflammatory
vascular response by providing new sites for leukocyte entry into the vascular tree, and
also by providing a new source of cytokines, chemokines, and growth factors (Cid,
2002 ).
Angiogenesis is controlled by a balance between the influx o f angiogenic and anti-
angiogenic factors, and the control of the expression o f relevant receptors. Several
molecules may promote angiogenic activity. These include growth factors, chemokines,
thymosins, acute-phase proteins and extracellular matrix protein fragments (Carmeliet
and Jain, 2000; Carmeliet and Collen, 2000). Several angiogenic factors have been
detected in temporal artery lesions from patients with GCA, although their functional
relevance is incompletely understood (Cid, 2002).
1.2.8 T cells and vasculitis
T cells are involved in the pathogenesis of vasculitis, but their role in different vasculitis
syndromes is less clearly defined. There seems to be no doubt that they play a role in the
formation of granulomas of WG (Sundy and Haynes, 1995), and the response of ANCA-
negative patients with MPA to monoclonal antibody therapy to T cells provides
103
compelling evidence that they are involved in the pathogenesis o f that entity (Mathieson
and Oliveira, 1995).
1.2.8.1 T cells and Giant cell arteritis
Much progress relating to the pathogenesis of GCA points to a central role for T cells in
this disorder (Weyand and Goronzy, 1996; Mohan and Kerr, 2000). Non-specific
immunological abnormalities include decreased circulating CD8+ lymphocytes, increased
circulating soluble IL-2 receptors, circulating soluble adhesion molecules, and cytokines
such as IL-6, all support the concept that immune mechanisms play an important role
(Weyand and Goronzy, 1996; Mohan and Kerr, 2000).
Arterial wall T cells in GCA have been found to have identical sequences at the third
complementary determining region (CDR3) of their T-cell receptors (Weyand,
Schonberger and others, 1994). This supports the hypothesis that GCA lesions may
represent an immune response toward an antigen present in the arterial wall. The inciting
antigen has not yet been identified, however. T cells enter the involved artery through the
vasa vasorum in the adventitial layer, and produce INF-y. A potent activator of
macrophages, IFN-y is crucial for the development o f granulomas and giant cells.
1.2.8.2 T cells and small vessel vasculitides
T cell-mediated immunity is thought to contribute to pathogenesis of SSV, and several
previous studies have documented the ability of peripheral blood T-cells fi*om patients
with either active or quiescent SSV to proliferate in response to PR3 or MPO (Brouwer,
104
Stegeman and others, 1994; Griffith, Coulthart, and Pusey, 1996; King, Brooks and
others, 1998). As previously discussed, circulating T cells are continuously activated,
even when small vessel vasculitides are in clinical remission (section 1.2.4.1.4).
To unequivocally establish a true effector role for antigen-specific T-cells, it would be
necessary to demonstrate the presence of such cells in lesional vasculitic tissue, and this
has not yet been achieved. Immuno-phenotyping of monocytic infiltrates in lesional
tissue in ANCA associated vasculitides has demonstrated CD4-positive and CDS-positive
T cells (Savage, Harper, and Holland, 2002). Moreover, many of the cell adhesion
molecules and cytokines that are required for T cell recruitment are upregulated in
lesional vasculitic tissue (section 1.2.7.2).
Cell-mediated immunity, including that of T-cells, macrophages, and fibrin in the
absence of immunoglobulin, has been demonstrated in the kidneys o f patients with pauci-
immune glomerulonephritis (Cunningham, Huang and others, 1999). In that study (of 15
patients) CD3-positive T cells, CD45RO-positive T cells, macrophages, fibrin, and
endothelial-associated tissue factor were all demonstrated to be prominent in glomeruli.
These mediators were absent in a group of 12 patients with thin basement membrane
disease, and only occasionally observed in a group o f eight patients with "humorally
mediated" (non-crescentic) glomerulonephritis. Thus, these data would suggest that
pauci-immune crescentic nephritis is most likely a manifestation o f T cell-directed
immune injury.
105
Further support for a Thl bias rich in INF-y and poor in IL-4 is provided by the study by
Csemok et al (Csemok, Trabandt and others, 1999). This was an interesting study
because it examined multiple body compartments to determine whether a specific
cytokine pattern (Thl or Th2) predominates in Wegener's granulomatosis (WG), by
evaluating INF-y and IL-4 expression in biopsied nasal mucosal tissue, bronchoalveolar
lavage fluid, and peripheral blood, and compared the findings with those in disease and
healthy control subjects. Patients with WG and chronic rhinitis were found to share in
situ production of messenger RNA (mRNA) specific for IFN-y (Thl). Only 2/13 patients
with WG expressed IL-4 in nasal mucosa, whereas IL-4 mRNA PCR products were
found in inflamed nasal mucosa of all the disease control patients. The granuloma-
derived T cells of WG patients produced only IFN-y, while CD4+ and CD8+ T cells from
bronchoalveolar lavage fluid and peripheral blood produced mainly IFN-y. The authors
concluded that a Thl cytokine pattern predominates in the granulomatous inflammation in
patients with WG.
1.2.8.3 T cells and Kawasaki disease
Perhaps the most compelling evidence that T cells are involved in the pathogenesis of
vasculitis relates to the observations in KD. While there is currently much debate
regarding a conventional antigen versus superantigen aetiopathogenesis, most authorities
agree that T cells are central to the pathogenesis. This argument, including an in-depth
review of the evidence for and against the role of superantigens in KD and other
vasculitides, will be the subject of the next section and will be the main theme followed
throughout the subsequent result chapters of this thesis.
106
1.2.9 Concluding remarks relating to the pathogenesis o f vasculitis
Many mechanisms have been described to explain the vascular injury associated with
vasculitis syndromes. Most of these are downstream events, and although important do
not explain aetiology in most cases. For the purposes of description, the various
mechanisms have been described in isolation. It is likely however that different
mechanisms will operate together (perhaps synergistically), with varying contributions
from each mechanism in individuals with vasculitis.
107
1.3 Superantigens and vasculitis
1.3.1 Introduction
The discussion from previous sections has established that there is considerable
heterogeneity among the various vasculitis syndromes that can affect children and adults.
In addition, many different infectious and immune processes can interact on the
background of host predisposing factors to culminate in vascular injury. It would be thus
overly simplistic to hypothesise that a single aetiopathogenetic factor, such as
superantigens, would be the cause o f all childhood vasculitic syndromes. A more realistic
question would be to ask if there is any evidence that superantigens could be involved at
some level in certain vasculitis syndromes, either as the main precipitating fector, or by
amplifying an inflammatory cascade already set up by other factors; and secondly to ask
what mechanisms superantigens might utilize to cause vascular injury.
This section of the thesis will describe what a superantigen is, outline how they are
processed by T cells at a molecular level, and describe the evidence for and against the
role of superantigens in KD and other vasculitides.
1.3.2 What is a superantigen?
Superantigens (SAgs) are a class of immuno-stimulatory proteins o f bacterial or viral
origin with the ability to activate large fractions (5-30%) o f the T cell population (Li,
Liera and others, 1999; Muller-Alouf, Camoy and others, 2001), and are responsible for
human toxic shock syndrome and some forms o f gastroenteritis. By contrast.
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conventional antigens stimulate 0.01-0.0001% o f T cells (Muller-Alouf, Carnoy and
others, 2001). SAg activation o f T cells requires interaction with a specific Vp segment
o f the T cell receptor (TCR) and with major histocompatibility complex (MHC) class II
molecules on the surface o f antigen presenting cells (figure 1.8) (Muller-Alouf, Carnoy
and others, 2001). The complete responding Vp repertoire has not been identified for
many o f the known SAgs, and some SAgs will bind to more than one Vp family (for
exam ple staphylococcal enterotoxin B [SEB] which typically activates T cells bearing
Vp3, V p l2 , and V p l7 as major-responding Vp families; and V p l4 , V p i 5 and Vp 20 as
lesser-responding families) (Cohen Tervaert, Popa, and Bos, 1999).
Contrary to the view o f some authors who have suggested that SAgs only stimulate CD4
T cells (Janeway CA, Travers P and others, 1999; Mingari, Cambiaggi and others, 1996),
SAgs can activate CD4, CDS and sometimes yÔ T cells (Herrmann, Baschieri and others,
1992; Muller-Alouf, Carnoy and others, 2001). The consequence o f T cell stimulation by
SAgs is massive release o f T cell derived cytokines such as INF-y, TN F-a and TNF-p
(Li, Liera and others, 1999; Choi, Lafferty and others, 1990; Choi, Kotzin and others,
1989; Fraser, Arcus and others, 2000; Kappler, Kotzin and others, 1989), following
which the stimulated T cells are generally deleted or become anergic (see below). Since
SAg-producing organisms such as Staphylococcus aureus and Streptococci are
ubiquitous among the population (including the paediatric population (Lindberg,
Nowrouzian and others, 2000)), there is considerable interest in SAgs as initiators or
modifiers o f autoimmune disease states (Fraser, Arcus and others, 2000; Schiffenbauer,
109
Figure 1.8: Conventional versus Superantigen presentation to T cells
APC APC
MHCn MHCn Conventional A g
Superantigen
TLy
I
1 in 10® T lymphocytes
1
1 in 10 T Lymphocytes
activated, but oniy those
activated bearing particuiar TCR Vp
sub-units
Soos, and Johnson, 1998; Torres and Johnson, 1998), including the systemic vasculitides
(Cohen Tervaert, Popa, and Bos, 1999).
1.3.2.1 Conventional antigen versus superantigen presentation
to T cells
Conventional antigens are processed (by intracellular degradation) by classical antigen
presenting cells (APC) into peptides that are presented in association with MHC class U
molecules expressed at the surface o f APC. The peptide-MHC molecules are then
specifically recognized by T cells through the groove formed by the a and p variable
domains of the TCR. Therefore, each antigen-processed peptide is destined to be
recognized in the context of self-MHC molecules by a specific cognate TCR structure
(Muller-Alouf, Camoy and others, 2001). As a result o f this when an antigen is
introduced into the host the number of T cells that would have the TCR that can
recognise the appropriate peptide epitope of the antigen is in the order of 1-100 cells per
million.
MHC class n molecules are expressed on the cell surface as a and p heterodimers. After
synthesis in the endoplasmic reticulum, MHC class II molecules bind to the invariant
chain (li) to form a complex between 3 a and P heterodimers, and an li trimer (Shoukry,
Lavoie and others, 1997). The li protects the peptide-binding groove of MHC class II
molecules during their transport in the golgi from loading with endogenously derived
peptides. Furthermore, the li acts as a chaperone that guides properly folded MHC class
n molecules to endosomal vesicles, where loading with antigenic peptide occurs. In the
111
endosomes, the li is cleaved, leaving behind a small peptide known as CLIP (class II-
associated invariant chain peptide) and thereafter the MHC class II molecule promotes
the release of CLIP (Shoukry, Lavoie and others, 1997). MHC class II molecules are then
loaded with antigenic peptides and reach the cell surface to be presented to T cells
(Shoukry, Lavoie and others, 1997).
MHC class n molecules are also required for the presentation o f SAgs. SAg presentation
to T cells requires MHC class II molecules and TCR cross-linking, as demonstrated by
the formation of TCR SAg MHC trimers in solution (Seth, Stem and others, 1994). Thus
SAgs differ from conventional antigens in that the former do not require intracellular
processing. Whereas the stimulation induced by the MHC class II and conventional
antigen complex in the periphery serves to activate and alert T cells against the presence
of foreign antigens, SAg stimulation results in early activation followed by anergy and
deletion (figure 1.9). From an evolutionary point o f view, this would have survival
advantages for the SAg-producing organism since depletion o f the host immune response
to that organism would almost certainly be the result. Multiple factors such as the
distribution o f the SAg to generative lymphoid tissues or presentation by an inappropriate
antigen presenting cell (APC) may contribute to this effect (Baccala, Vandekerckhove
and others, 1993). The major differences between SAg and conventional antigen
processing are summarised in table 1.3 (adapted fi’om Shoukry et a l 1997) (Shoukry,
Lavoie and others, 1997).
112
Figure 1.9: Fate of a T cell following SAg stimulation
Key: APC = Antigen presenting cell

T Ly = T lymphocyte
= Superantigen
APC
'm u c u anergy
Va
activation ► expansion
apoptosis ► deletion
Table 1.3 Conventional antigen versus superantigen presentation to T cells
SuperantigeiH Com cntional pepddc antigens

Internal processing D o not require processing Internally processed m vesicles
Binding to MHC class II Bind outside the antigen groove B ind in the peptide groove
Half-life association w ith class II V irtually irreversibly associated with class
molecules minutes to hours 11 molecules
Interaction with the TCR Does not necessarily require the cognate Presentation involves cognate recognition
recognition o f the M HC molecule by the betw een the TC R and the M HC
TCR
Functional outcome Activation and proliferation, followed by Activation and proliferation under
anergy or apoptosis conventional circumstances
HLA-DR alleles differ in their ability to present SAgs to T cells
A study by Herman et al demonstrated that different human MHC class H proteins
differed in their ability to bind staphylococcal enterotoxins and stimulate murine and
human T cells (Herman A, Croteau G and others, 1990). Since the HLA-DR proteins
share a common a chain, these results indicate that the polym orphic p chain plays an
important role in SAg binding and presentation to T cells. O f the different HLA DR
alleles tested, HLA-DR w53 was the poorest enterotoxin presenter. The overall MHC
class n hierarchy for supporting SAg stimulation, however, favoured HLA DR over
HLA-DQ and HLA DP: (highest to lowest ability: HLA-DR > HLA-DQ> HLA-DP)
(Herman A, Croteau G and others, 1990).
Mycoplasma SAgs are “partially Vp restricted”: SAgs masquerading as
conventional antigens
The work of Hodtsev et al has important implications for our understanding o f how SAgs
are presented to T cells, and in the interpretation o f data generated from studies
examining T cell Vp skewing (the “immunological footprint” left by a SAg) (Hodtsev
AS, Choi Y and others, 1998).
114
Mycoplasma arthritidis, which induces a chronic form of arthritis in rodents resembling
human rheumatoid arthritis, produces a 25-kDa superantigenic toxin called MAM
{Mycoplasma arthritidis mitogen). MAM, which was first described in the early eighties
strongly activates murine T cells bearing Vp6 and Vp8 (Hodtsev AS, Choi Y and others,
1998). MAM also stimulates human T cells through the Vpl7 region, but to a lesser
extent compared to its activation of murine T cells, or to the human T cells activated by
the staphylococcal enterotoxins (Cole and Atkin, 1991). Like Yersinia superantigenic
toxins, MAM does not have significant homology with staphylococcal and streptococcal
SAgs (Cole, Knudtson and others, 1996).
The binding domains recognized by MAM on the TCR and MHC class II molecules have
been extensively characterised (Hodtsev AS, Choi Y and others, 1998). Unlike other
superantigenic toxins, MAM recognises two distinct domains on the TCR. It interacts
with the Vp region of the TCR, but it also binds to the complementary-determining
region 3 (CDR3) of the p chain, which is central to the conventional peptide antigen
recognition by the TCR (Hodtsev AS, Choi Y and others, 1998). During conventional
antigen peptide recognition, Vp and V a domains o f the TCR form contacts with MHC
class n, and the complex is stabilised by CDR3-peptide interactions. Similarly,
recognition of MAM is Vp-dependent and is also apparently stabilised by direct contacts
with the CDR3-P region.
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Not all human V p l7+ T cells respond to MAM. The response is limited to those cells
expressing two appropriate residues at the base o f the CDR3-p loop (Hodtsev AS, Choi Y
and others, 1998). This suggests that T cell activation by MAM is dependent on TCR
junctional diversity, thus limiting the number o f potential MAM-reactive T cell clones.
The frequency of T cells responding to this superantigen therefore are significantly lower
than to other superantigens (for example SEB), but significantly higher than the T cell
response to conventional antigens. Thus, MAM represents a new type of ligand for TCR,
distinct from both conventional peptide antigens and other known superantigens:
effectively a SAg masquerading as a conventional antigen. Whether this characteristic is
limited to MAM or is a feature of other (as yet) poorly characterised SAgs is unknown.
The effect of SAgs can be blocked by peptide SAg antagonists
Any consideration of the biology of SAgs would be incomplete without some mention of
the exciting work of Arad et al. This group have developed a peptide antagonist of SEB,
which additionally has cross reactivity with other SAgs (Arad G, Levy R and others,
2000 ).
The investigators synthesised peptides containing SEB residues 150-161, which are
conserved among superantigens, yet are not known to be involved in binding to either to
the TCR or MHC class II molecules. In addition they synthesised two variants o f this
sequence (numbers in parentheses represent amino-acid positions in SEB), pl2(150-161)
and plO(l 52-161). When present in molar amounts 100- to 200-fold higher than SEB,
116
none of these peptides had substantial SEB agonist activity, as shown by their inability to
induce expression of mRNA encoding for the Thl cytokines IL-2 and IFN-y in human
peripheral blood mononuclear cells (PBMCs). The dodecapeptide p12(150-161)
(YNKKKATVQELD) was a particularly active antagonist, inhibiting expression of IL-2
mRNA by 18- Fold and that of IFN-y mRNA by 10-fold. This peptide is a variant of the
natural SEB sequence in pSEB(150-161) (TNKKKVTAQELD).
Furthermore, the investigators went on to test the ability o f p i2 to protect mice from
lethal challenge with various staphylococcal and streptococcal SAgs, including SEB.
Although 100% of control mice exposed to a lethal dose o f SEB were killed within about
48 hours, all survived lethal challenge with SEB when pl2 was administered
intravenously 30 minutes before challenge, and 70% survived when it was given
intraperitoneally. Mice exposed to pl2 alone stayed fully viable and showed no de
tectable side effects. Moreover, pl2 not only was protective when given before SEB
challenge but also was able to rescue mice undergoing lethal shock even when injected 3
hours after the toxin.
To determine whether the antagonist peptide provided broad-spectrum protective activity,
they next studied its effect during lethal challenge with the streptococcal toxin SPEA
(streptococcal pyrogenic exotoxin A), and with TSST-1 (which has only 6% overall
sequence homology with SEB). pl2 was also protective against these toxins. Moreover,
the surviving mice rapidly developed protective antibodies against SAg that rendered
them resistant to further lethal challenges, even with different superantigens.
117
Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that
inhibits their action at the beginning of the toxicity cascade, before activation o f T cells
takes place. It is also interesting to note that this group (from Israel) are now developing
the pi 2 technology to protect against possible germ warfare attacks (Kaempfer, Arad and
others, 2002).
118
1.3.2.2 Superantigens and vasculitis
SAgs are one of the environmental factors that have been proposed to modulate a number
of autoimmune diseases, including vasculitis. The most compelling evidence for
involvement of SAg in the pathogenesis of vasculitis relates to KD, but at the same time
this hypothesis has provided the most controversy (Barron, 2002). This section will
review the evidence for and against SAgs in the pathogenesis o f KD, and other
vasculitides.
Superantigens and Kawasaki disease
There are striking similarities between the clinical and immunological features of KD and
the superantigen toxin mediated staphylococcal and streptococcal toxic shock syndrome
(TSS) and scarlet fever (Curtis, Zheng and others, 1995; Leung, Meissner and others,
1995a; Leung, Giomo and others, 1995; Leung, Meissner and others, 1995b; Leung,
Meissner and others, 1995c). The difficulty in distinguishing these diseases has been
extensively documented (Hansen, 1983; Raimer, Tschen, and Walker, 1981; Hall, Hoyt
and others, 1999), and the exclusion of staphylococcal disease has been one o f the criteria
for the diagnosis of KD. The similarities between these diseases led to the suggestion that
KD is also caused by a bacterial superantigen toxin (Furukawa, Matsubara, and Yabuta,
1991; Levin, Tizard, and Dillon, 1991).
The epidemiology of KD is characteristic of an infectious disease (see section 1.1.3.3)
and suggest that KD is caused by an infectious agent to which everyone is exposed and to
which most acquire immunity in childhood. Those individuals who succumb to KD may
119
be genetically predisposed. Despite extensive research into different possible infectious
aetiological agents, none have been conclusively proven to be the cause of KD.
Bacteria producing superantigen toxins are ubiquitous amongst the commensal flora of
the respiratory and gastrointestinal tract (Lindberg, Nowrouzian and others, 2000).
Despite the frequency with which individuals carry these organisms, disease caused by
these commensals is rare. Pre-existing immunity is believed to be a major factor in
determining whether such toxin-producing bacteria cause disease, although other host
and bacterial factors are likely to be involved. These are likely to include SAg dose (high
doses causing TSS, lower doses causing vasculitis in experimental animals (Leung DYM,
Meissner HC and others, 1995); age of the patient- in the rabbit model shock occurs in
mature rabbits, whereas immature rabbits less than 6 months old are more resistant to
shock induced by TSST-1 (Leung DYM, Meissner HC and others, 1995); and perhaps
other poorly-defined immunological and/or genetic fectors (Leung DYM, Meissner HC
and others, 1995).
Infiltration of activated T cells expressing HLA-DR in skin biopsies and coronary
vascular lesions at autopsy has been reported (Terai, Kohno and others, 1990). However,
there are conflicting reports regarding whether peripheral blood T cells are activated in
acute KD, as some reports have provided evidence o f peripheral blood T cell activation
(Leung, Chu and others, 1983; Barron, DeCunto and others, 1988), whereas other reports
have suggested that there is only a low level o f activation of peripheral blood T cells
during acute KD (Furukawa, Matsubara, and Yabuta, 1992).
120
Perhaps some of the inconsistency relating to studies of T cell activation in peripheral
blood in KD relates to the fact that this compartment may not be the optimum site to
sample, with higher sensitivity possibly associated with sampling lesional tissue. Another
explanation will undoubtedly relate to the timing o f the blood sample in relation to
disease onset (Leung, Meissner C, and Schlievert, 1997). Nonetheless, these data do
indicate an important role for T cells in KD.
The study by Abe et al in 1992 was the first to describe selective expansion of Vp2 and
Vp8.1 T cells in KD (Abe, Kotzin and others, 1992), indicating T cell Vp skewing- the
hallmark of a SAg-mediated process. Since then, many similar studies have examined T
cell Vp repertoires in KD using a variety of techniques (mainly flow cytometry or semi-
quantitative polymerase chain reaction), or examined the prevalence o f serological
conversion or colonization with SAg-producing organisms. These studies are summarised
in table 1.4; the top half of the table (in white) are studies which support the SAg
hypothesis; the studies in the bottom half of the table (in grey) refute the SAg theory.
Clearly the jury is still out regarding whether or not SAgs are involved in the aetio
pathogenesis of KD.
121
Table 1.4: Studies for and against the SAg theory in KD
Study Patients Controls FACs PCR study Main findings in KD Other comments
study and patients andmicribiological/histological
Vp Abs findings
used
Abe, Kotzin, et 19 12 healthy adults; 5.1, 5.2Z5.3, Semi- Increased mean Vp2 &
al. 1992 8 healthy childroi; 6.7, 8.1/8.2, quantitative PCR Vp 8.1
10 f^ rile control 12 of22V p
children families
Abe, Kotzin, et 23 13 healthy children 2,5.1, CDR3 regions Increased mean Vp2
al. 1993 8 healthy adults 8.1/8,2,12 sequoiced, but and 8.1: extensive
not divided into CDR3 junctional
CD4 or CD8 diversity i.e. polyclonal
subs^ v p family expansion
Leung, 16 15 healthy children no no SAg-producing bacteria Sites cultured w oo throat, rectum,
Meissner, et al. isolated from 13/16 KD axilla, and groin
1993 patioits, but only 1/15
controls. TSST-1
isolated from 11/13
toxin positive patients;
SPEB & SPEC were
cultured from the other
2 patients
Curtis, Zheng, et 21 22 healthy adults; 28 2,5,8,12,19 no Increased mean Vp2
al. 1995 fd>rile control overall; 2 patients had
children significantly lower Vp2;
did not confirm
increased Vp8.1
suggested by Abe
Leung, Giomo, 1 nil 1,5.1,81 Examined CDR3 Increased VP2 in
et al. 1995 in Vp2T cells pa-ifjieral CD4 & CDS
T cells; found to be
polyclonal eiqiansion
based on CDR3
diversity; increased Vp2
T cells infiltrating
myocardium and
coronary artery
Leung, 27 nil no no TSST-secreting S.
Meissner, et al. aureus isolated from
1995 26/27 KD patients.
Study Patients Controls FACs PCR study Main findings in KD Other comments and
study and patients micribiological/histological findings
Vp Abs
used
Yamashiro et al 12 8 children with food 2,5a, no No skewing of Increased infiltration of Vp2 T cells in
1996 intolerance 5b,5c,6a,8a, peripheral blood T cell the small intestine but not the jejunum
12a Vp repertoire
Leung, Sullivan, 3 nil 2,8.1, 12 no Serial measurement of
etal. 1997 Vp repertoire: increased
Vp2 in 2 patients which
returned to normal in
convalescence; low Vp2
in one patient; all had
coronary artery lesions
Masuda, Takei, 45 24 healthy children; no no Investigated peripheral T cell responses
etal. 1998 13 disease control to SAgs: found T cell anergy (lower
children IL2 production and proliferation) in KD
in response to SPEC, but SPEA or
TSST-1. Did not examine for evidence
of infection with SPEC producing
organisms. The T cell response
normalised within 1 year.
Nomura, 10 10 healthy children no Reverse 25 Vp families Authors suggest a relationship between
Masuda, et al. transcription analysed: Deletion of deletions obsaved and KD
1998 polymerase Vp9 and V pi5 observed
chain reaction
Yoshioka T et al 22 10 healthy adults; 14 no Adfôr ligation Mean périmerai Vp2 or Serum antibodies to SPEC were higher
1999{Yoshic4ca, M)rile control polymerase Vp6.5 T cells in acute in KD patients than control groups
Matsutani, et al. children chain reaction KD higher than in the
1999 4 2 /id} convalescait phase.
This expansion was
polyclonal because
CDR3 DNA sequences
were different
Burgner et al 87 57 healthy children no no Similar nasal S. aureus 29% of isolates from KD patients were
2001{Burgner, colonisation rates (25%) mitogenic compared with 7% of
Curtis, et al. between KD patients controls
2001 6327/id} and controls
lo
u>
study Patients Controls FACs PC R study M ain findings in KD O ther comments and
Vp Abs
used
Nomura Y et al 15 (all less than 22 age matched no no Higher TSST-1 and Mothers of KD patients had lower
2001{Nomura 6 months old) infants; 40 healthy SEB antibody titres in antibody titres to TSST-1 than their
Y, Masuda K, et adults KD affected offspring. Authors conclude
al. 2001 6329 that that maternal antibody for TSST-1
/id} may protect from KD
Suzuki H et al 54 nil VP2 and Adaptor-ligation 74% of patients showed 22/35 paired KD samples showed
2001{Suzuki H, VP6.5; rest polymerase polyclonal expansion of higher IgM to SPEC tiian in
Muragaki Y, et not stated chain reaction Vp2 and/or Vp6.5 T convalescent phase
al. 2001 6328 cells
/id}
Pietra et al 28 12 healthy children 2, no No v p expansions
1994{Pietra, De 5.1,81/8.2, detected, nor changes in
Inocencio, et al. 12 HLA-DR, IL2-R; but
1994 9 1 9 /id} increased CD45RO in
CD8 population
Sakaguchi et al 20 18 healthy children; 2,8.1 no No v p expansions or
1995{Sakaguchi 20 healthy adults deletions detected
, Kato, et al.
1995 9 2 2 /id}
Marchette NJ 150 96 healthy children; no no No serological evidence
1995{Marchette 129 healthy adults of TSST-1 inK D
NJ, Cao X, et al. patients
1995 6323 /id}
Tristani-Firouzi 7 3 healthy children; no Reverse No selective expansion Immunoglobulin production in
et al 14 healtiby adults transcriptase ofV p2orV p8.1 response to polyclonal stimulants was
1995 {Tristani- polymerase suppressed in the acute phase of KD,
Firouzi M, chain reaction and normalised in convalescence
Kamanga-Sollo (?evidence of THl response)
E lP .etal. 1995
6324 /id}
Abe et al 20 20 febrile controls no Semi- Could not confirm that Authors postulate that the changes in
1995{AbeJ, quantitative PCR TSST-1 is specifically VP2 occur via an undefined
Takeda I , et al. associated with KD, or immunological process, not related to
1995 6325 /id} that the changes in the SAg
v p repertoire are due to
the SAgs isolated from
KD patients
to
Vp Abs
used
Nishiyori A et al 20 18 healthy children; 2, 8.1 no No expansion of Vp2 or
1995{Nishiyori 20 healthy adults VP8.1; No increase in
A, Sakaguchi M, antibodies to TSST-1 in
etal. 1995 6326 KD (acute or
/id} convalescent)
Todome et al 127 17 disease control no no No noticeable Authors conclude that the pathological
1995{Todome, children; 6 healthy differences between S. or aetiological role of a new TSST-1
Ohkuni, et al. controls aureus strains from KD secreting S. aureus clone in patients
1995 7 3 8 /id} patients and control with KD was not confirmed. Group A
children in the Streptococci could not be isolated from
production of either KD patients or controls.
staphylococcal
exotoxins A-E
Terai et al 26 22 healthy children no no Culture supernatants of In KD patients IgG seroconversion
1995{Terai, bacterial isolates from rates to TSST-1, SEA, SEE, and SEC
Miwa, et al. KD patients did not were 10%, 15%, 21% and 16%,
1995 7 4 /id} support involvement of respectively.
SAg-producing bacteria
Deresiewicz et 6 nil no no Found no evidence of an
al S. aureus strain or
1996{Deresiewi TSST-1 sequence
cz, Flaxenburg, uniquely associated with
etal. 1996 666 KD
/id}
to
LA
Table 1.4; Studies for and against the SAg theory in KD
Vp Abs
used
Monta et al 50 nil no no found a very low
1997 {Monta, frequency of detection
Imada, et al, of anti-superantigen
1997 7 1 6 /id} antibodies by ELISA
and no marked IgG
seroconversion to
several streptococcal
SAgs, indicating the
absence of a serological
relationship between
toxin-producing
streptococcal infection
and the onset of KD
Choi et al 26 13 children with 2,8 2 stage PCR to Several clonal Authors suggested that conventional
1997{Choi, seizures or pre estimate the expansions were found Ags rather than a SAg were involved in
Chwae, et al. operative admissions CDR3 size mainly in the CD8+ T the pathogenesis of acute KD
1997 6 5 7 /id} profile among T cells Âat disappeared
cells expressing during the long term
V p l,2 ,4 , 5, 8, follow-up period; only 2
14, 16, 17,18, patients had increased T
and 20 chains cells expressing VP8; no
difference in VP2.
Mancia L et al 25 24 healthy children 2 ,3 ,5 s l, no No abnormal usage of Did observe increase IL2R-expressing
1998{Mancia, 5s2, 5s3, any Vp family found in CD4 T cells
Wahlstrom, et 6 sl, KD.
al. 1998 9 1 6 /id} 6s78sl/8s2,
9, 11, 13sl,
13s6,14,
16, 17, 18,
20,21s3,
22, 23
K)
ON
Superantigens and other vasculitides
There are limited data examining this hypothesis, and most of it relates to vasculitides
affecting adults.
Simpson et al studied T cell Vp repertoires in 28 adults with miscellaneous vasculitides
(Simpson, Skinner and others, 1995). These included 12 with MPA, 10 with WG, and 6
with unclassifiable vasculitis. Controls comprised 28 age and sex-matched healthy adults.
The majority of the vasculitis patients had inactive disease at the time of sampling, and
only 8 patients had active disease. Using a reverse transcriptase PCR technique they
analysed Vp mRNA levels for 20 different Vp families. Overall they found increased
Vp2.1 mRNA expression in the vasculitis patients, and this was most marked for patients
with MPA. Moreover, this expansion seemed to be polyclonal, because there was little
conservation of the o f the TCR Vp2.1 junctional region. The authors concluded that their
data could be compatible with a SAg mediated immunopathogenesis. Interestingly, the
increased Vp2.1 usage appeared to be unaffected by disease activity.
Giscombe et al studied 11 patients with necrotizing vasculitides (10 with WG; 1 with
PAN) (Giscombe, Grünewald and others, 1995). 7 of the patients had active disease.
Controls comprised 16-57 healthy adults. This study used monoclonal antibodies and
flow cytometry to analyse 10 different Vp families. They found a higher number of T cell
Vp expansions within (predominantly) the CD4 population o f T cells in the vasculitis
group. In long-term studies of the T cell expansions (for up to 18 months), there was no
obvious correlation to clinical features such as disease activity or treatment. Moreover,
127
the expansions were heterogeneous, i.e. did not affect any single Vp family
predominantly. The authors concluded that their observations may, in the future, lead to
the identification of unknown antigens, but did not go as far as speculating on a SAg-
mediated immunopathogenesis.
Popa et al studied T cell Vp repertoires by flow cytometry in 27 patients with WG (Popa,
Stegeman and others, 1998). 11 had active disease, and 16 inactive disease. Controls
comprised 16 healthy adults. They observed Vp expansions in 21/27 patients, and 4/16
controls. Expansions were present in the CD4 and CDS T cell populations. Again, the
expansions appeared to be heterogeneous, although the authors did not state which
individual Vp families were affected. Interestingly, the senior author o f that study (which
was only published in abstract form) went on to state in a subsequent review that no SAg-
related Vp expansions could be detected in their WG population (Cohen Tervaert, 2002).
Stegeman et al studied nasal carriage of Staphylococci in 57 patients with WG
(Stegeman, Tervaert and others, 1994). Thirty-six o f the 57 patients were found to be
chronic nasal carriers of S. aureus. Nasal carriers of staphylococcus were nearly 8 times
more likely to relapse with their disease. The same group went on to confirm this
observation in a further 63 patients with WG, and moreover pin-pointed the risk to those
strains of Staphylococcus mreus capable of producing TSST-1 (Popa ER, van der Meer
B and others, 2002)
128
1.3.2.3 Concluding remarks regarding SAgs and vasculitis
There are data suggesting a role for the SAgs in the aetio-pathogenesis of the
vasculitides. This is strongest (and most controversial) for KD. It should be borne in mind
that SAg versus conventional antigen processes are not mutually exclusive: i.e. both
could be important, perhaps at different stages of the disease process. How SAgs could
contribute to vascular injury is a different question, and largely a matter of speculation so
far. This issue will be considered in chapter 4.
129
1.4 Aims of this thesis
The aims of this thesis were:
1. To examine the hypothesis that SAgs could be involved in the aetiopathogenesis of
vasculitis syndromes of the young.
2. To investigate whether endothelial cells can promote SAg-driven T cell responses.
3. To investigate the consequence of endothelial cell mediated SAg-driven T cell
activation.
130
Chapter 2: Materials and methods
2.1 Introduction
2.2 Subjects, classification of vasculitic syndromes, and

assessment of disease activity
2.3 Materials
2.4-2.6 Methods
131
2.1 Introduction
This section contains predominantly materials and methods that are used in more than
one chapter. Methods applicable to one chapter only, or those that have been developed
or adapted specifically for this study will be discussed in detail in the appropriate section.
2.2 Subjects, classification of vasculitic syndromes, and
assessment of disease activity
2.2.1 Subjects and classification o f vasculitic syndromes
A detailed description of patients is made in the relevant chapters, but some comment
regarding the classification of vasculitic syndromes used in this thesis is required. The
main classification system used in chapters 3 and 5 was that described by the Chapel Hill
consensus (Jennette, Falk and others, 1994), although in chapter 3 children were
classified using both the Chapel Hill criteria and the American College o f Rheumatology
(ACR) classification criteria (Hunder, Arend and others, 1990). Children were classified
using both systems because there is controversy over the most reliable classification
system for the childhood vasculitides (Hunder G, 1998; Ozen, Besbas and others, 1992;
Petty RE and Cassidy JT, 2001a; Besbas, Ozen and others, 2000) and there is a
considerable degree of "polyangiitis overlap" in the childhood vasculitides (Brogan and
Dillon, 2000a; Brogan, Davies and others, 2002; Leavitt RY and Fauci, 1986) such that
neither system is absolutely sensitive or specific (Jennette and Falk, 2000). Moreover,
neither system has been formally validated in children (Petty RE and Cassidy JT, 2001a).
Classification of vasculitic syndrome is particularly important when considering aetio-
132
pathogenesis, however, and hence in Chapter 3 (which examines a possible aetio-
pathogenetic mechanism of systemic vasculitis in childhood) both systems were used to
allow comparison with previously published data, which may have used either system.
Since Chapter 5 examines a marker for endothelial injury (which occurs in all vasculitis
syndromes irrespective o f classification) only the Chapel Hill criteria were used. It should
be emphasised, however, that neither of these systems comprehensively address vasculitis
in childhood, and are thus of limited usefulness. The Chapel Hill and ACR criteria are
shown in Appendix 1.
2.2.2 The assessment o f vasculitic disease activity
The assessment o f vasculitic disease activity was performed using the Birmingham
Vasculitis Activity Score (EVAS) (Luqmani, Bacon and others, 1994). This is a tool
which can provide uniform assessment of current disease activity in groups of patients
with different forms of vasculitis, and is not specifically designed for any single
vasculitic syndrome. It thus has obvious advantages when considering patients with
different vasculitic syndromes, or overlapping features of different vasculitic syndromes.
The BVAS is weighted towards objective and categorical or biochemical evidence of
active vasculitis. Furthermore, objective evidence o f organ involvement (e.g. gut or
kidneys) is weighted more heavily than symptoms that have a subjective element (e.g.
myalgia, arthralgia). Laboratory markers have attractions but also present problems- they
may be affected by chronic changes such as hypertension, or by concurrent infection.
Anti-neutrophil cytoplasmic antibody (ANCA) titres may be able to predict relapses of
133
disease (Savage, 2001), but many patients are ANCA-negative (especially children)
(Brogan and Dillon, 2000a; Brogan, Davies and others, 2002).
In the BVAS scoring system, only recent (i.e. within the previous month) activity is
measured. The observer must only record the presence of an abnormality if has occurred
afresh or deteriorated within the previous month and it is concluded that the abnormality
is caused by active vasculitis. If a particular statement is recorded as being true, the score
for that statement contributes towards the value for its relevant section. The score is
calculated as the total sum of the values obtained from each o f the nine sections. Each
section can only contribute a maximum score as indicated.
The main limitation of the BVAS is that it has never been formally validated in children.
Moreover, although it is designed to score different vasculitis syndromes, KD was not
included in the original series of patients in which the BVAS was validated. Nonetheless,
with this limitation taken into account, the criteria within the BVAS are entirely
appropriate and relevant to the paediatric vasculitides (including KD) with the possible
exception of the laboratory reference ranges quoted (which are adult ranges). The BVAS
proforma is shown in Appendix 2.
134
2.3 Materials
2.3.1: Fluorochrome-conjugated antibodies fo r flo w cytometry
The fluorochrome-conjugated monoclonal antibodies used are given in table 2.1. All
antibody dilutions were made with 0.01 M phosphate buffered saline with 0.1% sodium
azide, and checked by plotting a dilution curve against median fluorescence index, the
relevant dilution for each antibody corresponding to the shoulder o f the curve. Each
antibody was checked against an appropriate isotype-control antibody with the same
protein concentration, as per manufacturer recommendation.
135
Table 2.1: Fluorochrome-conjugated antibodies for flow cytometry.
Specificity Isotype Conjugate Clone Dilution Source

CDS Mouse IgGl Phycoerythrin SK7 1:20 Becton
(PE) Dickinson
CD4 Mouse IgGl Quantum Q4I20 1:20 Sigma
Red™(QR)
CD8 Mouse lgG2a QR UCHT-4 1:20 Sigma
vpi Rat IgGl FITC BL37.2 I :I0 Immunotech
VP2 Mouse IgGl FITC MPB2D5 I :I0 Immunotech
Vp3 Mouse IgM FITC CH92 I :I0 Immunotech
v p s.i Mouse IgG2a FITC IMMUI57 I :I0 Immunotech
VP5.2 Mouse IgGl FITC 36213 I :I0 Immunotech
vp? Mouse lgG2a FITC ZOE I :I0 Immunotech
VP8.1/8.2 Mouse IgG2a FITC 56C5 I :I0 Immunotech
v p ii Mouse IgG2a FITC C2I I :I0 Immunotech
VP12 Mouse IgG2a FITC VER2.32.I I :I0 Immunotech
VplS.l Mouse IgG2b FITC IMMU222 I :I0 Immunotech
VP13.6 Mouse IgGl FITC JU-74 I :I0 Immunotech
VP14 Mouse IgGl FITC CAS 1.1.3 I :I0 Immunotech
Vpl6 Mouse IgGl FITC TAMAYA I :I0 Immunotech
1.2
Vpl7 Mouse IgGl FITC EI7.5F3 I :I0 Immunotech
VP20 Mouse IgG FITC ELL 1.4 I :I0 Immunotech
VP21.3 Mouse IgG2a FITC IGI25 I :I0 Immunotech
VP22 Mouse IgGl FITC IMMU 546 I :I0 Immunotech
CD69 Mouse lgG2a PE CH/4 T.20 Insight
Biotecnology
Ltd
CD25 Mouse IgGl PE 2A3 1:10 Becton
heavy chain and Dickinson
Khght chain
HLA-DR, DP, Mouse IgG2a k FITC Tu39 1:20 BD
DQ Pharmingen
CD54 Mouse IgGI k Cy-chrome HA58 1:20 BD
Pharmingen
CD 106 Mouse IgG I k PE 5I-I0C9 1:20 BD
Pharmingen
CD62E Mouse IgG I k Cy-chrome 68-5HII 1:20 BD
Pharmingen
CD62P Mouse IgGI k PE AC 1.2 1:100 BD
Pharmingen
CD 105 Mouse IgGl PE SN6 1:15 Serotec
136
2.3,2 Cytokines and protein reagents
The following cytokines were used; recombinant TNF-a (Sigma, used at a concentration
of lOOng/ml); recombinant INF-y (Genzyme, used at a concentration o f 1000 U/ml). The
following SAgs were used: staphylococcal toxic shock toxin type 1 (TSST-1; Sigma,
used at a concentration of 10 ng/ml); staphylococcal enterotoxin type B (SEE; Sigma,
used at a concentration of 1OOng/ml). The following fluorochrome-conjugated proteins
were used: annexin-V (FITC; Annexin V-FITC kit. Bender MedSystems, 1:100 dilution);
Ulex europaeus I Agglutinin (UEA-I FITC; ICN Biomedicals, 1:20 dilution).
2.3.3 Tissue culture media
The following tissue culture media were used: RPMI-1640 culture medium containing 2
mM L-glutamine (Life Technologies, Gibco BRL) and supplemented with antibiotics
(1% penicillin/streptomycin/amphotericin); MCDB-131 with 10 mM L-glutamine (Life
Technologies, Gibco BRL) and antibiotics (1% penicillin/streptomycin/amphotericin);
foetal calf serum (heat inactivated at 56° C for 60 minutes to destroy complement).
2.4 Methods
2.4.1: Isolation ofperipheral blood mononuclear cells and T cells fro m whole blood
5-40 mis of blood from a single healthy adult donor was collected into sterile 20ml
Universal bottles containing 40 microlitres of preservative-free heparin (Monoparin, CP
Pharmaceuticals Ltd, 1000 U/ml). Peripheral blood mononuclear cells (PBMCs) were
separated from whole blood by Lymphoprep™ (Nycomed) centrifugation as follows. All
137
reagents, tubes and the centrifuge were pre-cooled to 4°C to minimise the activation of T
cells. The blood sample was diluted 1:1 with RPMI-1640 medium containing 2 mM L-
glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin. The diluted sample was
slowly and carefully overlaid, onto an equal volume o f lymphocyte separation medium
(Lymphoprep™) in an appropriate number o f 15ml conical tubes using a 10ml pipette.
The tubes were centrifuged at 2,000 rpm (1,110 g) with the brake off for 20 minutes at
4°C. A wide tipped Pasteur pipette was used to aspirate PBMCs from the interface into a
fresh 30 ml Universal container containing 10 ml RPMl to wash the cells. To allow
efficient sedimentation of cells, it was ensured that the interphase was diluted at least 3:1.
After further centrifuging at 1500 rpm (500 g) for 10 min at 4°C, the supernatant was
discarded by inversion of the tube and the cell pellet resuspended by gentle ‘flicking’ of
the container. The cells were then washed again in 10 or 20 ml RPMl followed by
centrifugation at 1200 rpm (280 g) for 10 min at 4°C. The cell pellet was resuspended in
5-20 ml o f RPMI-1640 medium (supplemented with 10% heat-inactivated PCS, 2 mM L-
glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin), aiming to give a
concentration above that required. PBMC were either used immediately in experiments,
or frozen suspended in PCS with 10% dimethyl sulfoxide (DMSO, Sigma).
2.4.1 Preparation ofpurified CD3^ T lymphocytes from PBMCs
T cells (CD3^ lymphocytes) were further purified from the PBMC population by using a
T cell negative isolation kit (Dynal) as per manufacturer recommendations. This system
utilises an antibody mix containing anti-HLA class II DR/DP, anti-CD 16 (a and b), anti-
CD56, and anti-CD 14 and depletion magnetic beads thus removing B cells, natural killer
138
(NK) cells, activated T cells, monocytes, and granulocytes (if present). The purified T
cell population routinely contained greater than 98% CD3^ cells, and 0.2% MHC class II
(DP/DQ/DR) as determined by flow cytometry (figures 2.1 and 2.2).
2.4.2 Calculation o f cell number and cell viability
10 pi of resuspended cells were removed and mixed with 10 pi o f trypan blue to
differentiate dead from live cells. The number of cells was counted using a Neubauer cell
haemocytometer. The cell viability (%) was calculated as 1OOx (live cells)/ total cells
(live and dead). The resuspended cells were then diluted further, based on the live cell
count, to give the desired concentration (usually 1x10^ /ml).
2.4.3 Storage o f cells
Unless cells were required fresh for immediate use, they were frozen at -70°C. Cells for
freezing were aliquoted in 2 ml flat microtubes (Sarstedt) at a concentration o f 10^
139
Figure 2.1: Purity of CD3 lymphocyte population after T cell negative isolation using
magnetic beads
n
O
>- o
u
ig
. - 98.4%
□
o
10* 10^ 10^
CD3 FITC
o
Figure 2.2: Percentage of contamination MHC Class II positive cells in the purified
CD3 T cell population
0 .32% 0 . 8%
u
!? ■ «n ^
< g
o
□
o
1023 1023
FSC-Heighl FSC-Height
PBMCs per 1.0 ml in PCS containing 10% DMSO. The cells were cooled stepwise to
prevent formation of ice crystals (4°C for 30 minutes, -20°C for 30 minutes, then to -
70°C). Cells for storage for periods greater than one month were stored in 2 ml cryogenic
vials (Nalgene) and transferred to liquid nitrogen after initial freezing to -70°C.
2.4.4 Flow cytometry o f PBM C or T cells
3-colour FACS analysis of PBMCs or T cells from experiments was performed as
follows. Cells were plated onto U-bottomed 96-well plates at a concentration of 1 x 10^
cells per ml, and incubated for 30 minutes at 4°C using fluorochrome-conjugated
monoclonal antibodies to CD4 (Quantum Red™), or CDS (Quantum Red™), CD69
(phycoerythrin), and one of the 17 FITC-conjugated T cell VP families antibodies.
Following incubation for 30 minutes, cells were washed 3 times with 0.01 M phosphate
buffered saline with 0.1% sodium azide, then fixed in buffer containing 10%
formaldehyde and 1% azide (CellFIX, Becton Dickinson). Flow cytometry was
performed on a FACScalibur flow cytometer (Becton Dickinson) using CellQuest
software with optimal compensation set for green, orange, and far-red fluorescence. T
cells were identified by gating for forward and light-scatter characteristics, CD4^ or CDS^
populations, and the different Vp families. Percentages o f T cells (CD3^CD4^ or
CD3^CD8^ expressing different Vp gene products were subsequently calculated using
quadrants set on dot-plots, with markers for positivity defined using isotype-control
antibodies. 20-40 thousand gated events were stored for each VP family analysis. Vp-
specific activation was determined in some experiments by gating on the individual Vp
family sub-population (CD4^ or CD8^ and Vp double-positives) and then plotting a
142
histogram of CD69 fluorescence, with determination o f the CD69 median fluorescence
index (MFI) for each Vp family in the CD4^ and CD8^ sub-population o f T cells. The
plate plans for determination of the Vp repertoire are shown in Appendix 3, and for
determination of Vp-specific CD69 expression in Appendix 4. Flow cytometry
instrument settings for PBMCs/T cells are given in Appendix 5.
2.4.5 In vitro technique fo r validation o f effect o f SAgs on T cell V p repertoire skewing
and T cell activation
The flow cytometry technique and antibodies were validated by examining the changes in
PBMCs from 2 healthy adults in response to 8 days incubation with the SAgs TSST-1 or
SEB. The technique used was the same as that described by Curtis et al (Curtis, Zheng
and others, 1995). PBMCs from a healthy adult donor were cultured at a density o f 2 X
lOVml in RPMl medium (Gibco) and 10 % fetal calf serum (heat inactivated) in the
presence or absence of 10 pg/ml anti-CD3,10 ng/ml TSST-1, or 10 ng/ml SEB. The cells
were cultured at 37°C and were stimulated with 10 units/ml of recombinant human
interleukin-2 (IL-2) (Recombinant Human IL-2; R &D Systems) on days 3 and 7. The
cells were then harvested on day 8 and the percentage of each of the Vp femilies within
the CD4 and CD8 populations, and the expression of T cell activation markers was
analysed as described above.
Stimulation of PBMCs fi’om healthy adults (n=2) with TSST-1 after 8 days resulted in an
increased percentage of T cells expressing Vp2 as compared with control (resting PBMC
143
i.e. cultured but unstimulated for 8 days). This was true o f both the CD4 and CDS
populations of T cells (figure 2.3a and 2.3b). In contrast, stimulation with SEB resulted in
an increased percentage of T cells expressing Vp3; again this was true of both the CD4
and CDS T cell populations (figure 2.4a and 2.4b). There was also a decrease in Vp2
observed in response to SEB within the CD4 T cell population, presumably reflecting
true “skewing” of the Vp repertoire (figure 2.4 a). In addition, incubation o f PBMC with
SAg (TSST-1 or SEB) resulted in T cell activation as determined by upregulation of
CD69 and CD25. This was true for both CD4 and CDS populations of T lymphocytes
(figure 2.5a-d).
144
Figure 2.3: Stimulation of healthy adult (n=3) PBMC with TSST-1
CD4 V-beta repertoire CD8 V-beta repertoire
20 1 25 J
■ Control Day 8 20 -{ ■ Control Day 8
□ TSST-1 Day 8
□ TSST-1 Day 8 15
10 -
H Ti 1- - - - - - - - - - - - - - 1- - - - - - - - - - - - - 1- - - - - - - - - - - - - 1 ~r
1 2 3 5.1 8 12 14
Figure 2.3a: CD4 Vb response to TSST-1 (mean, SEM) Figure 2.3b: CD8 Vb response to TSST-1 (mean, SEM)
Figure 2.4: Stimulation of healthy adult (n=3) PBMC with SEB
CD4 V-beta repertoire CD8 V-beta repertoire
16 22
14 20
18
12 16
10 14
■ Control Day 8 12 ■ Control Day 8
^ 8 -
10
mSEB Day 8 H SEB Day 8
6 - 8
4 6
4
2 2
0 0
1 2 3 5.1 8 12 14 1 2 3 5.1 8 12 14
Figure 2.4a: CD4 Vb response to SEB (mean, SEM) Figure 2.4b: CD4 Vb response to SEB (mean, SEM)
Figure 2.5: T cell activation in response to Sags (mean CD69 MFI, SEM of 3
experiments)
Figure 2.5a: CD4CD69 Figure 2.5b:CD8CD69
200 1 150
150
o>
to
■ Control Day 8 O) 100 ■ Control Day 8
<D
a 100 □ TSST-1 Day 8 Q □ TSST-1 Day 8
Ü O
M- El SEB Day 8 00 50 mSEB Day 8
Q 50 Q
Ü X O X
0 0
Figure 2.5c: CD4CD25 2.5d: CD8CD25
200 1 200
U_
150 I 150
■ Control Day 8 ■ Control Day 8
8 100 □ TSST-1 Day 8 I 100 □ TSST-1 Day 8

Tf H SEB Day 8 CO
iii □ SEB Day 8
Q 50 - Û 50 -
Ü O
0 0
2.4.6 Endothelial cell culture
Endothelial cells can be isolated and cultured from a wide variety o f sources, including
different anatomical sites and different species. Each type requires specific techniques for
isolation and growth. They may require various growth factors and special media. In
general, endothelial cells require media which are rich in amino acids and various sugar
moieties, and also require high quality FCS at concentrations up to 20%.
Human umbilical vein cells (HUVEC) are an ideal source o f endothelial cells since they
are derived from a plentiful and renewable resource that would otherwise be discarded.
The basic method employed is as described in 1973 (Jaffe, Nachman and others, 1973).
2.4.7 HUVEC culture media
The media used are listed in section 2.3.3. RPMl 1640 medium, containing lOmM L-
glutamine, 80pg gentamicin, 100 units penicillin/streptomycin was used for collection
and storage of umbilical cords, and kept at 4° C in autoclaved polypropylene bottles.
Washing of umbilical cords was performed with RPMl with same supplements as above
but containing 5% FCS (HUVEC wash medium). Collagenase type H solution (0.1%)
comprised of Ig collagenase dissolved in IL of RPMI-1640 which was then passed
through a 0.2 micron tissue culture filter and stored in aliquots at -2 0 ° C until required.
Primary and secondary cultures used a specialised media for growth of endothelial cells;
MCDB-131, supplemented with lOmM L-glutamine, 100 U/ml penicillin/streptomycin,
amphotericin B, and 20% FCS.
148
2.4.8 Foetal calf serum
The quality and batch of FCS was found to be a critical determinant o f yield of HUVEC
from either primary culture or growth in secondary cultures. For this reason, batches of
FCS were tested for quality. Specific batches, which proved to be most effective at
sustaining growth of HUVEC, were ordered in large quantities and were used solely for
HUVEC culture. All FCS was heat inactivated at 56° C for 60 minutes and stored at-20°
C in aliquots until required.
2.4.9 Protocolfor isolation o f HUVEC
Only fresh, intact cords were used, always within 48 hours o f collection (and usually
within 24 hours o f collection). Cords that were heavily meconium stained were discarded.
All the procedures described below were performed in a Class II safety cabinet using
aseptic technique. Autoclaved glassware and metal instruments were used. Culture and
wash media, and enzymes were pre-warmed to 37° C in a water bath.
1. The cords were inspected for damage such as cuts, or needle punctures from cord
blood sampling. Suitable cords were then sprayed with 70% IMS, and blood
expressed into a collection pot. Washed cords were then placed in a sterile Duran
bottle containing RPMI-1640 medium with antibiotics as described above.
149
2. Single cords were then cut at least 1 cm from both ends. One end was clamped with an
artery forceps. The other end was then inspected and the vein identified. The vein was
dilated with blunt forceps, and cannulated with a sterile plastic filling tube (Kwill).
The filling tube was secured with sterile suture and finally a small artery forceps.
3. The vein was then infused with warmed RPMI-1640 medium with 5% FCS (HUVEC
wash medium). Sites of leakage were clamped as necessary.
4. This process was repeated for a maximum o f three cords at one time.
5. The vein was flushed with wash medium to remove excess blood, and then filled with
0.1% Collagenase II solution.
6. The cord was then incubated at 37° C in 5% C02 for 15 minutes.
7. After incubation, the digest was removed from the cord into sterile Duran bottles, and
flushed through with equal volume of wash medium. This was transferred to a sterile,
50ml conical tube.
The digest was centrifuged at 200g for 7 minutes at room temperature. The supernatant
was discarded, and the pellet resuspended in MCDB-131 medium with 10 mM L-
glutamine, 20 % FCS and antibiotics (HUVEC culture medium).
2.4.10 HUVEC primary culture and sub-culture
The digest was transferred to tissue culture flasks. These were always surface modified,
polystyrene flasks designed for culture of adherent cells. 25 or 75 cm^ flasks were used
depending on requirements. In general, digest from a large cord (enough to take 20 to 30
150
ml o f collagenase solution) was resuspended in 10ml o f culture medium and transferred
to 25cm^ flask. These were incubated at 37°C and 5% CO2 Cells were washed in fresh
culture media the following day and inspected under phase contrast microscopy.
HUVEC could be identified as small clusters of oval adherent cells. Cultures were
inspected each day for growth. When cells were approaching confluence (which usually
occurred after 72 hours culture), they were sub-cultured. Cells that grew poorly, or were
contaminated with smooth muscle or fibroblastic cell overgrowth were discarded.
HUVEC that were near-confluent were then washed three times in warmed PBS to
remove non-adherent cells and protein in FCS. Cells were washed once in 0.5 ml
Trypsin-EDTA solution (per 25-cm^ flask) which was then removed and a further 0.5-ml
Trypsin-EDTA solution added. Cells were inspected under phase-contrast microscopy,
and when rounding and becoming dislodged (usually after 30 seconds), the flask was
tapped against the bench top to aid removal. The cells were quickly resuspended in
warmed HUVEC culture medium and transferred to a 50-ml sterile conical tube. These
were then seeded into 48 or 24 well flat-bottom tissue culture plates. Tissue culture plates
were then incubated at 37°C in 5% CO2 . Details of specific conditions used to grow
HUVEC for various experimental procedures are mentioned in the relevant sections in
subsequent chapters. HUVEC morphology and phenotype, especially in terms of
induction o f cell adhesion molecules in response to pro-inflammatory stimuli may alter
after serial passages. The cells used throughout this study were therefore always first to
151
third sub-culture passage. Generally, cells were used within one week o f passaging.
Medium was replaced every 3 days.
2.4.11 Identification o f HUVEC
Endothelial cells were inspected under phase-contrast microscopy prior to use for
characteristic cobblestone appearance (figure 2.6). In particular, care was taken to avoid
cultures where there was significant overgrowth o f smooth muscle-like cells (which are
characteristically spindle-shaped and can be seen to arch over endothelial monolayers).
Additionally, cells were identified by the characteristic forward and side scatter of
HUVEC, and by the binding of high levels of FITC-conjugated UEA-I by flow
cytometry, as shown in Figure 2.7.
2.4.12 Protocolfor detection o f cell adhesion molecule expression by flo w cytometry
Single colour FACS analysis of HUVEC was performed as follows. HUVEC from
experiments were re-suspended in 0.01 M phosphate buffered saline with 0.1% sodium
azide and 5% FCS, and plated onto U-bottomed 96-well plates. The cells were then
incubated for 30 minutes with fluorochrome-conjugated antibodies to MHC class H,
ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), or P-selectin (CD62P).
Following 3 washes (with PBS supplemented with 5% FCS; centrifuged at 200g for 5
152
Figure 2.6: Phase contrast light microscopy of resting HUVEC (figure 2.6a) and
HUVEC following stimulation with lOOOU/ml of INF-y for 48 hours (figure 2.6b)
■
Figure 2.7: Forward and side scatter characteristics of HUVEC, and characterization
by binding of UEA-I-FITC
Control
UEA-I FITC: 99%
10 ^ 10 ^
UEA-I FITC binding

10^
FSC-Height
I/,
minutes and the supernatant discarded by flicking and blotting the plate), FACS analysis
was performed immediately by gating on the typical forward and light scatter
characteristics of HUVEC, and histograms plotted for each antibody to determine the
median fluorescence index. In addition, percentage positivity for each antibody was
determined using isotype control antibodies with a cut-off for positivity at 2%.
Endothelial cells were analysed on a FACScalibur flow cytometer using CellQuest
software. 5000 events within endothelial gate were collected. Flow cytometer instrument
settings for endothelial cells are given in Appendix 5.
2.4.13 Assay fo r endotoxin contamination o f HUVEC
When considering experiments involving the activation of HUVEC, an important
consideration (as with all tissue culture) is the possibility of endotoxin contamination.
Certainly this was a complication that was encountered when the initial HUVEC culture
and sub-culture was being set up. The offending agent was found to be a batch of
endothelial cell attachment factor (ECAF) which was used in early tissue culture
experiments, but eventually abandoned because of this risk.
To assess for the possibility of endotoxin contamination, a timed-gel formation endotoxin
kit was utilised (Sigma; product number TGF-1). This kit is designed for the rapid
determination of endotoxin levels in foetal bovine serum, but can be used for any tissue
culture reagent. This kit provides semi-quantitative results and as such should not be used
to determine exact endotoxin levels. The Timed Gel Formation test is based on the
observation that higher levels of endotoxin will shorten the incubation time required for
155
gel formation. The test relies on predetermined gel formation times for samples
containing known amounts o f endotoxin as determined by the LAL gel clot method and is
based on Control Standard Endotoxin.
Test Protocol: Contact/contamination o f the dilution tubes was avoided.
1. Labelled one baked dilution tube and two LAL single test vials for each sample to be
tested. Placed LAL vials in ice bath until ready for incubation.
2. Dispensed I ml o f endotoxin-free water into each dilution tube.
3. Added 0.5 ml o f test sample to the labelled dilution tube containing endotoxin-free
water.
4. Vortexed and placed tubes in boiling water for 5 minutes. Exercised care to avoid
contamination o f tubes.
5. Removed tubes from boiling water and allowed heated samples to cool to touch
[30°C]. Dispensed 0.2 ml o f cooled test sample through septum cap into labelled LAL
vials using 0.5 ml syringe. Swirled gently to dissolve LAL completely. Care taken not
to agitate vials vigorously.
6. Placed vials upright in 37°C water bath and began timing.
7. Incubated the test samples for the time period indicated on the Endotoxin Standard
Chart or Curve (see product information), sufficient to detect the expected endotoxin
level. (Comment: care should be taken not to disturb vials during the incubation
period since this may interfere with formation o f the gel resulting in a false negative.
Once removed from the water bath, a sample should not be returned to incubation).
56
8. At the indicated time, gently removed vials from water bath and slowly inverted 180°.
A solid gel that did not slide down the side o f the vial was a positive result. A liquid,
cloudy or soft gel, or a gel that slid down the vial when inverted was considered a
negative result.
9. A positive result indicated an endotoxin concentration greater than or equal to the
indicated level on the Endotoxin Standard Chart or Curve.
Possible sources of error included:
1. Incomplete solubilization at start of test.
2. Mixing too vigorously.
3. Disturbing the vial during incubation.
4. Disturbing a formed gel during inversion of the LAL vial at the end o f incubation.
5. Incorrect incubation temperature.
6. Incorrect incubation time.
7. Neglecting to boil serum samples (if they are the sample to be tested, to denature
proteases which may impair gel formation).
157
2.5 Isolation and analysis of whole blood cellular and platelet
microparticles
2.5.1 Preparation ofplatelet poor plasm a fo r microparticle analysis
1.4-5 mis of whole blood was collected into bottles containing 3.2% trisodium citrate
(Becton Dickinson). Platelet poor plasma was obtained by immediate centrifugation of
the whole blood at 5000G for 5 minutes twice. Plasma was then stored at -70° Celcius
until use.
2.5.2 Isolation o f microparticles from platelet poor plasm a
Platelet poor plasma was defrosted in a water bath at 37 ° Celcius. Exact volumes of
plasma (400-800 microlitres) were then centrifuged at 17000 G for 60 minutes and the
supernatant decanted to obtain the microparticle (MP) pellet. The MP were then
reconstituted in 350 microlitres of annexin V buffer (Bender Medsystems), and divided
into ten 3 5-microlitre aliquots plated onto the first 10 wells of a 96 well U-bottomed
plate.
2.5.3: Labelling o f microparticles with annexin V and monoclonal antibodies
The labelling and quantification of MP was achieved as follows. 5 microlitres of a 1 in 10
dilution o f FITC-conjugated annexin V in buffer (Bender Medsystems) was added to
every well. In addition, antibodies against platelet or endothelial surface markers
conjugated to red (PE) or far-red (PERCP or CYC) fluorochromes were used to
158
differentiate MP of platelet or endothelial origin. Platelet markers examined were the
constitutively expressed platelet marker CD42a (mouse IgGl anti-human CD42a-
PERCP, Becton Dickinson), and the platelet activation marker P-selectin (mouse anti
human CD62P-PE, BD PharMingen). Endothelial surface markers examined were E-
selectin (mouse IgGl anti-human CD62E-CYC, BD PharMingen); CD 105 (“endoglin”;
mouse anti-human IgGl CD105-PE, Serotec); ICAM-1 (mouse IgGl anti-human CD54-
CYC, BD PharMingen); and VCAM-1 (mouse IgGl anti-human CD106-PE, BD
PharMingen). 10 microlitres of each antibody (diluted 1 in 3 with RPMI medium with
5% fetal calf serum) was added to individual wells. The final dilution o f the annexin V-
FITC was 1 in 100, and 1 in 15 for each of the conjugated antibodies. MP were incubated
with the labelled antibodies and annexin V for 10 minutes at room temperature with
gentle shaking. The incubation was then terminated by adding 200 micro litres of annexin
V buffer to each well, and the samples transferred to tubes prior to flow cytometry.
2.5.4: Flow cytometric analysis o f microparticles
All analysis was performed on a FACScalibur flow cytometer (Becton Dickinson). To
obtain optimal forward and side scatter instrument settings for MP, 0.8 micrometer and 3
micrometer latex beads (Sigma) were run and logarithmic forward and side scatter plots
obtained (Combes, Dignat-George and others, 1997). Gates were then set to include
particles less than approximately 1.5 micrometers, but to exclude the first forward scatter
channel containing maximal noise. Optimal compensation was set for green, red and far-
red fluorescence (Appendix 5). Specific binding for each antibody was determined using
159
isotype control antibodies with equal protein: fiuorochrome ratios, and at the same final
dilution as per manufacturer recommendation. Since annexin V is a protein and not an
antibody (and hence no isotype control antibody exists), the threshold for annexin V
binding was determined by using the fluorescence threshold established for MP in the
absence o f labelled annexin V. Particles less than 1.5 micrometers in size and binding
annexin V were then gated, and histograms obtained for this gated population for binding
to individual monoclonal antibodies to determine the cell o f origin o f the MP. The gate
was checked by examining MP derived from supernatants taken from monolayers of
HUVEC stimulated with 100 ng/ml o f TN F-a (Sigma) at 24 hours. MP samples were run
at medium flow rate with a cut-off time o f I minute, which resulted in capture o f
approximately 5000 gated bead events.
2.5.5 Determination o f absolute microparticle number per ml ofplasma
To convert flow cytometer counts to an estimate o f the number o f MP per ml o f plasma, a
predetermined number (always 200000, calculated as per manufacturer
recommendations, Appendix 6) o f 3 micrometer latex beads (Sigma) were run
concurrently with the microparticle samples. The absolute number o f annexin V binding
microparticles per ml o f plasma was then determined by using the proportion o f beads
counted and the exact volume o f plasma from which the microparticles were analysed, as
described by Combes et al in l997(Combes, Dignat-George and others, 1997). The
following equation was thus derived to convert flow cytometer counts to an estimate o f
the number o f MP per ml o f plasma (figure 2.8):
160
F IGURE 2.8 C o n v e r s io n e q u a t io n f o r c a l c u l a t io n o f MP n u m ber from flow cy to m eter counts
Total i»iiinfoer of MF/ ml of pWma
Z ## 0 J / (RQ.^MFoottflted perwellj^X
N um ber o f beads added per well The number o f wells

Usually 300-800 p L into which the sample
was divided
Usually 5000 beads
Since samples from each individual were run 10 times, microparticle counts from
individual subjects were expressed as the mean number per ml o f plasma, with standard
error of the mean based on 10 measurements. To determine the absolute number of MP
derived from different cellular populations (i.e. platelet, or endothelial) the absolute
number of total MP derived from the above equation was multiplied by the percentage
positivity for that particular marker. A representative set of flow cytometric plots, and the
gating protocol are shown in figure 2.9.
161
Figure 2.9: Flow cytometry gating protocol for microparticles
A nnexin V
binding 3 pm latex beads
m icroparticles
Control Antibody
E selectin Antibody
24% E selectin +
10 ’ 10 ^
10^ E Sel CYC
FSC-Height
O
KN
)
2.5,6 Validation o f the technique for the analysis o f microparticles
Whilst the technique used to extract MP from whole blood was adapted from a previously
validated and published technique (Combes, Dignat-George and others, 1997),
considerable time was spent modifying and validating the technique described to extract
and analyse microparticles in this thesis.
2.5.6.1: Reproducibility of MP analysis
Since the sample from individual subjects was divided into 10 wells, an estimate of the
total number of MP was derived 10 times, and standard error o f the mean thus derived
from 10 measurements for each subject. Using this approach, blood from a healthy adult
donor was divided into 3, and the microparticle analysis performed in triplicate (i.e. 3 X
10 wells). The results are shown in figure 2.10 (mean, SEM) and provide an estimate of
the reproducibility of the technique.
2.S.6.2: Effect of different centrifugation protocols on
microparticle analysis
Different centrifugation protocols for the preparation o f platelet poor plasma and
extraction of MP from plasma were examined, and were found to influence the absolute
number of MP measured, and the intra-subject reproducibility o f the MP count. There is a
trade-off between fast centrifugation protocols which would remove platelets more
effectively from whole blood but in theory could cause red cell or platelet fragmentation
(and thus create artefactual phosphatidylserine exposure with spuriously high MP
163
Figure 2.10: Reproducibility of MP analysis
0.6
(0 0.5
= 0.4
■ Analysis 1
L. 0.3 s Analysis 2
□ Analysis 3
.o
G
ON
counts), and slower centrifugation protocols which run the risk o f unacceptably high
platelet contamination and/or incomplete MP isolation from plasma potentially making
the MP analysis inaccurate. Four different whole blood centrifugation protocols were
investigated (Table 2.2).
Table 2.2: Centrifugation protocol fo r isolation o f M P from whole blood.
]|^rotocol 1: 5000G for 5 mmufes; supernatant then spun at 17000G for 60t«inutes
(dkcard supernatant: and analyse MP pellet).
Frofocol 2; 5000G for 5 minutes; supernatant then spun at 15000G for 60mmutes
(discard supernatant and analyse MP pellet).
Frotocoi 3: î 5WO for 15 minutes; supernatant then spun at î 7000G for î 5 minutes
(discard supernatant and analyse MP pellet).
Frolocol 4 :50Ô0G for 5 minutes; supernatant then re-spun at 5000G for 5 minutes;
supernatant then spun at 17000G for 60 minutes (discard supernatant and analyse
MP pellet)*
The results (mean total MP count, SEM o f 10 measurements) for each protocol are shown
in figure 2.11. As demonstrated, the least accurate reproducibility was that associated for
protocol 3, with little to choose from between the remaining protocols. The protocol used
m this thesis was protocol 4, since it appeared that this was reproducible and was the least
likely to be associated with platelet contamination since two sequential centrifugations
were employed to prepare the platelet poor plasma.
165
Figure 2.11: Effect of different centrifugation protocols on MP connt
■ Measurement 1
□ Measurement 2
t
Protocol Protocol Protocol Protocol
1 2 4
9.
2.S.6.3: Effect of freeze-thawing on microparticle analysis
The effect of 24 hours of freezing at -70°C followed by a rapid defrost (water bath at 37
°C for 1 minute) on the intra-subject reproducibility o f mean total MP number (SEM of
10 measurements) was assessed. The result is shown in figure 2.12. MP number was
unaffected by freeze-thaw.
167
Figure 2.12: Effect of freeze-thaw on MP number (mean, SEM)
re 3
re
a 2.5
X
E 2
Frozen
I 1.5
□ Non-frozen
r 1
Q)
n
I
C
0.5
Û.
0
ON
00
2.5 6.4: Effect of delayed separation of plasma from whole
blood on microparticle analysis
To assess the affect of delayed separation of the plasma from whole blood, the blood
sample taken from a healthy adult was left at room temperature, and the MP profile
analysed from aliquots of the sample at 0 ,1 , 2, 4, and 24 hours. The results are shown in
figure 2.13. Data for VCAM-1 MP are omitted because they were undetected at any time
point. From the figure it can be seen that delay in separation o f the blood sample for
longer than 2 hours profoundly affected the whole MP profile, but in particular the total
MP count and platelet MP expressing CD42a, presumably the result of platelet activation
and ultimately fragmentation with time. Hence in this thesis, all samples for MP analysis
were separated from whole blood within an hour, and usually within 10 minutes.
2.5.6.S: Properties of latex beads which affect microparticle
analysis
Uniform latex particles were first discovered in 1947 (Sigma product information). Since
then they have been utilized in a wide variety of applications including electron
microscopy and cell counter calibration, antibody mediated agglutination diagnostics, and
phagocytosis experiments.
Polystyrene microparticles are negative charge-stabilized colloidal particles, supplied as
aqueous suspensions with small amounts of surfactant to prevent agglutination. Particles
can be diluted with deionized water. However, some particles, especially at very dilute
169
Figure 2.13: Effect of delayed plasma separation from whole blood on MP profile
♦ Total num ber of AnV+ MPs

—■— CD42a MPs
— A — Psel MPs
s -H -IC A M -1 MPs
Q. CD105 MPs
0
—O—Esel MPs
1
W
c
o
o
E
3
C
Û.
o
Time (hours)
concentrations, may require additional surfactant to prevent clumping and uneven
dispersion. An early observation was that if the latex beads were left longer than 10
minutes or so, the number counted by the flow cytometer would fall very quickly. Since
the bead number is critical in the conversion o f flow cytometer counts and appears as a
denominator in the conversion equation (figure 2.8), a falsely low bead count would
result in a falsely elevated MP count. In this thesis, 6 pL of neat beads was added to 2
mis of 0.2 pm filtered de-ionized water. lOpL of the dilute beads thus contained 200 000
beads as determined using the equation provided by sigma (Appendix 7). Beads were
made afresh every 5 minutes as required. Using a medium flow rate on the flow
cytometer (with a cut-off time of 60 seconds or until 5000 bead events were collected)
resulted in 5000 bead events counted usually after approximately 58 seconds. Diluted
beads that were left for longer than an hour prior to use resulted in counts 2 or even 3-
fold less, profoundly affecting the MP calculation.
171
2.6 Statistics
A number o f parametric and non-parametric tests were used in this thesis, and a
description o f individual tests and their application is given in the relevant chapters.
Parametric tests were used wherever possible (i.e. when the data were normally
distributed, or could be easily transformed to normality). If the data could not be easily
transformed (e.g. Chapter 5), non-parametric tests were performed.
Parametric tests used were one-way analysis of variance (ANOVA) with Bonferroni and
Tamhane post-hoc tests; the F test of variance; and the 2-sample test o f proportion. In
chapter 3, all p values quoted are adjusted for multiple comparisons using the Bonferroni
technique. Non-parametric tests used were the Kruskall-Wallis test, the Wilcoxon signed-
ranks test, and Spearman rank correlation coefficients. Normality o f the data was
assessed by plotting histograms, and by the Kolmogorov-Smimov and the Shapiro-Wilks
tests of normality.
In addition, for assessment of the diagnostic test characteristics for endothelial
microparticles in Chapter 5, sensitivity, specificity, negative and positive predictive
value, and likelihood ratios were calculated (Gilbert R and Logan S, 2000). In addition, a
receiver operator characteristic curve (ROC curve) was plotted (Gilbert R and Logan S,
2000 ).
172
All graphs were plotted using Microsoft Excel 97, with the exception o f column dot-
plots, which were plotted using Graphpad Prism version 1.03. All statistics were
calculated using Microsoft Excel 97, and SPSS versions 8 and 10.0.
173
Chapter 3: T cell activation and VP repertoires in childhood
vasculi tides
3.1 Summary
3.2 Introduction
3.3 Patients and methods
3.4 Results
3.5 Discussion
174
3.1 Summary
Introduction: Superantigens (SAgs) are potent stimulators o f T cells bearing specific Vp
T cell receptors (TCR) and may play a role in the aetio-pathogènesis o f systemic
vasculitis, although this remains contentious.
Aims: To investigate the possible aetiological role o f SAgs, this study examined
peripheral blood T cell activation, and Vp repertoires in children with systemic vasculitis.
Methods: FACS analysis of 17 different peripheral blood T cell Vp families was
performed in 20 healthy control children; 27 disease control children with non-vasculitic
inflammatory disease; 25 children with primary systemic vasculitis; 6 patients with KD;
and 6 patients with Henoch-Schonlein purpura (HSP). T cell activation markers (CD69
and CD25) in the CD4 and CDS T cell subsets were also examined in these patients, and
in an additional 8 patients with PAN (5 with active disease, 3 with inactive disease), and
an additional 5 disease controls with viral sepsis.
Results: Children with active vasculitis had higher CD4 and CDS peripheral blood T cell
CD69 expression than those with inactive vasculitis and healthy controls, but not disease
controls. No difference in CD25 expression in either the CD4 or CDS T cell populations
was observed between active and inactive vasculitis groups, however. There was a
significantly increased variance of CD4 V pl2, and V p l7 and CDS V pi in the primary
systemic vasculitis group as compared to control and disease controls. Moreover, S0% of
the primary systemic vasculitis children had one or more CD4 Vp expansions or
deletions, as compared with 30% of controls (p<0.002), and 37% o f the disease controls
(p<0.002). In the KD group, the mean % of CD4 VP2 T cells was higher than in controls
175
or disease controls. In the HSP group, there was no consistent skewing of the T cell VP
repertoire.
Conclusion: The observation of peripheral T cell activation in systemic vasculitis of the
young may support a role for T cells in the pathogenesis of these disorders. Moreover,
there are differences in the T cell Vp repertoire in children with vasculitis over and above
those observed in disease controls. Whilst these data provide impetus for further research
into this contentious field, they do not unequivocally resolve the question o f the role of
SAgs in childhood vasculitic syndromes.
176
3.2 Introduction
As described, systemic vasculitis is characterised by the presence o f inflammation in and
around a blood-vessel wall (Petty and Cassidy, 2001a). In recent years, there has been
considerable interest in the role of superantigens (SAgs) in the aetio-pathogenesis of
several autoimmune diseases, including the systemic vasculitides (Cohen Tervaert, Popa,
and Bos, 1999; Giscombe, Grünewald and others, 1995; Schiffenbauer, Soos, and
Johnson, 1998; Simpson, Skinner and others, 1995; Torres and Johnson, 1998).
Following SAg activation, T cells rapidly proliferate resulting in T cell Vp "expansions"
(Choi, Kotzin and others, 1989; Fraser, Arcus and others, 2000; Kappler, Kotzin and
others, 1989; Shoukry, Lavoie and others, 1997). This is followed by T cell Vp-restricted
deletion from the peripheral circulation- a process mediated by Fas-Fas ligand (Fraser,
Arcus and others, 2000; Mingari, Cambiaggi and others, 1996; Webb, Hutchinson and
others, 1994). Thus the characteristic "immunological footprint" left by SAgs are the
presence of T cell Vp expansions and deletions in the peripheral circulation o f subjects
exposed to these toxins.
Abnormal expansions and deletions of T cells bearing particular Vp gene products have
been found in the peripheral blood of adults with primary systemic necrotizing vasculitis
(microscopic polyangiitis, Wegener's granulomatosis, giant cell arteritis, and polyarteritis
nodosa) (Cohen Tervaert, Popa, and Bos, 1999; Giscombe, Grünewald and others, 1995;
Popa, Stegeman and others, 1998; Simpson, Skinner and others, 1995), providing indirect
evidence of superantigenic involvement in the aetio-pathogenesis o f these vasculitides.
177
Furthermore, an important observation in Wegener's granulomatosis in adults is that
those with the disease who are nasal carriers of superantigen-producing staphylococci are
significantly more likely to have relapses of vasculitis compared with non-carriers
(Cohen Tervaert, Stegeman and others, 1998; Cohen Tervaert, Popa, and Bos, 1999).
In some studies, children with KD also show non-clonal expansion o f peripheral T cells
bearing Vp2 (Abe, Kotzin and others, 1992; Curtis, Zheng and others, 1995; Leung,
Meissner C, and Schlievert, 1997), although other workers have not confirmed this
observation, and argue against a SAg-mediated pathogenesis (Choi, Chwae and others,
1997; Morita, Imada and others, 1997; Pietra, De Inocencio and others, 1994; Rowley,
Shulman and others, 2001; Terai, Miwa and others, 1995). Furthermore, T cells
infiltrating the walls of coronary arterial aneurysms (Leung, Giomo and others, 1995),
and the intestinal mucosa of patients with KD show a skewed T cell VP profile, with
increased numbers of cells expressing Vp2 (Yamashiro, Nagata and others, 1996). These
results have led to speculation that SAgs play an important aetio-pathogenic role in this
vasculitic illness. Similar studies on vasculitis in children, apart from KD, are however
lacking.
To investigate the possible aetiological role of SAgs this study examined peripheral blood
TCR Vp repertoires in children with primary systemic vasculitis, Kawasaki disease, and
Henoch-Schonlein purpura.
178
3.3 Patients and methods
3.3,1 VASCULITIS PATIENTS
37 children with vasculitis at various stages of disease activity and treatment had VP
repertoires studied. The diagnosis of vasculitis was established in all on the basis of
clinical features of vasculitis in addition to suggestive selective visceral angiography
and/or tissue biopsy. The vasculitis was subsequently classified using the American
College of Rheumatology (ACR) 1990 classification criteria (Lightfoot, Jr., Michel and
others, 1990), and the Chapel Hill consensus (Jennette, Falk and others, 1994). Children
were classified using both systems because there is controversy over the most reliable
classification system for the childhood vasculitides (Besbas, Ozen and others, 2000;
Hunder G, 1998; Ozen, Besbas and others, 1992; Petty RE and Cassidy JT, 2001a), and
there is a considerable degree o f "polyangiitis overlap" in the childhood vasculitides
(Brogan and Dillon, 2000a; Leavitt RY and Fauci, 1986a), such that neither system is
absolutely sensitive or specific (Jennette and Falk, 2000b). Neither system, however, has
been formally validated in children (Petty and Cassidy, 2001a).
25 children were classified as primary systemic vasculitis. The mean age was 10.1 years
(range 2.5-15.6 years), and the male to female ratio was 2.1:1. Of this group, using the
ACR classification criteria, 19 had polyarteritis nodosa (PAN), 1 had Wegener's
granulomatosis (WG), 2 had hypersensitivity vasculitis, and 3 had unclassifiable
vasculitis. Using the Chapel Hill consensus 16 had PAN, 6 had microscopic polyangiitis
179
(MPA), and 3 had unclassifiable vasculitis. Clinical details o f these patients are
summarised in table 3.1. Additionally, longitudinal studies were performed on paired
samples collected from patients before and after induction o f remission of vasculitis
(n=6). Remission was defined as patients with a Birmingham vasculitis activity score of
zero (Luqmani, Bacon and others, 1994) (Appendix 2).
180
Table 3.1 Clinical features ofpatients with primary systemic vasculitis
181
P a tie n t Sex/age C lin ic al fe a tu re s Selective v isceral a n g io g ra p h y B iopsy ANCA A ctive disease a t V a sc u litis cla ssification, a n d system used f o r
num ber (yrs) (HF) tim e o f sampling classification
1 M/11.4 FevCT, purpura, «escentic Renal & hqratic perfusion Skin biopsy -ve for neg N 1. PAN (♦ACR)
nq>hritis, arthritis, defects; aneurysms of medium vasculitis; 33% crescentic 2. Microscopic polyangiitis (Chapel Hill
intestinal inflammation and small hepatic arteries nephritis, pauci-immune consensus)
2 M/2.7 Periodic fever, failure to thrive, Small aneurysms and caliber Lung: medium sized artery neg Y 1. PAN (ACR)
testicular infarction, pulmonary variation of *SMA and its poivascular & vascular 2. PAN ( C h ^ l Hill consensus)
vasculitis branches monocytic and
granulocytic infiltrate;
spleen: non-specific
inflammatory changes;
testis; infarction only (no
vasculitis demonstrated)
3 F/13.6 Fever, arthralgia, orbital Not done Orbital granulomatous neg Y 1. WG(ACR)
granuloma, microscopic inflammation compatible 2. Unclassified (Chapel Hill consaisus)
haematuria withWG
4 M/8 FevCT, myalgia, weight loss Renal perfusion defects; SMA Not done neg Y 1. PAN (ACR)
aneurysms; "corkscrew" changes 2. PAN ( C h ^ l Hill consensus)
of medium and small-sized
hepatic arteries
S F/7.5 Fever, myalgia, livedo Large aneurysms affecting Skin biopsy -ve for neg Y 1. PAN (ACR)
reticularis, brain stem infarction, predominantly medium-sized vasculitis 2. PAN (Ch^rel Hill consensus)
hypertension, renal impairment renal arteries
6 F/3.8 Failure to thrive, Not done Brain biopsy- non-specific neg Y Unclassified using either ACR or C hêl hill
encq>halopathy, livedo reactive changes only consensus criteria
reticularis, MR brain consistent
with cerebral vasculitis
7 F/14.5 Fever, weight loss, myalgia, Small aneurysms and caliber Indeterminate uppo- and neg Y 1. PAN (ACR)
intestinal inflammation, variation of medium-sized renal lowo* gastrointestinal 2. PAN ( C h ^ l Hill consensus)
arthralgia arteries; pruning of small renal inflammation
arteries; pafusion defects of
kidneys, tortuous small hepatic
arteries
8 M/6.2 Fever, arthritis, livedo Normal Skin biopsy: vasculitis neg N 1. PAN (ACR)
reticularis, myalgia affecting medium and 2. PAN (Chqjel Hill consensus)
small arteries
9 M/15 FevCT, weight loss, livedo Small aneurysms affecting Note done neg N 1. PAN (ACR)
reticularis, testicular pain medium and small renal and 2. PAN ( C h ^ l Hill consensus)
hepatic arteries
10 M/9.8 Fevo", urticaria, palpable Not done Skin- leucocytoclastic neg N 1. Hypersensitivity vasculitis (ACR)
purpura, myalgia, raynaud*s vasculitis 2. Microscopic polyangiitis OR cutaneous
leukocytoclastic angiitis (Chapel Hill consensus)
11 M/13.6 Fever, myalgia, weight loss, Caliber change and beading of Not done neg N 1. PAN (ACR)
livedo reticularis medium and small renal arteries 2. PAN (Chqiel Hill consensus)
12 M/14.1 F cvct, weight loss, myalgia Small aneurysms and caliber Not done neg N 1. PAN (ACR)
variation of medium and small 2. PAN (Oiîçel Hill consensus)
renal arteries and branches of
SMA
*ACR: American College of Rheumatology; #SMA; siq>a-ior mesenteric artery, 4>IMA: inferior mesoiteric artery IcANCA; cytoplasmic anti-neutroj^ cytoplasmic antibody fpANCA; painuclear
anti-neutrophil cytoplasmic antibody.
8
Patient Sex/age Clinical features Selective visceral autography Biopsy ANCA Active disease at Vasculitis classification, and system used for
number (yrs) (m o time of classification
sampling
13 F/9.7 FevCT, anaemia, myalgia, Small aneurysms affecting SMA Not done neg Y 1. PAN (ACR)
arthritis, weight loss and branches; small aneurysms 2. PAN ( C h ^ l Hill consensa)
and stenoses of medium to small
ratal arteries
14 M/14 Fevo-, weight loss, testicular small aneurysms of SMA Skin: perivascular neg N 1. PAN (ACR)
pain, myalgia, intestinal brancha monocytic infiltrate in 2. PAN (Chqrel Hill consensa)
inflammation, subcutaneous small arteria
nodules
IS M/11.2 Fevo-, weight loss, myalgia, Not done Liver: perivascular ^cANCA N 1. Unclasified (ACR)
deranged liver function, no grantilaomatous 2. Unclasified (Chapel Hill consensa)
urinary sediment inflammation
16 F/10.9 Fcvct, arthralgia, macular rash, Not Done >50% crescentic *pANCA N 1. Unclassified (A Q l)
nephritic-nephrotic glomerulone;*ritis 2. Microscopic polyangiitis (Chapel Hill
consensus)
17 M/13.1 Fever, heavy proteinuria, Small aneurysms and caliber Rertal biopsy normal neg V 1. PAN (ACR)
myalgia, weight loss, urticaria, variation of medium and small 2. Microscopic polyangiitis ( C h ^ l Hill
pericarditis, negative ratal arteria and SMA consaisus)
streptococcal serology
18 M/2.9 Fever, w e i^ t loss, intestinal Small aneurysms artd caliber SkirH perivascular neg Y 1. PAN (ACR)
inflammation, bullous rash, chartge affecting SMA & *IMA monocytic infiltration of 2. Microscopic polyangiitis (Chapel Hill
arthritis brancha, small anetrrysms and small arteria consensa)
pruning of mediirm and small
renal arteria
19 M/6.2 Fever, myalgia, abdominal pain, Small anetrrysms and caliber Upper GI biopsy normal neg Y 1. PAN (ACR)
w e i^ t loss variation with cut-off of medittm 2. PAN (Chapel Hill consensa)
and small renal a r ta ia and SMA
20 F/2.5 Fevo-, w e i^ t loss, myalgia. Small aneurysms and caliber Not done neg Y 1. PAN (ACR)- initial classification of KD
Polymorphous rash, peelii% of variation of medium and small changed to PAN due to multiple relapses artd
extronities, conjunctival renal and mesenteric arteria chronicity o f symptoms
iiÿection- chronic symptoms 2. PAN (C hêl Hill coaensa)
pasisting for >12 months;
coronary arterial aneurysms on
echocardiography
21 F/15.6 Fcva, weight loss, myalgia Small aneurysms affecting Not done neg N 1. PAN (ACR)
medium and small renal and 2. PAN (Chîçrel Hill conseaa)
hepatic arteria
22 M/8.2 Feva, myalgia, weight loss, Small aneurysms affecting Not done neg N 1. PAN (ACR)
hypatension, livedo reticularis medittm and small rertal and 2. PAN ( Q i ^ l Hill coaensa)
hepatic arteria
23 M/12 Feva, myalgia, polymoprphous Not done Skin- leucocytoclastic neg Y 1. Hypersotsitivity vasculitis (ACR)
rash, lymphadenop^y, vaculitis 2. Nficroscopic polyartgiitis (Chapel Hill
diarrhoea, conjunctival injection, consensa)
weight loss, abdominal pain
24 M/13.9 Feva, weight loss, oral Renal perfusion defects and rertal Liva- non-specific neg Y 1. PAN (ACR)
ulceration, myalgia, abnormal arterial prtming; small aneurysms inflammatory changa 2. PAN (Chryel Hill consensa)
liv a function of meditim-sized hepatic arteria
25 M/12.5 Feva, palpable purpura, myalgia Small aneurysms and caliber Skirt: perivascular neg Y 1. PAN (ACR)
variation affecting branches of lympjiocytic infiltrate in 2. PAN ( C h ^ l Hill conseaa)
SMA artd meditrm and small small arteries, but not
hepatic arteria arteriola
6 children (5 boys) fulfilled completely the diagnostic criteria for KD (Dajani, Taubert
and others, 1993; Petty RE and Cassidy JT, 2001c). The mean age was 1.7 years (range
0.25-5.5 years). 4 had coronary arterial aneurysms, and one had axillary arterial aneurysm
formation in addition to coronary arterial aneurysms. All patients were in the second
week of the illness, and 4 had already received intravenous immunoglobulin (IVIG) at the
time o f blood sampling. The patients with KD were described separately from those with
primary systemic vasculitis since KD differs from these latter vasculitides in that it has a
distinct clinical phenotype with unique association with the muco-cutaneous lymph node
syndrome (Brogan and Dillon, 2000a; Brogan, Bose and others, 2002) (although there are
overlapping clinical features with classical "infantile PAN", perhaps now regarded by
some as a severe form of KD), is usually self-limiting (unlike PAN, WG, and MPA), and
has epidemiological features suggestive of an infectious aetiology, unlike PAN, WG or
MPA.
6 children (2 boys) were classified as Henoch-Schonlein purpura (HSP) using both ACR
(Lightfoot, Michel and others, 1990) (Appendix 2) and Chapel Hill consensus criteria.
The mean age was 11.1 years (range 7-15 years). 4 had renal biopsy evidence
demonstrating typical glomerulonephritis with IgA-predominant immune deposits, and
one had skin biopsy again demonstrating typical IgA-dominant immune deposits in a
small arterial wall.
184
3.3,2 CONTROLS AND DISEASE CONTROLS
Blood samples were obtained from 20 control children undergoing venepuncture for pre
operative assessment prior to minor orthopaedic procedures. Children with syndromic
diagnoses, underlying inflammatory disease, on regular medication, or with intercurrent
infection were excluded from this group. The mean age was 10.6 years (range 0.5-19
years), and the male to female ratio was 1.5:1.
Disease controls comprised 27 children with bacterial or viral sepsis (n=10), autoimmune
disease without vasculitis (n=6: one ulcerative colitis, one juvenile dermatomyositis, one
CINCA syndrome (chronic idiopathic neurological cutaneous and articular syndrome),
one Sneddon syndrome, one transient post-viral anti-phospholipid syndrome, and one
focal and segmental glomerulosclerosis); or recipients of renal allografts (n=16, 8
children with dysplastic kidneys; 8 children with posterior urethral valves). These
recipients of renal allografts were included since they were on a similar spectrum of
immunosuppressive therapy (prednisolone, azathioprine, and cyclosporin) as the primary
systemic vasculitis group, but did not have a prior history o f vasculitis or other
autoimmune disease and hence would control for any effect on the T cell repertoire
mediated by immunosuppressive drugs. The mean age o f this group was 11.3 years
(range 4.5-17.2 years), with male to female ratio of 1.5:1.
Positive controls comprised 2 children with the toxic shock syndrome (TSS). The first
was a 3-week-old baby who developed the staphylococcal TSS secondary to a post
operative wound infection following repair of gastroschisis. The organism responsible
185
was a Staphylococcus aureus producing the superantigens TSSTl and staphylococcal
enterotoxins A, G, and I. Blood was obtained for T cell analysis from this patient
approximately 10 days into the illness. The second patient was a 2-year-old boy who
developed streptococcal TSS secondary to an infected gastrostomy site. The responsible
organism was a Lancefield group A Streptococcus, although this was not sent for toxin
typing. Blood was obtained from this patient 12 hours into the illness.
In addition to the above patients who had Vp repertoire studies performed, T cell
activation markers were studied in the above patients plus another 8 patients with PAN (5
with active disease, 3 with inactive disease), and 5 patients with viral infections (upper
respiratory tract infection).
Written, informed consent was obtained from all parents, and older children involved in
the study. The study was approved by the local hospital research ethics committee.
3.3.3 ANALYSIS OF PERIPHERAL BLOOD T CELL ACTIVATION MARKERS
AND T CELL V p REPERTOIRES
This was performed as described in Chapter 2, sections 2.4.1, 2.4.4 and Appendix 3. In
addition to the analysis of CD4 and CDS T cell Vp repertoires, the T cell activation
markers CD69 and CD25 (table 2.1) were analysed within the CD4 and CDS T cell
populations.
186
3.3.4 IN VITRO TECHNIQUE FOR VALIDATION OF EFFECT OF SAgs ON T
CELL v p REPERTOIRE SKEWING
In addition to the in vitro validation technique described in Chapter 2 (section 2.4.5),
analysis of the T cell Vp repertoire was performed in 2 children with TSS (described
above) to check the validity of the technique in vivo.
3.3.5 STATISTICS
Comparison of group demographics was performed using the Kruskall-Wallis test. T cell
activation between the groups was assessed using the Kruskall-Wallis and Mann-Whitney
tests.
Assessment of skewing of the T cell Vp repertoire between the groups of children was
performed by comparison of mean percentages for each Vp family using ANOVA, and
all p values adjusted for multiple between-group comparisons using the Bonferroni
method when the groups had equal variance, or the Tamhane method when significant
differences in the variance for individual Vp families was found. In addition, to further
protect fi"om the effect of multiple comparisons, biological significance was only inferred
from the statistical results when the difference in the vasculitis group was consistent for a
particular Vp femily between both the control and disease control groups. Vp skewing
was also assessed by comparison o f the variance of each Vp family between the groups
using the F test, with significance adjusted for 17 comparisons (i.e. the number o f Vp
families examined). To check the validity of the F test, normality of the data was assessed
187
using the Kolmogorov-Smimov and the Shapiro-Wilks tests, and no evidence for non
normality was demonstrated in any Vp family.
For comparison, T cell Vp skewing was also assessed using previously published but
arbitrarily-defined definitions of T cell Vp expansions and deletions, with the proportion
of children in each group having an expansion or deletion compared using the two-
sample test o f proportion. Thus, a Vp expansion was defined as a value more than the
control group mean plus 2 standard deviations for an individual Vp &mily, and a deletion
was a value less than the control group mean minus 2 standard deviations(Popa,
Stegeman and others, 1998; Wedderbum LR, Maini MK and others, 1998).
Paired data derived from the longitudinal study of 6 of the vasculitis patients before and
after induction of remission were analysed using the Wilcoxon signed-rank test.
Statistical significance was defined at p<0.05 (adjusted for multiple comparisons), and is
implied in the results wherever differences are highlighted. All statistics were performed
using Microsoft Excel 97 and SPSS 10.0 for Windows.
3.4 Results
3.4.1 PATIENT DEMOGRAPHICS
Children with KD were younger than children with other vasculitides, controls, and
disease controls (p=0.001). Otherwise there were no significant age or sex differences
between the groups of children. The median time o f blood sampling from disease onset
188
was 12 months (range: 0.6-72 months) in the primary systemic vasculitis group, and 1.5
months (range 0.5-6 months) in the HSP group. All the patients in the KD group were
sampled in the second week of the illness.
3.4,2 T CELL ACTIVATION IN PATIENTS AND CONTROLS
Since there is a linear pattern of CD69 staining in CD4 and CDS T cells the level of
expression was determined by analysis of both the median fluorescence index (MFI), and
by determining the percentage of cells with “high” CD69 expression (using an arbitrary
cut-off between CD69 “high” and CD69 “low” expression).
Overall, patients with active vasculitis (all types of vasculitis; mean BVAS 9.4; range 3-
20) had higher levels of peripheral CD4+ T cell activation as determined by CD69
expression compared with those with inactive vasculitis (BVAS=0 by definition),
(p=0.01 for CD69 MFI; p=0.005 for percentage of high CD69); and healthy controls
(p=0.04 for CD69 MFI; p=0.007 for percentage of high CD69). There was no difference
in CD4+ CD69 expression between the active vasculitis group and the disease controls
(p=0.4 for CD69 MFI; p=0.37 for percentage o f high CD69). These data are summarised
in figure 3.1.
189
Figure 3.1: CD4 T cell CD69 Expression
p=0.4
p=0.04
p=0.01
200-1
■ Active vasculitis
* inactive vasculitis
Li_
s » Healthy controls
S * Disease controls
3 100 -
'ît
û
ü
p=0.7
p=0.001
P=0.37
100-1
s
P =0.007 ■ Active vasculitis
P=0.005
A Inactive vasculitis
au 75-
▼ Healthy controls
8€)
♦ Disease controls
50-
o %
ss 2 ■ ♦
a. 2 5 - ♦♦
s
P=0.53
P=0.003
190
Regarding the CDS T cell population, patients with active vasculitis (all types of
vasculitis) had higher CD8+ CD69 expression than those with inactive vasculitis (p=0.04
for CD69 MFI; p=0.01 for percentage of high CD69). CD69 expression was only higher
in the active vasculitis compared with healthy controls when the results were expressed
as percentage o f high CD69 (p=0.007), and not when the result was expressed as CD69
MFI (p=0.11). There was no difference in CD8+ CD69 expression between the active
vasculitis group and disease controls (p=0.5 for CD69 MFI; p=0.3 for percentage of high
CD69). These data are summarised in figure 3.2.
To examine T cell activation profiles within different types o f vasculitis, the vasculitis
patients were sub-divided into a primary systemic vasculitis group (inclusive o f the
necrotizing vasculitides PAN, MPA, and WG); HSP; and KD. There were several
missing data points from these latter 2 groups, and so no statistical analysis was
performed for these sub-categories. Again, there was higher CD4+CD69 expression (MFI
data only shown) in the active primary systemic vasculitis group compared with inactive
vasculitis (p=0.007); and healthy controls (p=0.02); but there was no difference between
active vasculitis and disease controls (p=0.92). These data and the data regarding the HSP
and KD patients are summarised in figure 3.3. CD8+CD69 expression was higher in the
active primary systemic vasculitis group than in the inactive group (p=0.042), but no
difference was observed between the active primary systemic vasculitis group and the
healthy controls or disease controls (p=0.13 and p=0.4 respectively). These data are
summarised in figure 3.4.
191
Figure 3.2: CD8 T cell CD69 expression
P=0.5
P=0.11
P=Q.Q4
75-1
LL
^ Inactive vasculitis
^ Healthy controls
m 50-
(D ♦ Disease controls
Û ♦♦
O
00 ♦♦
0 25-
P=0.5
P =0.007
P=0.3
P =0.007
V P=0.01 ^ T
■■
■
* Inactive vasculitis
♦
» Healthy controls
■
■■ ♦ Disease controls
r ^r
P =0.05
P=0.007
192
Figure 3.3: CD4 T cell CD69 expression by vasculitis type
p=0.92
p=0.02
p=0.007
200-1
■ PSV active
A PSV inactive
V Healthy controls
s ♦ Disease controls
0 100 -
Q
O
jim !
p=0.76
D=0.001
200-1
■ HSP active
A HSP inactive
100 -
♦♦
200-1
■ KD active
A KD inactive
S ♦ Disease controls
8 100
Û
o
♦♦♦
♦
u
0 ▼Tw^ wT
193
Figure 3.4: CD8 T cell CD69 expression by vasculitis type
p=0.4
p=0.042 p=0.13
75 n
■ PSV active
U.
A PSV inactive
S ▼ Healthy controls
50-
S ♦ Disease controls
o ♦♦
o
00
O 25-
O
%
t:
0
p=0.55
i
p=0.000
75-1
■ HSP active
A HSP inactive
O) 50-
V Healthy controls
♦♦
♦♦
2 5-
75n
■ KD active
LL
A KD inactive
i(O 50 :
^ Healthy controls
O ♦♦
Ü
CO
25-
Ü
▼ ♦♦♦♦♦
194
There was no difference in CD4+CD25 or CD8+CD25 expression between the active
vasculitis and inactive vasculitis groups (figures 3.5 and 3.6). CD25 expression was not
measured in the controls or disease control groups.
3,4.4 T CELL Vp REPERTOIRES FROM PATIENTS AND CONTROLS:
COMPARISON OF MEANS
Figures 3.7a and 3.71b summarise the mean percentage o f CD4 and CDS T cells
expressing each of the individual Vp gene products for controls, disease controls, and
children with vasculitis (PSV, KD, and HSP). For comparison, the T cell Vp repertoire
from the 2 positive control children with TSS compared with controls and disease
controls is shown in figures 3.8a and 3.8b.
3.4.4.1 PRIMARY SYSTEMIC VASCULITIS
For CD4 T cells, the mean percentage of Vp 13.1 was higher in the primary systemic
vasculitis group than controls (p=0.02) but not disease controls (p=0.2). For the CD8 T
cells, the mean percentage of Vp2 was lower in the disease controls versus the controls
(p=0.03), although there was no difference between vasculitis and controls or disease
controls. The mean percentage of CD8 Vp7 T cells was higher in the primary systemic
vasculitis group than the controls (p=0.01) but not the disease controls (p=0.1). The mean
percentage of CD8 V p l7 T cells was lower in the primary systemic vasculitis group than
controls (p=0.03) but not disease controls (p=0.1).
195
Figure 3.5: CD4 CD25 expression
r ~ p=0.48
i
150-1
100 -
Q
O
S 50-
O
UT T T W W T T T ---------------------
Active vasculitis Inactive vasculitis
p=0.71
lOOn
in
CM
^ Q
SU O 75-
50-
c
(0
(0
o
® 25- A.
Q.
▲▲
g
0
196
Figure 3.6: CD8 CD25 expression
p=0.25
30n
20 -
U)
CM
O
ü
g
▲A
10 -
lOOn p=0.53
U)
CM
O
75-
O) 50-
c
(0
(0
o OR-

197
F ig ure 3.7 a and 3.7 b : T cell V(3 repertoires in study groups
Mean (SEM) CD4 and CDS Vp repertoire from healthy control children, disease control
children, and children with vasculitis (primary systemic vasculitis, Kawasaki disease, and
Henoch Schonlein purpura). All comparisons were performed using ANOVA, and all p
values adjusted to account for multiple comparisons (see text).
198
Figure 3.7a and 3.7b: Mean percentage ofC D 4 and CD8 T cells expressing individual Vp
receptors for controls, disease controls, and the 3 groups o f children with vasculitis.
Figure 3.7a: CD4 T cell vbeta repertoire

(mean.sem)
■ Control m ean
a D is e a s e control m ean
□ Primary S ystem ic Vasculitis m ean
□ Kawasaki D is e a s e m ean
0 H S P m ean
vb
Figure 3.7b: CDS T cell vbeta repertoire

(mean,sem) ■ Control m ean
B D is e a s e control m ean
□ Primary S y stem ic Vasculitis m ean
□ Kawasaki D is e a s e m ean
□ H SP m ean
vb
199
F ig u r e 3 .8 a and 3 .8 b :
CD4 and CDS Vp repertoire from 2 children with toxic shock syndrome. The first patient
with staphylococcal TSS shows an expansion ofCD4 and CDS Vp2 T cells. The second
patient with streptococcal TSS shows an expansion of CD4 VP5.1 T cells.
200
Figure 3.8a and 3.8b; CD4 and CD8 repertoire from 2 children with TSS, compared with
controls and disease control children.
Figure 3.8a: CD4 T cell V b eta repertoire in T S S patients
25 □ staphylococcal TSS
□ Streptococcal TSS
■ Control mean
B D isease control mean
3 5.1 5.2 7 8 11 12 13.1 13.6 14 16 17 20 21.3 22

CD4 V b e ta
Figure 3.8b: CD8 T cell V beta repertoire in T S S patients
40 -1
□ staphylococcal TSS
35 1
30 -I □ Streptococcal TSS
I
■ Control mean
25 -
g Disease control mean
20 -
15 -
10 -
5 4
0
5.1 5.2 11 12 13.1 13.6 14 16 17 20 21.3 22
CD8 V beta
20 1
S.4.4.2 KAWASAKI DISEASE
In the KD group the mean percentage of CD4 Vp2 cells was higher than in both the
control and disease control groups (p=0.03, and p=0.01 respectively). The mean
percentage of CDS Vp2 cells was higher in the KD group than in the disease controls
(p=0.02) but not the controls (p=0.9). The mean percentage o f CDS Vp7 T cells was
higher in the KD group than both the controls (p=O.OOS) and disease controls (p=0.03).
3A A 3 HENOCH SCHÔNLEIN PURPURA
In the HSP group the mean percentage of CD4 V pl T cells was lower than in both the
control (p=0.02), and disease control groups (p=0.02). The mean percentage of CD4
V p l4 was lower in the HSP group than in the controls (p=0.01) but not the disease
controls (p=0.3). The mean percentage o f CDS V pi was lower in the HSP group than in
the controls (p=0.007), but not the disease controls (p=0.2).
3.4.5 T CELL VpREPERTOIRES: COMPARISON OF VARIANCE
Figures 3.9a and 3.9b summarise the ratios of the variance (F ratio) o f each Vp family for
CD4 and CDS T cells from the primary systemic vasculitis group and the controls and
disease controls. F ratios were obtained by dividing the variance ("spread") for each Vp
family in the vasculitis group by the corresponding Vp family variance in the control, and
disease control groups. If the variances are similar, the F ratio is close to 1. If the variance
of any Vp family is greatly increased in the vasculitis group, the corresponding F ratio is
much greater than 1. P values for the F ratios thus obtained between the groups were then
202
F ig u r e 3 .9 a and 3 .9 b : v a r ia n c e r a t io s f o r e a c h vp f a m il y
Variance ratios (F ratios) of each Vp family for CD4 and CDS T cells. F ratios were
obtained by dividing the variance ("spread") for each Vp family in the vasculitis group by
the corresponding Vp family variance in the control, and disease control groups. If the
variances are similar, the F ratio is close to 1. If the variance of any Vp family is greatly
increased in the vasculitis group, the corresponding F ratio is much greater than 1
(adjusted p values asterisked).
203
Figures 3.9a and 3.9b; Variance ratios (F ratios) of each Vp family for CD4 and CDS T
cells.
Figure 3.9a: Ratio of variance of CD4 v beta

repertoire
100 □ Vasculitis v s controls
□ Vasculitis v s d is e a se controls
10
■fO
i 1 J] fL jH .
.2 1 2 3 5^ 5.2 IT 8 11 12 13.1 14 IB" 17 20 21.3 22
c5
>
0.1 J *p<0.05 (adjusted for 17 comparisons)
for both vasculitis versus controls
and vasculitis versus disease
controls
0.01
V b e ta
Figure 3.9b: Ratio of variance of CDS v beta

repertoire □ vasculitis v s controls
100
^ vasculitis v s d is e a se controls
ê
2
5.1 20 21.3
*p<0.05 (adjusted for 17

comparisons) for both
vasculitis versus controls
and vasculitis versus disease
0.01 V beta controls
204
derived using the F test, with alpha adjusted for 17 comparisons. Only when the variance
ratio was statistically significantly increased for both vasculitis versus controls and
vasculitis versus disease controls was it regarded as biologically significant.
The variance was increased in the vasculitis group compared with both the controls and
disease controls for CD4 V p l2 (vasculitis versus controls adjusted p<0.00001; vasculitis
versus disease controls adjusted p<0.00001); CD4 Vp 17 (vasculitis versus controls
adjusted p=0.005; vasculitis versus disease controls adjusted p=0.0005); and CDS V pi
(vasculitis versus controls adjusted p=0.005; vasculitis versus disease controls adjusted
p=0.0001).
To minimise the effect of patient heterogeneity within the primary systemic vasculitis
group the data were reanalysed comparing only the 18 patients with classical PAN as
defined by the ACR criteria, and who had diagnostic visceral angiography for PAN (one
patient satisfying criteria for PAN with positive skin biopsy had a normal visceral
angiogram, and so was excluded fi*om this analysis). Again, the variance was increased
in the PAN group compared with both control and disease control groups for CD4 V p i2
(vasculitis versus controls adjusted p<0.00001 ; vasculitis versus disease controls adjusted
p<0.00001); CD4 V p l7 (vasculitis versus controls adjusted p=0.001; vasculitis versus
disease controls adjusted p=0.0001); and CDS V pl (vasculitis versus controls adjusted
p=0.001; vasculitis versus disease controls adjusted p<0.00001).
205
In the KD and HSP groups, there were no consistent differences in the variance compared
with controls and disease controls.
3.4,6 T CELL V p EXPANSIONS AND DELETIONS
Using the arbitrary definitions of VP expansion and deletion described previously, 80%
of the primary systemic vasculitis children had one or more CD4 Vp expansions or
deletions compared with 30% of the controls (95% Cl o f the difference 21-79%,
p<0.002) and 37% o f the disease controls (95%CI of the difference 16-70%, p<0.002).
15/18 (83%) of the patients with classical PAN (ACR defined, and confirmed on visceral
angiography) had one or more CD4 VP expansions or deletions, this percentage being
significantly higher than the controls (p< 0.002) and disease controls (p=0.002), although
there was no clear relationship between the duration o f vasculitis prior to blood sampling
and the presence of expansions or deletions. There were no differences in the number of
expansions or deletions in the CD8 T cell population between the vasculitis and
control/disease control groups.
In the KD group, 67% of the patients had one or more CD4 Vp expansions or deletions,
no different from the controls (95% Cl of the difference from controls -7-81%, p=0.1;
95% Cl of the difference from disease controls -14-73%, p=0.1). There were no
differences in the number o f expansions or deletions in the KD CD8 T cell population.
In the HSP group there were no differences in the number o f CD4 or CD8 expansions or
deletions compared with either the control or disease control groups.
206
3.4.7 LONGITUDINAL STUDIES ON PRIM ARY SYSTEMIC VASCULITIS
CHILDREN
Overall, there were no differences in the mean percentage o f individual Vp families in
either the CD4 or CDS population of T cells when paired samples from 6 primary
systemic vasculitis pre and post-induction of remission were compared. However, there
was a greater variance of certain VP families prior to induction of remission: CD4 V pl 2
(F ratio 190, adjusted p=0.00001), CD4 V p l4 (F ratio 296, adjusted p=0.00005), and
CDS V pl (F ratio SI, adjusted p=0.001). Moreover, 5 out o f 6 of the active vasculitis
group had 1 or more CD4 T cell Vp expansions or deletions (4 patients with one or more
expansions, and one patient with one expansion and one deletion), compared with 1 out
o f 6 in the remission group (95% Cl of observed difference 4-100%, p=0.02). No
difference in the numbers of expansions or deletions in the CDS Vp repertoire was
observed pre and post induction of remission.
3.5 Discussion
There are limited data suggesting that SAgs play an important role in the initiation and/or
subsequent relapse o f the systemic vasculitides in adults (Cohen Tervaert, Popa, and Bos,
1999; Cohen Tervaert, 2002). No such data exist for the primary systemic vasculitides
(PAN, WG, MPA) in children, although a recent small series o f 6 children with HSP
nephritis in association with staphylococcus infection demonstrated increased
percentages in the peripheral blood of T cells bearing Vp5.2, Vp5.3, and Vp8, suggesting
207
superantigenic stimulation o f the immune system in these patients (Hirayama, Kobayashi
and others, 2001).
The response of a T cell to superantigenic stimulation is complex, and the observed
"skewing" of the Vp repertoire, which typically follows superantigenic stimulation
(figures 2.3 and 2.4) is dependent on several factors. Firstly, timing o f the blood sample
from the onset of superantigenic exposure is critical, since initially there is Vp restricted
T cell activation, followed by specific T cell Vp proliferation, and finally Vp deletion
(Leung, Meissner C, and Schlievert, 1997; Shoukry, Lavoie and others, 1997). It is
reported that this peripheral VP deletion following superantigenic stimulation is the result
of apoptosis of activated T cells, although other possibilities including internalisation of
T cell receptors (resulting in a state of anergy) (Fraser, Arcus and others, 2000), or
sequestration o f specific activated Vp families into sites o f tissue inflammation (Leung,
Giomo and others, 1995; Yamashiro, Nagata and others, 1996) may also contribute to
removal o f certain Vp families from the peripheral circulation. Other influences such as
the HLA type of an individual (Herman A, Croteau G and others, 1990), and the recent
description of "partially Vp-restricted" SAgs (Hodtsev AS, Choi Y and others, 1998)
could also theoretically affect the ability to detect peripheral Vp skewing following SAg
exposure in some studies. It is therefore feasible that depending on the timing of blood
sampling in relation to SAg exposure, or other SAg or host determinants, peripheral T
cell Vp skewing may not be observed, perhaps explaining some o f the conflicting data
emerging from studies in KD.
208
The flow cytometric technique for the analysis o f T cell activation and T cell Vp
repertoires used in this study was able to demonstrate the classical response o f T cells to
SAg stimulation in vitro: there was expansion of CD4 and CDS T cells expressing Vp2
for TSST-1, and Vp3 for SEE. This observation was in agreement with what is known
about the major responding Vp families for these 2 SAgs (Cohen Tervaert, Popa, and
Bos, 1999; Curtis, Zheng and others, 1995). Furthermore, there was a high degree of T
cell activation in response to SAgs at 8 days, as compared to control PBMCs left cultured
but unstimulated for 8 days. Further evidence for the validity o f this technique was
provided rather serendipitously by the presentation of 2 children with classical TSS (one
staphylococcal and one streptococcal; the author wishes to add that both children were
treated successfully and made complete recoveries). Analysis o f the Vp repertoires in
these children revealed expansions o f T cell Vp families compatible with the
staphylococcal and streptococcal toxic shock syndromes (Vp2 and VpS.l respectively
(Watanabe-Ohnishi, Low and others, 1995)).
T cell activation as determined by CD69 expression was higher in patients with active
vasculitis (all types) and in those with primary systemic vasculitis compared with those
with inactive disease, and healthy controls. This result was in contrast to the findings of
Christensson et aï who showed that adult patients with ANCA-positive vasculitis show
an increased expression of T cell activation markers (CD69) irrespective of
immunosuppressive therapy or disease activity (Christensson, Betters son and others,
2000). The reason for this difference may be related to the nature o f the vasculitic
syndromes examined - ANC A associated vasculitides comprised the minority of subjects
209
in the study presented in this chapter (table 3.1). Another possibility may relate to age-
related maturational changes in the immune system. It was interesting to note that there
was no difference between the active and inactive vasculitis groups in terms of CD25
expression. Thus, in support of the concept of persistent T cell activation by Christensson
et al there was some evidence of high CD25 expression within the CD4 and CDS T cell
populations in a few of the patients with a BVAS of zero. These data would support a
role for T cells in the pathogenesis o f primary systemic vasculitis affecting children.
These results also emphasise, however, that observations in adults with vasculitis may not
hold true for vasculitis syndromes affecting children.
Despite the inherent limitations of examining peripheral blood T cell Vp repertoires in
patients with long-standing chronic inflammatory disease for evidence o f previous SAg
exposure, it was possible to demonstrate changes in the Vp repertoire o f certain vasculitis
patients. Thus, in the primary systemic vasculitis group (PAN, WG, MPA, and
unclassifiable vasculitis), there were minor deviations in the mean percentages of CD4
and CDS Vp families, although no consistent difference in the mean Vp percentages for
vasculitis versus both controls and disease controls was observed. Comparison of the Vp
variance, however, revealed significantly increased variance in the primary systemic
vasculitis patients for some VP femilies as compared to controls and disease controls:
CD4 V p l2 and CD4 V p l7, and CDS V pl. Additionally, significantly more "expansions"
and "deletions" were observed in the primary systemic vasculitis group than in the
control and disease control groups.
210
As documented in table 3.1, there was a degree of heterogeneity within the primary
systemic vasculitis group. These patients were analysed as one group because evidence
for superantigenic involvement has been suggested for adult patients with PAN, MPA,
and WG. Moreover, there is a greater degree o f polyangiitis overlap in children making
classification problematic. Nonetheless we were able to demonstrate skewing of the Vp
repertoire when these patients were compared with controls and disease controls.
Furthermore, when the 18 patients satisfying classification criteria for classical PAN
inclusive of diagnostic angiography were analysed separately, the result was unchanged
suggesting that the observed skewing of the Vp repertoire is not a consequence of patient
heterogeneity.
In the KD group, although patient numbers were small, in accordance with the findings of
others (Abe, Kotzin and others, 1992; Curtis, Zheng and others, 1995) there was a
significantly increased percentage of CD4 Vp2 in the second week o f the illness as
compared to controls and disease controls. Interestingly, there was also a significantly
increased percentage in CDS Vp7 in the KD group. It is suggested that this observation
was unlikely to be the result of the presence o f genetic polymorphisms, which have been
shown to affect Vp7.2 (Zhao, Whitaker, and Robinson, 1994), since the antibody clone
used in this study (ZOE) recognises Vp7.1. That said, some cross reactivity between this
antibody and Vp7.2 was not formally excluded in this study. In the HSP group, again
although patient numbers were small, CD4 V pl T cells were found to be lower in the
peripheral circulation than that observed in control and disease control children, but no
differences in variance or numbers of Vp expansions or deletions were observed.
211
Lastly, paired samples obtained from 6 vasculitis patients (all with classical PAN) before
and after induction of remission of vasculitis showed "normalisation" o f the Vp repertoire
following treatment. Thus, the variance of CD4 12, 14, and CDS V pl was significantly
greater in the group prior to treatment as compared to those following induction of
remission. Also, there were significantly more Vp expansions in the pre-treatment
vasculitis group than the post-treatment group.
Whilst these observations could be compatible with an effect o f SAgs on the immune
system in patients with vasculitis, it cannot be concluded from this study that SAgs are
directly involved in the initiation or relapse of vasculitis. Indeed, the T cell Vp skewing
observed could be an epiphenomenon in these patients. Potential confounding fectors
include a non-specific skewing of the Vp repertoire as a result o f chronic inflammation,
the use of glucocorticoids and immunosupressants, and prolonged periods o f in-patient
care (potentially altering carriage of bacterial commensals) in the vasculitis group.
Attempts were made to control for these factors, however, by the inclusion o f disease
controls who were renal allograft recipients with no prior history o f vasculitis or other
autoimmune disease, and who were treated with a similar (but not identical) range of
glucocorticoid and immunosuppressive therapy as the vasculitis children. These children
were treated for lengthy periods of time as in-patients at the same institution as the
vasculitis group, and thus theoretically exposed to the same range of microbial
commensals. Moreover, the disease control group also included children with
miscellaneous non-vasculitic autoimmune disease and sepsis. Even with this (arguably)
212
over-controlled disease control group, it was possible to demonstrate CD4 Vp skewing in
the children with vasculitis that was not observed in the disease controls, even though the
latter group displayed a comparable degree of T cell activation (figures 3.1 and 3.2).
Another interesting observation was the lack of similarity between the CD4 and CDS Vp
changes observed in the vasculitis patients, since SAgs are known to affect both T cell
subsets (as in the patient with staphylococcal TSS, and in the in vitro responses of T cells
to SAgs). Whilst this observation may mitigate against SAgs as a major influence in these
disease states, it should be borne in mind that the signaling events governing the T cell
response to SAgs (including activation, expansion, deletion, anergy or tissue
sequestration) may be different for CD4 and CDS T cells, and could vary with time
within these T cell subsets.
In conclusion the data presented in this study support a role for T cells in the
pathogenesis of primary systemic vasculitides in childhood. Moreover, there were
changes in the T cell Vp repertoire in children with vasculitis over and above those
observed in disease controls (who exhibited significant T cell activation, but no T cell Vp
repertoire skewing). Whilst these data provide impetus for further research into this
contentious field, they do not unequivocally resolve the question o f the role of SAgs in
childhood vasculitic syndromes. The results described in this study did however provide
enough “smoking-gun” evidence to prompt further study of the mechanisms that SAgs
may utilize to cause vascular injury- the subject o f Chapter 4.
213
Chapter 4: Vp-restricted T cell adherence to endothelial cells:
a mechanism for superantigen dependent vascular injury.
4.1 Summary
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussion
214
4.1 Summary
Introduction and aims: To investigate the potential for endothelial cells (EC) to operate
as a superantigen (SAg)-presenting cell for T cells (TC), and the potential for such an
interaction to cause EC activation.
Methods: MHC class iT HUVEC were co-cultured for 4 hours with purified TC and the
SAgs SEB or TSST-1. After staining with fluorescent conjugated monoclonal antibodies
flow cytometric analysis was performed on the HUVEC and TC to examine Vp-restricted
TC adherence to the EC monolayer; Vp-restricted TC activation (CD69 upregulation);
and surface expression of EC activation markers.
Results: Co-culture of purified TC with MHC class iT HUVEC and TSST-1 or SEB
resulted in Vp-restricted CD4 and CDS CD69 upregulation (Vp2 activation for TSST-1;
Vp3, Vp 5.1 and V p l2 activation for SEB). Additionally, there was CD4 and CDS TC
Vp-restricted adherence to the HUVEC monolayer at 4 hours, which was partially
abrogated by blockade of the integrin VLA-4, but not LFA-I. ICAM-I, E-selectin, and
VCAM-I expression were upregulated on the MHC class iT HUVEC following exposure
to SAg in the presence of T cells.
Conclusion: MHC class iT EC operate as competent SAg-presenting cells for CD4 and
CDS lymphocytes in vitro. Dual signalling between the EC and TC results in Vp-
restricted activation and adherence to endothelial monolayers, and EC activation, it is
proposed that this mechanism could account in part for the vasculitic injury associated
with SAg-mediated diseases.
215
4.2 Introduction
In Chapter 3 data were presented supporting the concept that T cells are involved in the
pathogenesis of vasculitis syndromes affecting the young. Moreover, there were changes
observed within the CD4 and CDS T cell Vp repertoires over and above those observed
in disease controls. These observations could be compatible with a SAg-mediated effect,
but by no means confirm this beyond all doubt.
An important observation is that T cells infiltrating the walls of coronary arterial
aneurysms and the intestinal mucosa of patients with KD show a skewed T cell Vp
repertoire, with increased numbers of cells expressing Vp2 (Leung, Giomo and others,
1995; Yamashiro Y, Nagata S and others, 1996).
Whilst there are many observational clinical studies providing indirect evidence of
superantigenic involvement in the vascular inflammatory process (Barron, 2002), there
are only a limited number examining the mechanisms that SAgs may utilize to cause
vascular injury. Proposed mechanisms for SAg-induced vascular injury include: 1. direct
activation of autoreactive T cells resulting in vessel wall destruction; 2. activation of
autoreactive B cells resulting in production o f autoantibodies such as anti-neutrophil
Cytoplasmic antibodies (ANCA) or anti-endothelial cell antibodies (AECA), which are
known to result in vessel injury; and 3. direct binding o f SAg to endothelial cells via
charge interactions with subsequent antibody binding and immune complex vasculitis
(Cohen Tervaert, Popa, and Bos, 1999).
216
Although the exact aetio-pathogenesis of most vasculitis syndromes is unknown, it is
now clearly established that endothelial cells actively participate in the pathogenesis of
systemic vasculitis, both as a target for injury, and as active protagonists of the
inflammatory process (Cid, 2002). Limited experimental data exist demonstrating that
the endothelial cell, following stimulation with interferon-y (ENF-y) to upregulate MHC
class n, can operate as a SAg-presenting cell for T cells resulting in T cell activation and
adherence to endothelial monolayers in vitro (hnanishi, Akatsuka and others, 1995; Kita,
Eguchi and others, 1996; Uchiyama, Araake and others, 1992). Furthermore, only one
study has demonstrated that a consequence of the interaction between the endothelial cell,
CD4 T lymphocytes, and the SAg Staphylococcal enterotoxin type B (SEB) is
upregulation of the cell adhesion molecules intercellular adhesion molecule type 1
(ICAM-1), vascular cell adhesion molecule type 1 (VCAM-1), and E-selectin on the
endothelial cell (Baum, Yaron, and Yellin, 1998). In that study, it was possible to block
the SAg-dependent T cell mediated endothelial cell activation using monoclonal
antibodies against tumour necrosis factor-a (TNF-a), thus implicating this cytokine as an
important mediator of the observed endothelial activation (Baum, Yaron, and Yellin,
1998). This observation, although important since it demonstrated the significance o f the
endothelial cell both as a SAg-presenting cell and as an amplifier of the vascular
inflammatory response, was limited because it did not demonstrate that the endothelial
cell activation was the result of Vp-restricted T cell activation (the hallmark o f a SAg-
mediated process) and adherence to the endothelium, and precluded the potential
217
importance of CDS T cells in SAg-dependent T cell mediated endothelial activation and
injury.
In this study the potential for the endothelial cell to operate as a SAg-presenting cell for T
cells, and in particular the effect of such an interaction on the T cell in terms of Vp-
restricted activation and adherence to the endothelium was studied in further detail.
Furthermore, the effect on the endothelium was assessed by measuring cell adhesion
molecule expression.
4.3 Materials and methods
4.3,1 Antibodies, cytokines, and protein reagents
The monoclonal antibodies used in these experiments are described in detail in table 2.1.
Also used were blocking antibodies against the a subunit o f the integrin LFA-1
(lymphocyte function associated molecule 1, GDI la; clone 38 [a kind gift from Professor
Nancy Hogg, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund] mouse
IgG2a anti-human CD 11 a, used at a concentration o f 1.8 pg/ml), or against the a subunit
of VLA-4 (CD49d, very late antigen-4; clone MCA697, Serotec, mouse IgGl anti-human
CD49d, used at a concentration o f 10 pg/ml). The control antibody for anti-LFA-1 was a
mouse IgG2a antibody (clone 4U, a kind gift from Professor Nancy Hogg, used at a
concentration of 1.8 pg/ml), and the control for anti-VLA-4 was a mouse IgGl antibody
(clone MCA928, Serotec, used at a concentration of 10 fig/ml). Again, as previously
218
described all antibody dilutions were made with 0.01 M phosphate buffered saline with
0.1% sodium azide, and checked by plotting a dilution curve.
4.3.2 Isolation o f human umbilical vein endothelial cells (HUVEC)
HUVEC were prepared as described in sections 2.4.6-2.4.13. In experiments where
HUVEC were stimulated with INF-y for 48-72 hours, this cytokine was added when the
HUVEC reached approximately 80% confluency, and the medium containing INF-y
removed prior to experiments. All HUVEC used in experiments were first to third
passage.
4.3.3 PBM C and T cell incubation with SAg: determination o f Vfi-specijic activation
To examine the Vp-specific activation of T cells to SAgs and the requirement of MHC
class J f cells for SAg presentation to T cells, PBMCs or purified T cells were incubated
in 6 or 24-well tissue culture plates in the presence or absence o f either TSST-1
(lOng/ml) or SEB (lOOng/ml) for 4 hours at 37°C. These experiments were different from
the experiments described in section 2.4.5, which determined changes in the percentages
o f the T cell Vp families in response to SAgs after 8 days. Again, the concentrations of
SAg used were derived from previous studies (Baum, Yaron, and Yellin, 1998; Curtis,
Zheng and others, 1995).
Analysis of VP-specific activation was then determined by flow cytometric analysis of
CD69 expression within individual Vp families as described below in section 4.3.5. In
some experiments the incubation was continued for 24 hours in order to assess the
219
relevance of the remaining 0.2% of cells expressing MHC class H in the purified T cell
population.
4.3.4 HUVEC-T cell co-culture experiments
Monolayers o f HUVEC (with and without pre-treatment with INF-y 1000 U/ml for 48-72
hours to upregulate MHC class D) were co-cultured in the presence o f T cells (1
million/ml), T cells and SAg (TSST-1 10 ng/ml; or SEB 100 ng/ml), or TNF-a 100 ng/ml
for 4 hours at 37°C. The HUVEC monolayer was then dispersed using trypsin/EDTA for
1 minute and the HUVEC prepared for flow cytometry as described in section 2.4.12. To
examine T cell adherence, the HUVEC monolayer was washed 4 times with PBS to
remove non-adherent T cells, and again the HUVEC-adherent T cell monolayer dispersed
with trypsin/EDTA and prepared for flow cytometry. Supernatants (including PBS
washes) containing the non-adherent T cell population were centrifuged at 500G for 8
minutes and the non-adherent T cell population frozen at -80 °C in FCS containing 10 %
DMSO for further analysis. The cell-free supernatants were also frozen for analysis of
endothelial microparticles (see below). The plate plan for these co-culture experiments is
shown in Appendix 7.
4.3.5 Flow cytometry o f T cells and HUVEC
3-colour FACS analysis of PBMCs or T cells from experiments was performed as
previously described. In these experiments, an abbreviated panel o f Vp-specific
antibodies was used (V pi, Vp2, Vp3, Vp5.1, Vp8.1, and V p l2) which included the
220
known major responding Vp families to TSST-1 and SEB. As described, T cells were
identified by gating for forward and light-scatter characteristics, CD4^ or CD8^
populations, and the different Vp families. Vp-specific activation was then determined by
gating on the individual Vp family sub-population (CD4 or CDS and Vp double
positives) and then plotting a histogram of CD69 fluorescence, with determination o f the
CD69 median fluorescence index (MFI) for each VP family in the CD4 and CDS sub
population of T cells. Single colour FACS analysis o f HUVEC was as described in
section 2.4.12.
4.3.6 Integrin blockade o f SAg-mediated T cell adherence to HUVEC
To investigate potential molecular mechanisms of the SAg mediated Vp-specific T cell
adhesion to HUVEC, MHC class HUVEC were again co-cultured with purified T
cells, but this time the T cells were pre-incubated for 30 minutes with monoclonal
antibodies against the a subunit of the integrin LFA-1 (lymphocyte function associated
molecule 1, C D lla; clone 3S [a kind gift fi"om Professor Nancy Hogg, Leukocyte
Adhesion Laboratory, Imperial Cancer Research Fund] mouse IgG2a anti-human CDl 1a,
used at a concentration of I S pg/ml), or against the a subunit o f VLA-4 (CD49d, very
late antigen-4; clone MCA697, Serotec, mouse IgGl anti-human CD49d, used at a
concentration of 10 pg/ml). Pre-incubation with isotype control antibodies used at the
same concentrations as the monoclonal antibodies against the a integrins served as
control conditions. Thus the control antibody for anti-LFA-1 was a mouse IgG2a
antibody (clone 4U, a kind gift from Professor Nancy Hogg, used at a concentration of
221
1.8 fxg/ml), and the control for anti-VLA-4 was a mouse IgGl antibody (clone MCA928,
Serotec, used at a concentration of 10 p,g/ml). Following the pre-incubation period, the
blocking or control antibodies were left in the co-culture, and thus the T cells (at an exact
concentration of 10^ /ml) plus blocking antibodies were added in the presence or absence
of the SAgs TSST-1 or SEB. Vp-specific T cell adherence after 4 hours incubation was
assessed as described above, and the effect of the blocking antibodies was assessed in 4
ways: 1. By phase-contrast light microscopy; 2. By counting the absolute number of total
adherent lymphocytes using flow cytometry; 3. By analysis o f the adherent Vp
repertoires expression (expressed as the percentage of the CD4 or CD8 populations); and
4. By analysis of Vp-specific CD69 expression within the adherent population of T cells.
The plate plan for these experiments is given in Appendix 8. These sets o f experiments
were repeated a total of 3 times for LFA-1 blockade: twice for TSST-1, and once for
SEB; and once for VLA-4 blockade (TSST-1 only). As such, no statistics were performed
on the data generated from these experiments.
4.3.7 Statistics
Results from different experimental conditions were compared using the Kruskall Wallis
and Mann-Whitney tests, with significance set at p<0.05. All statistics were performed
using SPSS version 10.0.
222
4.4 Results
4.4.1 SAgs activate T cells in a V^specific manner, and require M H C class II
expressing cells.
To confirm the ability of SAgs to activate T cells in a Vp-specific manner, and the
requirement of MHC class II -expressing cells to act as SAg-presenting cells for T cells,
PBMCs (containing T cells and class H expressing monocytes) or purified T lymphocytes
(greater than 98% CD3 positive, and containing approximately 0.2% MHC class II
positive cells) were incubated with the SAgs TSST-1 (10 ng/ml) or SEB (100 ng/ml) for
4 hours at 37°C. The CD4 and CDS T cell populations were then analysed using 3-colour
FACS analysis for Vp-specific activation as determined by expression of the T cell
activation induction molecule CD69. Incubation o f PBMCs with TSST-1 resulted in
upregulation of CD69 on CD4VP2 (figure 4.1a) and CD8Vp2 T cells (figure 4.1b),
whereas SEB resulted in upregulation of CD69 on CD4Vp3 and V p l2 (figure 4.1a), and
CD8Vp3 and V p l2 T cells, with a lesser (but reproducible) response in CD8Vp5.1
(figure 4.1b). Incubation of purified T cells (devoid of MHC class II -expressing cells)
with SAg for 4 hours, however, did not result in Vp-specific CD4 or CD8 T cell
upregulation of CD69 (figure 4.2a and 4.2b).
223
F i g u r e 4.1 PBMC i n c u b a t i o n w i t h SAg (TSST-1 or SEB) r e s u l t s in v p r e s t r i c t e d CD69
UPREGULATION AT 4 HOURS
Bold arrows indicate major responding Vp families within the CD4 (figure 4.1a) and
CDS (figure 4.1b) populations o f lymphocytes.
224
Figure 4.1: CD69 expression on lymphocytes after incubation of peripheral blood
mononuclear cells (PBMC) with SAg ( 4 hours)
it 400 n
■ control
□ TSST-1 (10 ng/m l)4 hrs
I
§ 250
U S E B (100 ng/ml) 4 hrs
1
2
200
i
150
I 100
O)
(O 50 -
O
Ü
vb1 vb2 vb3 vb5.1 vb8 vb12 vbl vb2 vb3 vb5.1 vb8 vb12
Figure 4.1 A: PBMC incubation with SAg: CD4 T cell CD69 expression Figure 4.18: PBMC incubation witli SAg: CD8 T cell CD69 expression
F ig u re 4.2 P u r i f i e d ly m p h o c y te p o p u la tio n (d e v o id o f c e l l s e x p r e s s in g MHC c la s s II)
INCUBATION WITH SAG AT 4 HOURS
Absence of Vp restricted CD69 upregulation.
226
Figure 4.2: CD69 expression on lymphocytes after incubation of purified T cells (CD3 )
with SAg ( 4 hours)
400
M control 40 0
350 □ TSST-1 (10 ng/ml) 4 hrs

3 50
□ SEB (100 ng/ml) 4 hrs
300
300
^ 250
^ 250
S 200 S 200
O
O
^ 150
^ 150
100
100
50
50
0 Ea_ j ü r t i m
0
vb1 vb2 vb3 vb5.1 vb8 vb12
vb1 vb2 vb3 vb5.1 vb8 vb12
Figure 4.2 A; 4 hour incubation of purified T cells with SAg: Figure 4.2B: 4 hour incubation of purified T cells witli SAg:
CD4 T cell expression of CD69 CD8 T cell expression of CD69
K )
'-J
4.4.2 HUVEC upregulate M H C class II in response to IN F -y and become competent
SAg-presenting cells fo r T cells resulting in Vp-specific T cell activation and adherence
to HUVEC monolayers
Pre-treatment of HUVEC with INF-y (1000 lU/ml) resulted in upregulation of MHC class
n at 48-72 hours (figure 4.3). To further assess the competence o f class Il-expressing
HUVEC to present SAg to T cells, monolayers of HUVEC with and without pre
treatment with INF-y for 48 hours were co-cultured in the presence or absence of purified
T cells for 4 hours with TSST-1 (10 ng/ml) or SEB (100 ng/ml). After the incubation
non-adherent T cells were removed by washing 4 times with PBS with 10% FCS. FACS
analysis of the adherent and non-adherent T cell populations was then performed
specifically examining for CD4 and CD8 vP-specific activation, and VP-specific
adherence to the HUVEC monolayer.
Only HUVEC which had been pre-treated with ENF-y were able to present SAg to T cells
resulting in Vp-specific CD4 and CD8 upregulation of CD69 on the T cells which were
adherent to the HUVEC monolayer: for TSST-1 there was activation o f CD4VP2
(p=0.017) and CD8Vp2 activation (p=0.028) (figures 4.4a and 4.4b); for SEB there was
activation of CD4VPS (p=0.012), CD4VpS.l (p=0.025), CD4Vpi2 (p=0.016), CD8Vp3
(p=0.004), and CD8Vpl2 (p=0.025) (figures 4.4c and 4.4d). Non-adherent T cells (CD4
or CD8) did not demonstrate Vp-specific upregulation of CD69 (p= non-significant for
all CD4 and CD8VP families; figures 4.5a-d).
228
Figure 4.3: Upregulation of MHC class II on HUVEC by INF-y
N o INF-y (c ontrol)
INF-y 4 8 hours
0>
o
o
(D
£
3
z
HUVEC MHC class II Phycoer>1hrin Fluorescence
to
to
F ig u r e 4.4 v p s p e c i f i c CD4 a n d CDS T l y m p h o c y t e a c t iv a t io n w ith in t h e a d h e r e n t p o p u la t io n
OF T CELLS ( m e a n , SEM OF 4 EXPERIMENTS).
Co-culture of MHC class II positive HUVEC with T cells in the presence o f SAgs TSST-
1 (figures 4.4a and 4.4b) or SEB (figures 4.4c and 4.4d) resulted in upregulation of CD69
on CD4VP2^ and CD8Vp2*cells (TSST-1), and CD4Vp3, CD4Vp5.1 and CD4Vpi2,
and CD8VP3 and CD8Vpi2 (SEB). Bold arrows indicate statistical significance
(p<0.05).
230
Figure 4.4: Vp specific CD4 and CD8 T lympfiocyte activation witliin tlie adherent
population of T cells (mean, SEM of 4 experiments)
I ■ C l a s s II n e g H U V E C & T c e l l s
□ C l a s s II p o s H U V E C & T c e l l s
300 - a C l a s s II n e g H U V E C , T c e l l s & T S S T - 1
H C l a s s II p o s H U V E C , T c e l l s & T S S T - 1
O 100
vb3 vb5.1 vb1 vb2 vb3 vb5.1 vb8 vb12

Figure 4.4A: v p specific CD4 activation of adlierent Figure 4.4B: Vp specific CDS activation of adherent
T cell population in response to TSST-1 T cell population in response to TSST-1
B C l a s s II n e g H U V E C & T c e l l s
□ C l a s s II p o s H U V E C & T c e l l s
0 C l a s s II n e g H U V E C , T c e l l s & S E B
a C la s s II p o s H U V E C , T c e l l s & S E B
600
5 00
u_
S 400
05
S 30 0 3 00
Q
O 200
100
vb1 vb2 vb3 vb5.1 vb8 vb12 vb1 vb2 vb3 vb5.1 vb8 vb12
Figiu-e 4.4C; Vp specific CD4 activation of adherent Figure 4.4D: Vp specific CDS activation of adlierent
T cell population in response to SEB T cell population in response to SEB
F ig u r e 4.5 Vp CD4 a n d CDS T l y m p h o c y t e a c t i v a t i o n
sp e c if ic w it h in t h e n o n -a d h e r e n t
POPULATION OF T CELLS (MEAN, SEM OF 4 EXPERIMENTS).
Co-culture of MHC class II positive HUVEC with T cells in the presence of SAgs TSST-
1 (figures 4.5a and 4.5b) or SEB (figures 4.5c and 4.5d) did not result in Vp specific
activation of T cells (CD4 or CDS) within the non-adherent population (p=non-
significant for all Vp families).
232
Figure 4.5:Vp specific CD4 and CDS T lymphocyte activation within the non-adherent
population of T cells (mean, SEIM of 4 experiments)
■ C la s s II n eg HUVEC & T c e lls
500 □ C la s s II p o s HUVEC & T ce lls
n C la s s II n eg HUVEC, T c e lls & TSST -1 500
400 n C la s s II p o s HUVEC, T c e lls & T SST -1
LL _ 400
u_
s 300
o> ^ 300
<r> o>
Q 200 Q 200
O
O
100 100
0 0
vb1 vb2 vb3 vb5.1 vb8 vb12 vb1 vb2 vb3 vb5.1 vb8 vb12
Figure 4.5A: Vp specific CD4 activation of non-adherent Figure 4.5B: Vp specific CD8 activation of non-adherent
T ceil population in response to TSST-1 T cell population in response to TSST-1
■ C la s s II n eg HUVEC & T ce lls

□ C la s s II p o s HUVEC & T c e lls
B C la s s II n eg HUVEC, T c e lls & SE B
0 C la s s II p o s HUVEC, T c e lls & SE B
500 500
_ 400 - 400
u_
S 300 ^ 300
o> O)
g 200 Q 200
Ü T
^ 100 100
0
w
w
U) vb1 vb2 vb3 vb5.1 vb8 vb12 vb1 vb2 vb3 vb5.1 vb8 vb12
Figure 4.5C; Vp specific CD4 activation of non-adlierent Figure 4.5D: Vp specific CD8 activation of non-adherent
T cell population in response to SEB T cell population in response to SEB
Moreover, T cell Vp-specifîc adherence to class II expressing HUVEC was observed
(again Vp2 for TSST-1, and Vp3 and V p l2 for SEB), and although this was true for both
CD4 and CDS T cells, Vp-specific adherence was greater for CD4 T cells than CDS T
cells (figures 4.6a-d). In those experimental conditions where there was Vp-specific
adherence to the HUVEC monolayer, there was a concomitant decrease in the
corresponding Vp family in the non-adherent population of CD4 and CDS T cells. These
data are summarised as “Vp ratios”, i.e. the ratio o f the percentage o f adherent to non
adherent v p families for each experimental condition. Thus, if there was no specific Vp
adherence to the HUVEC monolayer, the Vp ratio was close to unity; if there was
increased adherence to the HUVEC monolayer (and conversely a decrease in the
percentage of that VP family within the non-adherent population) the Vp ratio was
greater than 1. In response to co-culture of T cells with MHC class HUVEC in the
presence of TSST-1, the mean Vp ratio was increased for CD4VP2, and CD8Vp2 and
CD8Vp5.1 (figures 4.7a and 4.7b). In response to co-culture of T cells with MHC class
HUVEC in the presence of SEB the mean Vp ratio was increased for VpS and V pi2
(true for both CD4 and CD8 lymphocytes; Figures 4.7c and 4.7d).
234
Figure 4.6: VP-specific T cell adherence to HUVEC

Q C la s s II p o s HUVEC & T c e lls gINF
□ C la s s II n eg HUVEC. T c e lls & TSST-1 30
30 H C la s s II p o s HUVEC, T c e lls & T SST -1
25
25
20
20
15 15
10 10
5 5
0 0
Vbl Vb2 Vb3 Vb5.1 Vb8 Vb12 Vb1 Vb2 Vb3 VbS.I Vb8 Vbl 2
vb
vb
Figure 4.6A; TSST-1 induced Vp specific CD4 adherence to HUVEC monolayer Figure 4.6B; TSST-1 induced Vp specific CDS adherence to HUVEC monolayer

□ C la s s II p o s HUVEC & T c e lls gINF
□ C la s s II n eg HUVEC, T ce lls & SE B
□ C la s s II p o s HUVEC, T c e lls & S E B
14
12
10
8
6
4
2
0
Vbl Vb2 Vb3 VbS.I Vb8 Vbl 2
Vbl Vb2 Vb3 Vb5.1 Vb8 Vbl 2
Figure 4.6C: SEB induced Vp specific CD4 adherence to HUVEC monolayer Figure 4.6D: SEB induced Vp specific CDS adherence to HUVEC monolayer
F ig ure 4.7: CD4 a n d CDS V|3 ratios in response to TSST-1 (figures 4.7 a and 4.7 b ) and SEB
(fig u res 4.7 c and 4.7 d ) (m ea n , SEM of 3 experim en ts ).
The v p ratio was calculated by calculating the ratio of the percentage of adherent to non
adherent Vp families for each experimental condition. Thus, if there was no specific VP
adherence to the HUVEC monolayer, the VP ratio was close to unity; if there was
increased adherence to the HUVEC monolayer the Vp ratio was greater than 1. Bold
arrows indicate statistical significance (all p= 0.01). The broken arrow indicates statistical
significance (p= 0.01) but a lesser-responding Vp family.
236
Figure 4.7: CD4 and CDS Vp ratios in response to TSST-1 (figures 4.7a and 4.7b) and
SEB (figures 4.7c and 4.7d)

□ C la s s II p o s HUVEC & T c e lls
□ C la s s II n eg HUVEC. T c e lls & TSST-1
□ C la s s II p o s HUVEC. T c e lls & TSST-1
6 I 10
5
8
!
4 I
3 0 6
2 1 4
1 > 2
0 + 0 B -d S j
Vb1 Vb2 Vb3 Vb5.1 Vb8 Vb12 Vb1 Vb2 Vb3 Vb5.1 Vb8 Vb12
Figure 4.7A: CD4 V(3 ratio in response to TSST-1 Figure 4.7B; CD8Vp ratio in response to TSST-1
■ C la s s II n eg HUVEC & T c e lls

□ C la s s II p o s HUVEC & T ce lls
□ C la s s II n eg HUVEC. T ce lls & SEB
8 □ C la s s II p o s HUVEC. T c e lls & SE B 5
6 4
3
4 o
2 ? 2
n 2
> g 1
0 0
to
O Vb1 Vb2 Vb3 Vb5.1 Vb8 Vb12 Vb1 Vb2 Vb3 Vb5.1 Vb8 Vb12
-JJ
Figure 4.7C; CD4 VP ratio in response to SEB Figiue 4.7D; CD8 v p ratio in response to SEB
4.4.3 SAg-dependent T cell-mediated endothelial cell activation: upregulation o f cell
adhesion molecules
To assess the effect on the endothelial cell of Vp specific T cell activation and adherence
to the HUVEC monolayer, HUVEC cell adhesion molecule expression was determined in
the different T cell co-culture experimental conditions described above using flow
cytometry. Incubation of HUVEC with TNF-a at 100 ng/ml served as a positive control
in these experiments. At 4 hours there was upregulation o f E-selectin (4.4-fold), ICAM-1
(1.2-fold), and VCAM-1 (3.3-fold) on HUVEC which had been pre-incubated with JNF-y
and co-cultured with SAg (TSST-1 or SEB) and T lymphocytes which was o f a
comparable or greater magnitude to that observed in response to incubation with
1OOng/ml of TNF-a (figure 4.8- data for TSST-1 shown). No upregulation of P-selectin
on HUVEC was observed at 4 hours in any experimental condition (data not shown). No
upregulation of E-selectin, ICAM-1 or VCAM-1 was observed on HUVEC that had not
been pre-incubated with INF-y. There was a small increase in VCAM-1 expression on
HUVEC pre-treated with INF-y and incubated with T cells alone (1.6-fold), but this was
less than the VCAM-1 upregulation that was observed in the presence o f T cells and SAg
(either TSST-1 or SEB; data for TSST-1 shown only, figure 4.8).
238
F ig u r e 4 .8 F l o w c y t o m e t r y d o t -p l o t s o f c e l l a d h e s io n m o l e c u l e e x p r e s s io n o n HUVEC co-
cultured WITH PURIFIED C D S '" T CELLS IN THE PRESENCE OR ABSENCE OF TSST-1.
Up-regulation of E-selectin, VCAM-1, and to a lesser extent ICAM-1 (expressed as
percentage “high expression” and median fluorescence index [MFI]) only occurred on
MHC class HUVEC in the presence of TSST-1 (10 ng/ml) and T cells: thus the cell
adhesion molecule expression was TSST-1 dependent and T cell-mediated. Incubation
with TNF-a (100 ng/ml) served as a positive control.
239
Figure 4.8: TSST-1 dependent T cell-mediated endothelial cell activation
E-selectin ICAM-1 VCAM-1
14% 16%
M FI 1.4 M FI 10.0 M FI 3.0
H U V E C (MHC class II-) g
10' "10' 10' 10' 10' 10' "lO' 10' 10'

I
13% 35% 18%
M FI 2.1 M FI 9.0 M FI 3.6
H U V E C (MHC class II-) g

+ T cells
)' 10' 10^ 10’ 10 10' ID' to ’ 10*
18% 37% 27%

M FI 2.7 M FI 14.0 M FI 8.2
H U V E C (MHC class 11+)
g
+ T cells
"io* no" lb' 10'
12% 36% 18%

M FI 1.9 M FI 9.3 M FI 3.6
H U V E C (MHC class II )
g
+ T cells !
+ TSST-1
62% 47% 53%

M FI 22.5 M FI 17 M FI 19.0
H U V E C (MHC class 11+)
+ T cells !
+ TSST-1
40% . 48% 3 0%
M FI 12 M FI 17 M FI 9.0
H U V E C (MHC class II-) Ï;

+TNF-a 100 ng/ml I
0 1 1
240
4.4.4 Integrin blockade o f SAg-mediated T cell adherence to HUVEC
Pre-incubation of T cells with anti-LFA-1 resulted in a decrease in the absolute number
of T cells adherent to the MHC class HUVEC monolayer (true for both TSST-1 and
SEB), but an increase in the percentage of adherent Vp2 for TSST-1, or Vp3 and V pi2
for SEB. This was true o f both the CD4 and CDS lymphocyte populations and consistent
over all three experiments (twice for TSST-1 and once for SEB), and suggested that the
integrin LFA-1 (the ligand for ICAM-1) blocked predominantly T cell adhesion caused
by HLA mismatch between the T cells and HUVEC, but not the true SAg-mediated
adhesion. Figure 4.9 illustrates these results from a single representative experiment
using phase-contrast light microscopy (data for TSST-1 shown). Figure 4.10
241
Legend for figure 4.9: Phase-contrast light microscopy (medium power) demonstrating
adherent T cells on a background o f HUVEC. The HUVEC appear spindle-shaped
because they have been incubated with INF-y for 48 hours to upregulate MHC class II.
Blockade o f LFA-1 or VLA-4 by pre-incubation o f T cells with monoclonal antibodies
against these integrins resulted in decreased T cell adhesion (figure 4.98 and 4.9D), but
in the case o f LFA-1 blockade the percentage o f T cells expressing Vp2 (for TSST-1) or
Vp3 and V p l 2 (for SEB) increased. This result suggested that LFA-1 blocked non
specific T cell adherence caused by HLA-mismatch and did not affect SAg-mediated T
cell adherence.
242
Figure 4.9: Integrin blockade of TSST-1 mediated T cell adherence to MHC ClassII^
HUVEC (order of pictures corresponds to plate plan given in Appendix 8)
4.9A: MHC class 11+ HUVEC, T cells, TSST-1 (lOng/ml)

4.9B: MHC class 11+ HUVEC, T cells. TSST-1 (10
ng/ml), anti-LFA-1 (1.8 mcg/ml)
4.9C: MHC class 11+ HUVEC, T cells, TSST-1 (10
ng/ml), control antibody for anti-LFA-1 (1.8 mcg/ml)
4.9D: MHC class 11+ HUVEC, T cells, TSST-1 (10
ng/ml), anti-VLA-4 (10 mcg/ml)
4.9E: MHC class 11+ HUVEC, T cells, TSST-1 (10
ng/ml), control antibody for anti-VLA-4 (10 mcg/ml)
K)
Figure 4.10: Absolute number of adherent T eells in response to 4 hour eo-culture of
MHC elass 11+ HUVEC with SAg (TSST-1 or SEB): effect of LFA-1 blockade.
Figure 4.1 OA: Absolute number of adherent T cells:

TSST-1
c
2 300000 n
£
0)
■O 250000 - ■ TSST-1: 4 hours
<0
*0 (/) 200000
0)
.o 150000 -
□ TSST-1 & LFA-1
8 blockade: 4hours
E h-
3 100000 -
C a TSST-1 & control
50000 - antibody: 4 hours
.a 0 -
<
Figure 4.10B: Absolute num ber of a d h eren t T cells:

SEB
■£ 400000
0)
0) 350000 -
T3
(Q 300000 ■ S E B : 4 hours
0 250000
i2
Ï a> 200000 -
□ SEB & LFA-1 blockade:
4hours
E Ü
3 150000
C □ SEB & control antibody:
«
NJ 100000 - 4 hours
4^ Ô
U) 50000
< 0 -
demonstrates the absolute adherent T cell counts using flow cytometry and confirms the
visual representation o f these data given in 4.9 (data for TSST-1 and SEB given, single
representative experiments each). Figure 4 .1 1 illustrates the Vp repertoire o f the adherent
T cell population (from a single representative experiment). In addition, the T cells which
were adherent displayed Vp-specific activation as determined by CD69 expression (Vp2
for TSST-1; Vp 3, Vpi 2, and to a lesser extent V p5.l for SEB). Again this was true for
both CD4 and CDS lymphocyte populations. These data are summarised in figure 4.12.
In contrast, pre-incubation o f T cells with anti-VLA-4 resulted in a decrease in the
absolute number o f adherent T cells (CD4 and CDS) in response to TSST-1 (figure 4.13),
with no concomitant increase in the percentage o f adherent Vp2 T cells (figure 4.14). In
addition, analysis o f the Vp-specific activation profile following VLA-4 blockade
revealed a decrease in adherent T cell Vp2 CD69 expression (figure 4.15). Taken
together, these two observations suggested that blockade o f VLA-4 resulted in a decrease
o f both non-specific T cell adherence (occurring as a result o f HLA-mismatch) and
TSST-1 mediated T cell adhesion.
245
Figure 4.11: Vp repertoire of adherent T cells in response to 4 hour co-culture of MHC
class 11+ HUVEC with SAg (TSST-1 or SEB): effect of LFA-1 blockade.
Figure 4.11 A; CD4 V-beta repertoire of adherent T Figure 4.1 IB: CD8 V-beta repertoire of adherent T
cells cells
40 15
30 TSST-1 I TSST-1
10
20 □ TSST-1 and anti-LFA1-1 □ TSST-1 and anti-LFA-1
□ TSST-1 and control Ab 5 @ TSST-1 and control Ab
10
0 0
1 2 3 5.1 8 12 2 3 5.1 8 12
V-beta V-beta
Figure 4.11C: CD4 V-beta repertoire of adherent T cell Figure 4.1 ID: CD8 V-beta repertoire of adherent I cell
population population
20 15
15 SEB ■ SEB
10
10 □ SEB and anti-LFA-1 □ SEB and anti-LFA-1
0 SEB and control Ab 5 □ SEB and control Ab
5
0 0
1 2 3 5.1 8 12 1 2 3 5 1 8 12
V-beta V-beta
On
Figure 4.12: Vp-specific CD69 expression of adherent T eells in response to 4 hour eo-
euiture of MHC elass 11+ HUVEC with SAg (TSST-lor SEB): effeet of LFA-1
blockade.
Figure 4.12A: Adherent CD4 CD69 MFI: TSST-1 Figure 4.12B: Adherent CD8 CD69 MFI: TSST-1
500 400
■ TSST-1 & T cells ■ TSST-1 & T cells
400 300
300 D TSST-1 & T cells & LFA- □ TSST-1 & T cells & LFA-
200
200 1 block 1 block
E3 TSST-1 & T cells & 100 □ TSST-1 & T cells &
100
control Ab control Ab
0 0
3 5.1 8 12
V-beta V-beta
Figure 4.12C: Adherent CD4 CD69 MFI: SEB Figure 4.12D: Adherent CD8 CD69 MFI: SEB
600
500 ■ SEB & T cells ■ SE B & T cells
400
I 300
O S E S & T cells & LFA-1 Ü: 400 □ SE B & T cells & LFA-1
block block
200
□ SEB & T cells & control mSE B & T cells & control
100 Ab Ab
1 2 3 5.1 8 12
V-beta v-beta
Figure 4.13: Absolute number of adherent T cells in response to 4 hour co-culture of
MHC class 11+ HUVEC with TSST-1: effect of VLA-4 blockade.
= 120000
S
100000
2
■ TSST-1: 4 hours
^ 80000
"O
(0
60000 □ TSST-1 & VLA-4

o blockade: 4hours
JQ
E 40000 m TSST-1 & control
3 antibody: 4 hours
C
a 20000
3
8
0
<
00
Figure 4.14: Vp repertoire of adherent T eells in response to 4 hour eo-eulture of MHC
elass 11+ HUVEC with TSST-1: effeet of VLA-4 blockade.
Figure 4.14A: CD4 V-beta repertoire of adherent T

cells
25
20
■ TSST-1
15
□ TSST-1 an d anti VLAr4
10
E3 TSST-1 an d control Ab
5
0
1 2 3 5.1 12
V -beta
Figure 4.14B: CD8 V-beta repertoire of adherent T

cells
12
10
8 I TSST-1
6 □ TSST-1 and anti VLA-4
4 □ TSST-1 and control Ab
2
0
1 2 3 5.1 8 12
V -beta
'O
Figure 4.15: Vp-specific CD69 expression of adherent T eells in response to 4 hour eo
culture of MHC class 11+ HUVEC with TSST-1: effect of VLA-4 blockade.
Figure 4.15A: Adherent CD4 CD69 MPI: TSST-1
250
iZ 200
■ TSST-1
S 150 4
o □ TSST-1 and anti VLA-4
u 100 4
m TSST-1 and control Ab
O 50
3 5.1 12
V -b eta
Figure 4.15B: CDS CD69 MFI: TSST-1
250
E 200
■ TSST-1
S 150
□ TSST-1 and anti VLA-4
u 100
E3 TSST-1 and control Ab
O 50
K
U)
Ol
V -b eta
4.5 Discussion
The prototypic SAg-mediated disease is the toxic shock syndrome (TSS), a condition first
described in 1978 associated with infection with Staphylococcus aureus (Todd, Fishaut
and others, 1978). This entity is characterised by acute onset o f fever, shock, and multi
organ dysfimction- the consequence of massive T cell activation and cytokine production
in response to SAgs. Histological examination of tissue fi'om patients with this disease
typically reveals perivascular lymphocytic infiltration (Larkin, Williams and others,
1982; Paris, Herwaldt and others, 1982). More recently, there has been increased interest
in SAgs as modifiers of several autoimmune disease states, including the systemic
vasculitides. The evidence for this presently remains circumstantial. However, the best
evidence has been provided by the observation of peripheral blood and lesional tissue T
cell VP skewing in KD (Abe, Kotzin and others, 1992; Curtis, Zheng and others, 1995;
Leung, Giomo and others, 1995; Leung, Meissner C, and Schlievert, 1997; Yamashiro Y,
Nagata S and others, 1996), and peripheral blood T cell Vp skewing in microscopic
polyangiitis (Giscombe, Grünewald and others, 1995; Simpson, Skinner and others,
1995).
The results of the present study outlined in this chapter provide one possible mechanism
whereby SAgs may initiate and maintain vascular injury, namely by utilising the
endothelial cell as a SAg- presenting cell for T lymphocytes. This mechanism is attractive
because it would result in large numbers of T cells becoming activated directly on the
251
surface of the endothelium, and could explain the observation of upregulated cell
adhesion molecules on endothelial cells observed in most vasculitis syndromes, and the
persistence of the skewed Vp T cell infiltrate in the perivascular compartment in lesional
tissue from KD patients.
Perhaps under-emphasised in the literature is the role that cytotoxic CDS T lymphocytes
may play in the immune response to SAgs. A common misconception is that CDS
lymphocytes do not respond to SAgs since they classically recognise conventional
antigens presented on the surface of cells in association with MHC class I molecules
(Janeway CA, Travers P and others, 1999; Mingari, Cambiaggi and others, 1996). This
study, in accordance with others (Herrmann, Baschieri and others, 1992) and in keeping
with the results presented in Chapter 3, has clearly demonstrated that CDS lymphocytes
do become activated in a Vp-restricted manner (and also will proliferate in a Vp-
restricted manner- Chapter 3) in response to SAgs, and that this is dependent upon the
presence of S Ag-presenting cells (monocytes or endothelial cells) expressing MHC class
n. This result is worthy of emphasis since some workers have suggested that the presence
of activated cytotoxic CDS lymphocytes, which have been observed in lesional tissue in
patients with KD, is evidence of a conventional antigenic-driven process and precludes a
superantigenic-driven process (Brown, Crawford and others, 2001). Injection o f SEB into
mice leads to a strong and transient activation o f both CD4 and CDS lymphocytes
(Herrmann, Baschieri and others, 1992). These CDS lymphocytes are highly cytotoxic
towards MHC class n expressing cells between 2 and 3 days after injection o f SEB ex
252
vivo. It is thus now clear that CDS lymphocytes also play an important role in the
immune response to SAgs, and may be responsible in part for the vascular injury
associated with SAg-mediated diseases.
The data presented in this chapter also demonstrate that the endothelial cell can operate as
a competent SAg-presenting cell following upregulation of MHC class II by INF-y.
Moreover, the consequence of this interaction is CD4 and CDS T cell Vp-restricted
activation and adherence to the HUVEC monolayer, with subsequent upregulation of
HUVEC cell adhesion molecules, a situation which could conceivably result in a state of
chronic endothelial cell activation and ultimately vascular injury. Observations in vivo
which support such a mechanism include the presence o f increased numbers of Vp2
lymphocytes in lesional tissue in KD (Leung, Giomo and others, 1995; Yamashiro Y,
Nagata S and others, 1996), and upregulation of MHC class II on endothelial cells in the
lesional tissue of many chronic inflammatory syndromes (Barkley, Allard and others,
1989; Koretz, Momburg and others, 1987), including KD (Naoe S, personal
communication).
The results o f this study which demonstrate Vp-restricted T cell adherence to HUVEC
are in accordance with a previous in vitro study which demonstrated a preferential
adhesion of murine V p8.1,2 splenic T cells to a human endothelial cell line (Kita, Eguchi
and others, 1996). In that study, the response was mainly within the CD4 population o f T
cells. This process was dependent on the expression of MHC class II (DR and DQ) on the
253
endothelial cells, and the authors were able to inhibit the enhanced Vp-restricted
adhesion with monoclonal antibodies directed against human ICAM-1, but not with
antibodies directed against human VCAM-1. This result was in contrast to the findings of
Siegelman and others who demonstrated that the binding o f CD44 on activated
lymphocytes to endothelial hyaluronan (HA) mediates primary adhesion under sheer-
stress, followed by further firm adhesion involving predominantly the integrin VLA-4 (on
T cells activated with SEB) and VCAM-1 interaction, and with only a minor contribution
from the integrin LFA-1 (Siegelman, Stanescu, and Estess, 2000).
Because of time constraints only a limited number o f experiments examining the
blockade of SAg-mediated T cell adhesion were performed in this study. Nonetheless,
each single experiment inherently had several built-in controlling conditions since several
non-SAg responding Vp families were examined in each experiment. Thus, although
these data would constitute preliminary results, they are worthy o f some description and
comment. Using monoclonal antibodies against the a integrins LFA-1 (the ligand for
ICAM-1, ICAM-2, and ICAM-3), and against the a integrin VLA-4 (the ligand for
VCAM-1 and fibronectin) it was possible to block some o f the TSST-1-mediated
adhesion of T cells to MHC class iC HUVEC as determined by a decrease in the absolute
number of T cells adherent, with little change in the percentage o f adherent Vp2 CD4 or
CDS lymphocytes. Moreover, of those Vp2 T cells that were adherent, there was a
decrease in the CD69 expression of these cells following blockade o f VLA-4. That there
was no decrease in the percentage of Vp2 adherent T cells does imply, however, that
254
blockade of VLA-4 did not completely abrogate the TSST-1-mediated T cell adhesion
and points to other mechanisms mediating T cell adhesion caused by SAgs. This result
was in contrast to the effect of blockade of LFA-1 which resulted in a decreased absolute
number of adherent T cells, but a definite rise in the percentage of SAg-specific Vp
adhesion- a result that was observed in all 3 experiments performed examining this
situation (twice for TSST-1, and once for SEB). Thus in summary, these observations
would suggest that the integrin VLA-4 (but not LFA-1) on activated CD4 and CDS T
cells interacts with VCAM-1 (or fibronectin) on activated HUVEC and contributes in part
to the T cell adhesion mediated by SAgs in this non-flow model.
The data derived from these attempts at integrin blockade o f T cell adhesion also
emphasise an important limitation of the model described in this study, namely that the T
cells and HUVEC co-cultured were not HLA-matched. Thus any results obtained are
occurring on the background of a mixed lymphocyte-endothelial cell reaction. That said,
all the experiments described in this chapter were designed specifically to control for this
important confounding factor. Thus, at 4 hours, the data were relatively “clean” and in
keeping with the finding of others (Baum, Yaron, and Yellin, 1998). At 24 hours,
however, there was evidence of HUVEC activation and injury that may not have been
entirely caused by SAg-dependent T cell mediated mechanisms, and it is likely that some
of the “noise” in the model was the result of the mixed T cell-endothelial reaction.
255
The data presented here are also in keeping with previous work demonstrating that INF-y
upregulates MHC class H on HUVEC thus inferring Staphylococcal enterotoxin A
(SEA)-binding activity on the HUVEC for activation o f human T cells (Uchiyama,
Araake and others, 1992). In that study, antibodies against MHC class II blocked the
increased SEA-binding activity on HUVEC in response to INF-y.
Thus it is now apparent that the interaction between T cells (CD4 and CDS) and
endothelial cells in the presence of SAg results in adherence o f T cells to the endothelium
via complex interactions involving more than one mechanism. So far, some o f the
previously published experimental data are conflicting although a thorough
understanding of the mechanisms of T cell adherence and activation following SAg
stimulation may add to the armamentarium of molecular blockade o f potential injurious
pathways culminating in vascular injury in SAg-mediated disease states. For example, it
remains to be seen what contribution the newly-discovered SAg-peptide antagonists
(Arad G, Levy R and others, 2000) may have in the blockade of S Ag-dependent T cell
mediated endothelial activation and endothelial adherence.
In conclusion, based on the results presented in this chapter and accepting important
limitations to the in vitro model described, it is proposed that MHC class Il-expressing
endothelial cells operate as competent SAg-presenting cells for CD4 and CDS
lymphocytes. This results in increased T cell adherence to endothelial monolayers in vitro
256
mediated in part by VLA-4A^CAM-1 interactions, and Vp-restricted CD4 and CDS T cell
activation within the adherent population of lymphocytes. The consequence of this
interaction is upregulation of endothelial cell adhesion molecules, although, care must be
taken not to overly extrapolate the grossly unphysiological environment o f the in vitro
model to the in vivo situation.
Whilst this observation is potentially important, the finding o f endothelial cell activation
may not correlate with endothelial injury, per se. To further investigate this matter
further. Chapter 5 considers in further detail the consequence to the endothelial cell
following the interaction between SAgs and T cells, namely the generation of a recently
described surrogate marker of endothelial injury- endothelial microparticles (EMP).
257
Chapter 5: Endothelial and platelet microparticles in
childhood vasculitides: a window to the activated endothelium
in vasculitis of the young.
5.1 Summary
5.2 Introduction and background on EMP
5.3 Methods
5.4 Results
5.5 Discussion
258
5.1 Summary
Introduction and aims: Microparticles (MP) are released from endothelial cells in
response to a variety of injurious stimuli and recently have been shown to be increased in
a number of diseases associated with endothelial dysfunction. In chapter 4 it was
established that HUVEC activation occurred in response to SAg-T cell interactions. In
this chapter, it is demonstrated that endothelial microparticles (EMP) were released from
HUVEC into the supernatant as a consequence o f SAg-dependent T cell mediated
endothelial cell activation. This observation led to the in vivo studies presented in this
chapter, which examined EMP and platelet MP (PMP) profiles in children with systemic
vasculitis (SV) to test the hypothesis that EMP may provide a tool for the diagnosis and
monitoring o f disease activity, and could provide insight into the endothelial injury
associated with childhood vasculitis syndromes.
Methods: Supernatants from the HUVEC-T cell co-culture experiments described in
section 4.3.4 were analysed for EMP. Patients studied were 39 children with SV at
various stages of disease activity, 24 febrile disease control children, and a control group
of 43 healthy subjects. Supernatants from in vitro experiments, or plasma from subjects
was ultracentrifuged at 17000G for 60 minutes, and the MP pellet examined using flow
cytometry.
Results: In addition to HUVEC activation in response to co-culture with T cells and SAg
at 4 hours, there was increased EMP release from the activated HUVEC which occurred
earlier and was of a greater magnitude to that observed in response to TNF-a. Plasma
from patients with active SV contained a 8.5-fold elevation of E-selectin positive EMP
compared with patients in remission (p=0.000), a 5.6 fold elevation compared with
259
controls (p=0.000), and a 10.4-foid elevation compared with disease controls (p=0.000).
A similar result was obtained for EMP expressing the marker CD 105 (endoglin). EMP
correlated with the Birmingham Vasculitis Activity Score and acute phase reactants in the
patients with SV, but there was overall a poor correlation between EMP and acute phase
reactants in the disease controls. The test characteristics of EMP for the diagnosis of
active vasculitis were superior to previously published data regarding visceral
angiography in childhood PAN.
Conclusion: EMP may provide a "window" to the activated endothelium, and these data
suggest that they may be useful diagnostically and for the monitoring of disease activity
in SV of childhood.
260
5.2 Introduction
In Chapter 4 it was established that MHC class 11^ HUVEC could operate as competent
SAg-presenting cells resulting in T cell activation and adherence to HUVEC monolayers.
HUVEC, in turn, become activated and upregulate cell adhesion molecules. Whether this
endothelial activation correlates with injury, however, is unclear.
The studies outlined in this chapter examined in more detail the consequence to the
endothelial cell of SAg-dependent T cell mediated endothelial cell activation by
examining a novel surrogate marker of endothelial injury; the generation of endothelial
microparticles (EMP). The in vitro experiments and observations described in this
chapter led to the in vivo study of EMP in children with vasculitis subsequently
described. Some further background information on the biology of cellular and platelet
microparticle formation is first required.
5.2,1 Cellular membrane phospholipid asymmetry: pathophysiological implications
Membrane phospholipid asymmetry is a feature o f all eukaryotic cells, and is tightly
regulated by several energy dependent enzymatic processes (Zwaal and Schroit, 1997).
The outer leaflet of eukaryotic cell membranes is formed predominantly by the
cholinephospholipids (sphingomyelin [SM] and phosphatidylcholine [PC]), whereas the
inner leaflet is largely comprised of the aminophospholipids (phosphatidylserine [PS] and
phosphatidylethanolamine [PE]) (Zwaal and Schroit, 1997). It is now apparent that cells
invest energy to maintain this phospholipid asymmetry, and the process is controlled by 3
enzymes- aminophospholipid translocase, floppase, and lipid scramblase. The first 2
261
enzymes are ATP-dependent, and the last enzyme is calcium dependent, but ATP
independent (Zwaal and Schroit, 1997).
A general feature of all activated cells and cells undergoing apoptosis is a loss of normal
cell membrane phospholipid asymmetry resulting in an increase in phosphatidylserine
(PS) on the outer leaflet of the bi-lipid membrane layer (Martin, Reutelingsperger and
others, 1995). Surface exposure of PS has important physiological and
pathophysiological implications. For example, surface exposure of PS in platelet
membranes ensures proficient propagation and control of the haemostatic process since
PS on aggregated platelets can both restrict and promote thrombin formation at the site of
injury by providing a catalytic membrane surface for procoagulant (prothrombinase and
tenase) and anticoagulant (protein C) reactions (Zwaal and Schroit, 1997). The
pathophysiological significance of this phenomenon is illustrated by the genetic disorder
Scott’s syndrome- a bleeding disorder resulting fi'om the loss of function of the
scramblase enzyme, which normally serves to rapidly move PS fi'om the inner membrane
leaflet to the outer leaflet in response to injury (Toti, Satta and others, 1996).
Loss of cellular phospholipid asymmetry is often accompanied by blebbing and shedding
of lipid-symmetric microvesicles from the cell surface (Comfurius, Senden and others,
1990; Sims, Wiedmer and others, 1989; Wiedmer, Shattil and others, 1990). It has been
suggested that this microvesicle shedding is important to the cell to remove areas of
localised symmetrical cell membrane phospholipid, thus allowing the cell to return to a
resting (non-prothrombotic) state (Zwaal and Schroit, 1997). The process o f blebbing
262
appears to be mediated by calcium-dependent calpain activation (Wiedmer, Shattil and
others, 1990). Several agonists can induce platelet membrane asymmetry and
microparticle formation: Calcium-ionophore is the most effective followed by
complement membrane attack complex (C5b-9), collagen, and thrombin (Zwaal and
Schroit, 1997).
The physiological significance of platelet microvesicles (hereafter referred to as
microparticles [MP]) is unclear. Increased levels o f circulating PMP have been
demonstrated in patients with thrombotic disorders such as transient ischaemic attacks
and myocardial infarction (Jy, Horstman and others, 1995; Lee, Jy and others, 1993).
Since they are rich in surface exposed PS, it has been suggested that PMP may propagate
prothrombotic processes. PMP can also bind to and activate neutrophils, providing an
interesting link between haemostasis and inflammation (Jy, Mao and others, 1995).
5.2,2 Endothelial microparticles (EMP)
Whilst PMP have been extensively studied, until recently there has been very little
interest in microparticles of endothelial cell origin (EMP). EMP were first described by
Hamilton et al in 1990 (Hamilton, Hattori and others, 1990). In that study, the authors
demonstrated that assembly of the complement membrane attack complex (C5b-9) on
HUVEC resulted in the release of membrane-derived microparticles- particles less than 1
micron in diameter and expressing binding sites for activated factor V (factor Va). These
microparticles were not merely the result of cellular lysis in response to the complement
membrane attack complex. Subsequently, EMP release fi'om HUVEC has been
263
demonstrated to occur in vitro in response to TNF-a (Combes, Simon and others, 1999),
and in response to incubation of endothelial cells with serum o f patients with the anti
phospholipid syndrome (Combes, Simon and others, 1999), thrombotic
thrombocytopenic purpura (TTP) (Jimenez, Jy and others, 2001), and multiple sclerosis
(MS) (Minagar, Jy and others, 2001).
It is now becoming apparent that EMP may provide a window to the activated
endothelium in a number of disease states where endothelial injury is central to the
disease process including atherosclerosis (Mallat, Hugel and others, 1999), acute
coronary syndromes (Mallat, Benamer and others, 2000), antiphospholipid syndrome
(Combes, Simon and others, 1999), TTP (Jimenez, Jy and others, 2001), and MS
(Minagar, Jy and others, 2001). The functional significance of endothelial cell and
platelet PS externalisation and microparticle formation in these disease states has not
been fully elucidated, but one important pathophysiological consequence may be a
prothrombotic tendency mediated by activation of the extrinsic coagulation pathway (i.e.
the tissue factor/factor VU-dependent pathway) (Berckmans, Neiuwland and others,
2001; Combes, Simon and others, 1999; Joop, Berckmans and others, 2001; Mallat,
Hugel and others, 1999; Mallat, Benamer and others, 2000; Nieuwland, Berckmans and
others, 2000). Recently, it has been suggested that EMP may be useful diagnostically for
the detection of relapses of MS (Minagar, Jy and others, 2001), although others have
suggested that EMP may increase in a number o f autoimmune states other than MS
(Larkin, 2001).
264
5.2.3 EMP: relevance to the vasculitides
Diagnosis and monitoring of disease activity of the systemic vasculitides is dependent on
combinations of invasive tests such as tissue biopsy and visceral angiography, and
clinical criteria with less than optimal sensitivity and specificity (Albert, Silverstein and
others, 1988; Albert, Rimon, and Silverstein, 1988; Albert and Ekoe, 1983; Brogan,
Davies and others, 2002; Brogan, Bose and others, 2002). Although serial measurement
of anti-neutrophil cytoplasmic antibodies (ANCA) may play an important role in the
diagnosis and monitoring of disease activity in adults with vasculitis (Hagen, Daha and
others, 1998; Savage, 2001), so far there have been conflicting reports o f their usefulness
in this context (Kerr, Fleisher and others, 1993). Moreover, ANCA are present only in the
minority of young patients with systemic vasculitis and are therefore poorly sensitive as a
diagnostic marker for the majority of the vasculitides of childhood (Brogan and Dillon,
2000a; Brogan and Dillon, 2000b).
5.2.4 Study aims
1. To investigate the potential injurious response o f the endothelial cell to SAg-
dependent T cell mediated activation as determined by in vitro generation of
endothelial microparticles.
2. Based on these in vitro observations, to examine the hypothesis that circulating
EMP may be increased during active vasculitis, providing a non-invasive tool for
assessing endothelial injury.
265
5.3 Methods
5.3.1 In vitro studies: analysis o f endothelial microparticles in HUVEC-T cell co
culture supernatants
Supernatants from the experiments described in section 4.3.4 (with plate plan in
Appendix 7) were defrosted in a water bath at 37 ° C. Exact volumes of supernatant (400-
800 microlitres) were then centrifiiged at 17000 G for 60 minutes and the supernatant
decanted to obtain the microparticle pellet. The microparticles were then reconstituted in
350 microlitres of annexin-V buffer (Bender Medsystems), and divided into six 35-
microlitre aliquots plated onto the first 6 wells o f a 96-well U-bottomed plate. The
labelling and quantification of microparticles was then achieved as described in sections
2.5.3-2.5.5. Samples from each experiment were run 6 times and microparticle counts
from individual experimental conditions were expressed as the mean number per ml of
supernatant, with the intra-experiment standard error o f the mean (SEM) based on 6
measurements. Each microparticle experiment was repeated in triplicate, with the final
result reported as mean and SEM of these 3 experiments.
5.3.2 In vivo studies: Patients
A total of 39 children with primary systemic vasculitis were studied. The classification of
the type of vasculitic illness was defined using the Chapel Hill criteria (Jennette, Falk and
others, 1994).
29 children (14 males; mean age 7.24 years, range 0.7-15.8 years) had active systemic
vasculitis as defined by a Birmingham vasculitis activity score (BVAS) (Luqmani, Bacon
266
and others, 1994) of greater than zero (mean BVAS score 10.6/63; range 3-24/63) in the
absence o f intercurrent infection. 10 had polyarteritis nodosa (PAN); 3 had microscopic
polyangiitis (MPA- all with crescentic nephritis and perinuclear antineutrophil
cytoplasmic antibodies, [pANCA]); 1 had biopsy-proven Wegener's granulomatosis
(WG- with cytoplasmic antineutrophil cytoplasmic antibodies, [cANCA]); and 15 had
complete Kawasaki disease (KD) as defined by the American Heart Association criteria
(Dajani, Taubert and others, 1993). All KD patients were febrile (greater than 38 °C ),
and in the second week of illness; 2 had coronary arterial aneurysms, and 1 had
myocarditis with ventricular arrhythmia in the absence o f coronary arterial abnormalities.
In the KD group, all samples were obtained prior to treatment with intravenous
immunoglobulin.
10 children with inactive vasculitis (4 males; mean age 9.0 years, range 1.6-15.8 years)
were examined. In this group 4 had PAN, 3 had KD (post treatment, afebrile, and 4-7
weeks after the initial episode); 1 had WG (cANCA positive), and 2 had MPA (both
pANCA positive). All had a BVAS score of 0/63.
Control samples were taken fi'om 43 healthy subjects comprising 20 healthy children (10
males; mean age 10.3 years, range 2-15.1 years) and 23 young healthy adults (10 males;
mean age 29.3 years, range 23-40 years).
The disease control group comprised 24 age-matched febrile children (11 males; mean
age 7.16 years, range 0.6-15.2 years). 12 were attendees o f their General Practitioner
267
with fever and (presumed) viral rash (N=9), or upper respiratory tract infection (N=3). 8
were hospital inpatients with viral or bacterial sepsis (inclusive o f 2 patients with
meningococcal sepsis). 4 were hospital out-patient attendees with inactive systemic lupus
erythematosus (SLE) with fever secondary to upper respiratory tract infection, but in the
absence of clinical vasculitis or anti-phospholipid syndrome (including negative
anticardiolipin antibodies, and absence of lupus anticoagulant).
In addition, samples from 5 patients in the active vasculitis group were examined before
and after induction of remission of vasculitis. The diagnoses in these 5 children were
PAN (N=3), WG (N=l), and MPA (N=l).
Informed consent was obtained from the parents o f all children involved in the study, and
the study was approved by the local research ethics committee.
5.3.2 Preparation ofplatelet poor plasma
1.4-5 mis o f whole blood was collected into bottles containing 3.2% trisodium citrate
(Becton Dickinson). Platelet poor plasma was obtained by immediate centrifugation of
the whole blood at 5000G for 5 minutes twice. Plasma was then stored at -70° Celcius
until use.
268
5.3.3 Isolation o f microparticles from platelet poor plasma
Platelet poor plasma was defrosted in a water bath at 37 ° Celcius. Exact volumes of
plasma (400-800 microlitres) were then centrifuged at 17000 G for 60 minutes and the
supernatant decanted to obtain the microparticle pellet. The microparticles were then
reconstituted in 350 microlitres of annexin V buffer (Bender Medsystems), and divided
into ten 3 5-microlitre aliquots plated onto the first 10 wells o f a 96 well U-bottomed
plate. The labelling and quantification of microparticles (endothelial and platelet) was
then achieved as described in sections 2.5.3-2.5.5.
5.3.4 Statistical analysis
Comparison of mean platelet and endothelial microparticle numbers between the different
patient groups (independent analysis) was analysed using the Kruskal-Wallis test. Paired
data obtained from children before and after induction of remission were compared using
the Wilcoxon signed-rank test. Spearman rank correlation coefficients were calculated to
investigate the relationship between microparticles, BVAS, and other conventional acute
phase reactants. Non-parametric tests were used because the data were not easily
transformed to normality (some upward skew, some downward skew, and some
approaching “J-shaped” distributions). Receiver operator characteristic curves,
sensitivity, specificity, positive and negative predictive values, and likelihood ratios were
calculated to examine the diagnostic test characteristics o f endothelial microparticles for
active vasculitis.
269
5.4 Results
5.4.1 SAg-dependent T cell-mediated endothelial cell activation and injury: generation
o f endothelial microparticles (EMP)
To further assess endothelial cell injury in response to SAg-dependent T cell-mediated
activation, endothelial microparticles (EMPs) released by HUVEC into the supernatant
were analysed for the different experimental conditions (Appendix 7). TN F-a again
served as a positive control in these experiments since this cytokine is known to stimulate
microparticle formation from endothelial cell monolayers in vitro, and has been
implicated as the main cytokine involved in SAg-mediated T cell-dependent endothelial
cell activation. Supernatants from resting HUVEC monolayers, HUVEC stimulated with
TNF-a, or activated by the SAg-mediated T cell-dependent process described in previous
experiments, were sampled between 0 and 24 hours to investigate the kinetics o f the
formation o f EMPs in response to the experimental conditions.
There was a progressive rise over 24 hours in the total number of EMPs released into the
supernatant following stimulation with TN F-a as compared with resting HUVEC.
Following TNF-a stimulation, the total EMP number rose from a baseline o f zero at 0
hours to a mean o f 1.6 million/ml (SEM 0.7 million/ml) at 24 hours (mean, SEM of 3
experiments) as shown in figure 5.1a. Similarly, T N F-a resulted in a rise in CD 105
positive EMPs from 0 million/ml to 0.62 million/ml (SEM 0.4 millions/ml) at 24 hours
270
(figure 5.1b), ICAM-1 positive EMPs from 0 million/ml to 0.73 millions/ml (SEM 0.6
million/ml) at 24 hours (figure 5.1c), and E-selectin positive EMPs from 0 million/ml to
0.63 million/ml (SEM 0.4 million/ml) at 24 hours (figure 5.Id). Although the observed
rise in endothelial microparticle formation (for all the endothelial markers used) in
response to TNF-a was consistent in all 3 experiments performed, the magnitude of the
response in individual experiments varied considerably, as reflected in the wide error bars
(figures 5.1a-d).
In contrast to the response to TNF-a, which was maximal at 24 hours, endothelial
microparticle formation in response to SAg-mediated T cell-dependent stimulation
peaked earlier (at around 2 to 6 hours), and was overall o f a greater magnitude (figures
5.1a-d). Thus at 2 hours, there was a peak o f E-selectin positive EMPs rising from a
baseline of zero at 0 hours, to 1.76 million/ml (SEM 0.04 million /ml o f 3 experiments),
which subsequently dropped to 0.4 million/ml (SEM 0.07 million/ml) (figure 5.1 d). For
CD 105-positive EMPs, there was a rise from a baseline of 0.004 million/ml (SEM 0.002
million/ml) at 0 hours, which peaked at 6 hours (0.33 million/ml, SEM 0.2 million/ml)
and was sustained at 24 hours (0.33 million/ml, SEM 0.2 million/ml) (figure 5.1b).
Although, similar results were obtained for the total number o f annexin-V binding
microparticles, and microparticles expressing ICAM-1 (figures 5.1a and 5.1c), it was not
possible to attribute the origin of these microparticles entirely as endothelial since
activated lymphocytes can express ICAM-1 (Barclay AN, Brown MH and others, 1997a),
and were (necessarily) present in these co-culture experiments.
271
The peak o f E-selectin positive EMPs at 2-4 hours in these co-culture experiments was
SAg-dependent because no rise was observed in response to incubation with INF-y alone,
or with INF-y and T cells in the absence o f TSST-1 at 4 hours. There was, however, a
slight rise in E-selectin EMP formation from MHC class 11-negative HUVEC (i.e.
HUVEC not pre-incubated INF-y) which were incubated with T cells in the presence of
TSST-1 for 24 hours, but not at 4 hours. This was presumably a consequence o f the small
number o f MHC class II expressing monocytes (approximately 0.2%) contaminating the
purified T cell population. Indeed, it was confirmed that the purified T cell population did
become activated in a predominantly Vp restricted manner when incubated with SAg
(TSST-1 or SEB) at 24 hours, but not at 4 hours (figure 5.2), which could account for this
observation. At 24 hours there was also some increased CD69 expression o f CD4^ Vp i 2
(figure 5.2a). This is not one o f the classical responding Vp families for this SAg
suggesting that there could have been some bystander activation at 24 hours. This could
also explain the increased E selectin EMP formation at 24 hours by MHC class II
negative HUVEC in the presence o f T cells and TSST-1
272
Figure 5.1: Endothelial microparticle release from HUVEC in response to TNF-a, SAg
stimulation (co-culture of MHC class lU HUVEC with CD3 T lymphocytes in the
presence of TSST-1 10 ng/ml) or control (resting) HUVEC; (Mean, s e m of 3 experiments)
5 ■TNF 100 ng/ml 1.2
4 •SAg stimulation
■Control (resting HUVEC) E 0.8 ^

3 I
S 0-6 1
2 .= 0.4
S
1
0
0 10 20 30 0 10 20 30
hours hours
Figure 5.1 A: Total number of annexin-V binding microparticles Figure 5.IB: CD 105 positive endothelial microparticles
(millions/ml of supernatant) (millions/ml o f supernatant)
1.5
2 0.5 0.5
0
0 10 20 30 0 10 20 30
hours hours
U)
Figure 5.1C: ICAM-1 positive microparticles Figure 5. ID. E-selectin positive endothelial microparticles
(millions/ml of supernatant) (millions/ml o f supernatant)
F ig u r e 5.2 In c u b a t i o n o f p u r if ie d l y m p h o c y t e s w it h SAg a f t e r 24 h o u r s
Incubation of purified CD3^ lymphocytes with SAg after 24 hours resulted in Vp-
restricted upregulation of CD69, presumably the consequence o f contaminating MHC
class 11^ cells (approximately 0.2%).
274
Figure 5.2: Purified T lymphocyte incubation with SAg: V-beta specific upregulation of
CD69 after 24 hours
Figure 5.2a: CD4CD69MFI
50
40
control
o) 30
S □ TSST1
^ 20
eiSEB
Q
^ 10
vb1 vb2 vb5.1 vb8 vb12
Figure 5.2b: CD8 CD69 MFI
50
_ 40
ai 30 control
s □ TSST1
E3SEB
vbl vb2 \bS vb5.1 \b8 vb12

5.4.2 Comparison o f mean microparticle numbers between patient groups
The absolute numbers of microparticles (total, platelet, or endothelial origin) expressed in
millions per ml of plasma from the different patient groups are summarised in figures
5.3-5.5.
The mean total microparticle number expressed as millions per ml o f plasma for the
active vasculitis, inactive vasculitis, controls, and disease controls were: 6.48 million/ml
(SEM 1.07 million/ml), 1.09 million/ml (SEM 0.17 million/ml), 2.92 million/ml (SEM
0.63 million/ml), and 1.24 million/ml (SEM 0.25 million/ml) respectively (figure 5.3).
Thus, the mean total number o f annexin V binding microparticles in the children with
active vasculitis was 6.0-fold higher than in the inactive vasculitis group (p=0.000), 2.2-
fold higher than healthy controls (p=0.000), and 4.2 fold higher than in the disease
controls (p=0.000).
The number of endothelial microparticles as defined by E-selectin, and CD 105
expression was higher in the active vasculitis group than in the other patient groups
(figure 5.4a). The mean number of E-selectin positive microparticles in the active
vasculitis was 8.5-fold higher than in the inactive vasculitis group (p=0.000), 5.6-fold
higher than the healthy controls (p=0.000), and 10.4-fold greater than the disease controls
(p=0.000). The number of CD 105 positive microparticles in the active vasculitis group
was 6.2-fold higher than the inactive vasculitis group (p=0.001), 4.1-fold higher than the
healthy controls (p=0.000), and 11.4-fold higher than the disease controls (p=0.000)
276
(figure 5.4b). There was also a significant increase in platelet microparticles expressing
CD42a in the active vasculitis group as compared to the other groups. The number of
CD42a positive microparticles in the active vasculitis group was 8.4-fold higher than the
inactive vasculitis group (p=0.006), 1.6-fold higher than the healthy controls (p=0.04),
and 6.9-fold higher than the disease controls (p=0.000) (figure 5.5). There was no
difference in the number of platelet microparticles expressing P-selectin comparing the
active vasculitis, inactive vasculitis or healthy control groups, although the number o f P-
selectin microparticles was lower in the disease controls than the active vasculitis group
(p=0.003), inactive vasculitis group (p=0.01), and healthy controls (p=0.001) (figure 5.5).
277
F ig u r e 5 .3 : T o t a l m ic r o p a r t ic l e n u m b e r s
Column scatter plot of the total microparticle number taken from patients with active
vasculitis (n=29), inactive vasculitis (n=10), healthy controls (n=43), and disease controls
(n=24).
278
Figure 5.3: Total Microparticle Number
30n
Active Vasculitis
E
tf) Inactive Vasculitis
Healthy Controls
- E Disease Controls
CL (0
S Q.
<U
a
E
3
p = 0.000
p = 0.000
p = 0.000
279
F ig u r e 5 .4 E s e l e c t in a n d CD 10 5 m ic r o p a r t ic l e n u m b e r s
Column scatter plots o f E-selectin and CD 105 EMPs taken from patients with active
vasculitis (n=29), inactive vasculitis (n=10), healthy controls (n=43), and disease controls
(n=24).
280
Figure 5.4a: E Selectin Microparticle
Number
10.0n
o ■ Active Vasculitis
Ë A Inactive Vasculitis
c
o 7.5-
Healthy Controls
If
(0 (0
♦ Disease Controls
Q. (0 5.0-
S Q.
O
a 2.5- ■■■■
E _mm____
— — zttsâssL
p = 0.000
p = 0.000
p = 0.000
Figure 5.4b: CD105 Microparticle Number
4-1
3-
co
E
S. « 2
Q.
0)
.Q
E
3
p = 0.001
p = 0.000
p = 0.000
281
F i g u r e 5 .5 C o m p l e t e m ic r o p a r t i c l e p r o f il e
Bar chart of the complete microparticle profile from patients with active vasculitis
(n=29), inactive vasculitis (n=10), healthy controls (n=43), and disease controls (n=24). *
= microparticle population significantly higher in the active vasculitis group; t ~
microparticle population significantly lower in the disease control group.
282
Figure 5.5: Complete microparticle profile
*
*
2.5
(0
E
(0
to ■ Active vasculitis n= 29
Q.
□ inactive vasculitis n=10
1 1.5
□ Controls n=43
g
o m D isease controls n=24
0.5 :
X
0 .
CD42a Psel ICAM-1 CD105 VCAM-1 Esel
K)
00
w
There was no difference in the numbers o f microparticles expressing the adhesion
molecules ICAM-1 or VCAM-1 between the groups (figure 5.5).
5.4.2 Longitudinal analysis ofendothelial and platelet microparticle profiles before and
after induction o f remission o f vasculitis
Paired samples obtained fi'om 5 children before and after induction of remission of
vasculitis were analysed for changes in endothelial and platelet microparticle profiles. All
5 patients with active vasculitis demonstrated high levels o f endothelial MPs which fell to
normal following induction of remission (a 10.5-fold decrease for CD 105 MPs (p=0.04)
and an 8.7-fold decrease for E-selectin MPs (p=0.04). There was also a significant fall in
platelet microparticles expressing CD42a after induction o f remission (a 3.9-fold
decrease, p=0.04), but no significant change in microparticles expressing P-selectin.
There was no significant difference in microparticles expressing lCAM-1 or VCAM-1
before and after induction of remission of vasculitis. These data are summarised in figure
5.6.
5.4.3 Correlation o f EMPs with BVAS and conventional acute phase reactants
Correlation coefficients between microparticles and BVAS and conventional acute phase
markers are shown in table 5.1. There was a significant correlation between E-selectin
microparticles and the BVAS in the vasculitis patients as a whole (R=0.65, p=0.000;
active and inactive vasculitis patients combined); CD 105 microparticles (R=0.51,
p=0.001); and total microparticle number (R=0.65,
284
F ig u r e 5.6 C h a n g e s in MP p r o f il e b e f o r e a n d a f t e r in d u c t io n o f r e m i s s i o n o f v a s c u l it is
Changes in the endothelial (E-selectin and GDI 05) and platelet (CD42a) microparticle
profile before and after induction of remission in 5 children with vasculitis (PAN=3;
MPA=1; WG=1).
285
Figure 5.6a: E-selectin microparticles pre and post Figure 5.6b: CD105 microparticles pre and post
induction of remission induction of remission
P=0.04 P=0.04
0.5 -
0.1
Active Remission Active Remission
Figure 5.6c: CD42a microparticles pre and post

induction of remission
P=0.04
Active Remission
p=0.000). There was a weaker (but significant) correlation between CD42a microparticles
and the BVAS (R=0.42, p=0.01).
For the conventional acute phase laboratory markers there was a significant correlation
between the BVAS and the haemoglobin (R= -0.61, p= 0.000); total white cell count
(R=0.35, p= 0.038); ESR (R=0.72, p= 0.000); and plasma albumin (R= -0.50, p= 0.02).
In the patients with vasculitis (active or inactive) there was a correlation between E-
selectin and CD 105 positive microparticles and other conventional acute phase reactants
(table 5.1). In contrast, in the disease control group the strength o f the relationship
between microparticles (total, endothelial, or platelet) and conventional acute phase
reactants was overall weaker (table 5.1), with the notable exception o f P-selectin platelet
microparticles which were negatively correlated with plasma albumin levels (R= -0.69,
p=0.002). Notably, there was no significant correlation between the platelet count and
CD42a microparticles or P-selectin microparticles in either the vasculitis patients or the
disease controls (table 5.1).
5.4.4 Test characteristics o f EMPs fo r the diagnosis o f active vasculitis
The receiver operator characteristic curves (ROC curves) for EMPs expressing E-selectin
or CD 105 at varying definitions o f positivity are shown in figure 5.7. For comparison the
ROC curve for renal angiography in childhood PAN based on a previously published
series of 25 children is also shown (Brogan, Davies and others, 2002). Table 5.2
summarises the test characteristics of EMPs for the diagnosis of active vasculitis at
287
Table 5.1 Correlation between EMP and clinical indices o f vasculitic disease activity
Correlation between EMPs and conventional acute phase reactants in the vasculitis and
disease control patient groups. Hb= Haemoglobin; ESR= Erythrocyte sedimentation rate;
CRP= C-reactive protein; BVAS= Birmingham Vasculitis Activity Score.
288
Table 5.1: Correlation between EMP and clinical indices of vasculitic
disease activity
Table 5.1: Vasculitis patients: correlation between M P, laboratory markers and BVAS
Hb Total Platelets ESR CRP Albumin BVAS

w cc
Total MP R=-0.52 R=0.23 R=0.41 R=0 50 R= 0.25 R= -0.54 R=0.65
count (p=0.001) (p=NS) (p=0.01) (p=0.007) (P=NS) (p=0.009) (p=0.000)
E- selectin R= -0.50 R=0.05 R=0.30 R= 0.40 R= 0.06 R= -0.40 R=0.65
MPs (p=0.005) (p=NS) (p=NS) (p=0.02) (p=NS) (P=NS) (p=0.000)
CD105 R= -0.4 R=0.10 R=0.3 R=0.6 R=0.3 R= -0.6 R=0.51
MPs (p=0.01) (p=NS) (p=NS) (p-0.002) (p=NS) (p=0.007) (p=0 001)
CD42a R= -0.20 R=-0.03 R=0.21 R=0 30 R= -0.04 R= -0.2 R=0.42
MPs (P=NS) (P=NS) (p=NS) (p=NS) (p=NS) (P=NS) (p=0.01)
P-selectin R=-0.13 R= -0.26 R=0.21 R=-0.06 R=-0.23 R--0.14 R=-0.26
MPs (P=NS) (P=NS) (p=NS) (p=NS) (P=NS) (P=NS) (p=NS)
ICAM-1 RÔ.OO R=-0.12 R=-0.17 R= -0.34 R=-0.23 R= -0.32 R=-0.17
MPs (p=NS) (P-NS) (P=NS) (p=NS) (p=NS) (P=NS) (p=NS)
VCAM-1 R=0 29 R=0.06 R= -0.22 R= -0.05 R=-0.12 R=0 26 R= -0.22
MPs (p=NS) (P=NS) (p=NS) (p=NS) (p=NS) (p=NS) (P=NS)
Disease control patients: correlation between M P and laboratory markers
Hb Total Platelets ESR CRP Albumin BVAS

WCC
Total MP R=-0.04 R= -0.14 R--0.04 R=-0.09 R -0 16 R=0.24 NA
count (p=NS) (P=NS) (p=NS) (p=NS) (P=NS) (p=NS)
E- selectin R= -0.08 R=0.00 R= -0.20 R= 0.20 R= 0.30 R=0.50 NA
MPs (P=NS) (P=NS) (p=NS) (P=NS) (P=NS) (p=0.04)
CD105 R= -0.05 R=0.04 R= -0.08 R= -0.03 R= 0.00 R= -0.03 NA
MPs (p=NS) (p=NS) (p=NS) (p=NS) (P=NS) (p=NS)
CD42a R= -0.15 R=-0.05 R=0 08 R=0 35 R=0 23 R=-0.41 NA
MPs (p=NS) (P=NS) (p=NS) (p=NS) (P=NS) (P=NS)
P-selectin R=0.12 R=0.10 R= -0.04 R=0.50 R=0 19 R= -0.68 NA
MPs (P=NS) (P=NS) (P=NS) (P=NS) (p=NS) (p=0.002)
ICAM-1 R=-0.12 R-0.08 R--0.22 R=-0.12 R=-0.19 R-0.063 NA
MPs (p=NS) (p=NS) (p=NS) (P=NS) (P=NS) (P=NS)
VCAM-1 R= -0.07 R=0.00 R=0.06 R--0.03 R=0.032 R=0.09 NA
MPs (p=NS) (p=NS) (P=NS) (p=NS) (p=NS) (P=NS)
289
Table 5.2 Test characteristics o f EMPs for the diagnosis o f active vasculitis
PPV= positive predictive value; NPV= negative predictive value; LR+ = Likelihood ratio
for a positive test result; LR- = Likelihood ratio for a negative test result.
290
Table 5.2: Test characteristics of EMP for the diagnosis of active
vasculitis
Cut-off for test positivity Sensitivity % Specificity % PPV % NPV % LR+ LR-
E-selectin microparticles (95%CI) (95%CI) (95%CI) (95%CI)
(millions/ml of plasma)
0.1 9 7 (8 2 -1 0 0 ) 4 3 (3 2 -5 5 ) 3 9 (3 0 -4 8 ) 97 (9 4 -1 0 0 ) 1 .6 8 0 .0 8
0.3 9 0 (7 3 -9 8 ) 6 5 (5 3 -7 5 ) 4 9 (4 0 -5 9 ) 94 (9 0 -9 9 ) 2 .5 6 0 .1 6
0.5 9 0 (7 3 -9 8 ) 7 8 (6 7 -8 7 ) 6 0 (5 1 -7 0 ) 95 (9 1 -9 9 ) 4 .0 6 0 .1 3
0.7 7 9 (6 0 -9 2 ) 9 2 (8 4 -9 7 ) 7 9 (7 2 -8 7 ) 92 (8 7 -9 7 ) 1 0 .1 8 0 .2 2
0.9 6 6 (4 6 -8 2 ) 9 2 (8 4 -9 7 ) 7 6 (6 8 -8 4 ) 88 (8 1 -9 4 ) 8.41 0 .3 7
1.0 6 2 (4 2 -7 9 ) 9 5 (8 7 -9 9 ) 8 2 (7 4 -8 9 ) 87 (8 0 -9 3 ) 1 1 .9 5 0 .4
1.1 5 9 (3 9 -7 6 ) 9 5 (8 7 -9 9 ) 81 (7 3 -8 8 ) 86 (7 9 -9 3 ) 1 1 .2 8 0 .4 4
1.2 4 5 (2 6 -6 4 ) 9 5 (8 7 -9 9 ) 7 6 (7 4 -8 9 ) 82 (7 5 -8 9 ) 1 1 .3 6 0 .5 7
1.3 4 5 (2 6 -6 4 ) 9 5 (8 7 -9 9 ) 7 6 (6 8 -8 5 ) 82 (7 5 -8 9 ) 8 .6 3 0 .5 8
1.4 4 5 (2 6 -6 4 ) 9 6 (8 9 -9 9 ) 81 (7 4 -8 9 ) 82 (7 5 -9 0 ) 11.51 0 .5 7
1.5 41 (2 4 -6 1 ) 9 6 (8 9 -9 9 ) 8 0 (7 2 -8 8 ) 81 (7 4 -8 9 ) 1 0 .6 0.61
1.6 41 (2 4 -6 1 ) 9 7 (8 9 -9 9 ) 8 6 (7 9 -9 2 ) 82 (7 4 -8 9 ) 1 5 .9 3 0 .6
1.7 41 (2 4 -6 1 ) 9 7 (8 9 -9 9 ) 8 6 (7 9 -9 2 ) 82 (7 4 -8 9 ) 1 5 .9 3 0 .6
1.8 3 8 (2 1 -5 8 ) 9 9 (9 3 -1 0 0 ) 9 2 (8 6 -9 7 ) 81 (7 3 -8 8 ) 2 9 .2 0 .6 3
2.0 3 4 (1 8 -5 4 ) 9 9 (9 3 -1 0 0 ) 91 (8 5 -9 6 ) 80 (7 3 -8 8 ) 2 6 .9 0 .6 6
2.2 3 4 (1 8 -5 4 ) 1 0 0 (9 5 -1 0 0 ) 1 0 0 (9 7 -1 0 0 ) 80 (7 3 -8 8 ) infinity 0 .6 5
CD-105 microparticles Sensitivity % Specificity % PPV % NPV % LR+ LR-
(millions/ml of plasma) (95%CD (95%CD (95%CD (95%CD
0.1 8 6 (7 6 -9 1 ) 6 2 (5 3 -7 2 ) 4 6 (3 1 -5 0 ) 92 (8 1 -9 8 ) 2 .2 9 0 .2 2
0.3 6 2 (53-71 8 7 (8 1 -9 3 ) 6 2 (5 5 -7 3 ) 86 (7 6 -9 3 ) 4 .7 8 0 .4 4
0.5 4 5 (3 5 -5 4 ) 9 4 (8 9 -9 8 ) 7 2 (6 4 -8 1 ) 82 (7 2 -8 9 ) 6 .9 0 0 .5 9
0.7 31 (2 2 -4 0 ) 9 6 (8 9 -9 8 ) 7 5 (5 5 -7 3 ) 79 (6 9 -8 6 ) 7 .9 7 0 .7 2
0.9 21 (1 3 -2 8 ) 9 9 (9 7 -1 0 0 ) 8 6 (7 9 -9 2 ) 77 (6 7 -8 5 ) 1 5 .9 3 0 .8 0
1.0 1 7 (1 0 -2 4 ) 9 9 (9 1 -9 9 ) 8 3 (3 6 -9 9 ) 76 (6 6 -8 4 ) 1 3 .2 8 0 .8 4
1.1 1 7 (1 0 -2 4 ) 9 9 (9 7 -1 0 0 ) 8 3 (3 6 -9 9 ) 76 (6 6 -8 4 ) 1 3 .2 8 0 .8 4
1.2 1 7 (1 0 -2 4 ) 9 9 (9 7 -1 0 0 ) 8 3 (3 6 -9 9 ) 76 (6 6 -8 4 ) 1 3 .2 8 0 .8 4
1.3 1 7 (1 0 -2 4 ) 9 9 (9 7 -1 0 0 ) 8 3 (3 6 -9 9 ) 76 (6 6 -8 4 ) 1 3 .2 8 0 .8 4
1.4 1 7 (1 0 -2 4 ) 9 9 (9 7 -1 0 0 ) 8 3 (3 6 -9 9 ) 76 (6 6 -8 4 ) 1 3 .2 8 0 .8 4
1.5 1 7 (1 0 -2 4 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 76 (6 6 -8 4 ) infinity 0 .8 3
1.6 1 0 (5 -1 6 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 75 (6 6 -8 3 ) infinity 0 .9 0
1.7 7 (2 -1 2 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 74 (6 6 -8 2 ) infinity 0 .9 3
1.8 7 (2 -1 2 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 74 (6 6 -8 2 ) infinity 0 .9 3
2.0 7 (2 -1 2 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 74 (6 6 -8 2 ) infinity 0 .9 3
2.2 7 (2 -1 2 ) 1 0 0 (9 7 -1 0 0 ) 1 0 0 (4 8 -1 0 0 ) 74 (6 6 -8 2 ) infinity 0 .9 3
291
F ig u r e 5.7 R e c e i v e r o p e r a t o r c h a r a c t e r is t ic (ROC) c u r v e s for E - s e l e c t in and CD 105 EMPs f o r
THE DIAGNOSIS OF ACTIVE VASCULITIS.
For comparison is the ROC curve for the diagnosis o f polyarteritis nodosa in a previously
published cohort of children (Brogan, Davies, and others. 2002).
292
Figure 5.7:Receiver operator characteristic curves for endothelial microparticles
(E selectin and CD105) for the diagnosis of active vasculitis
100
E selectin EM Ps
C D 105 EM Ps
Visceral angiography
a 40
(JO
0 +
K)
VO 0 20 40 60 80 100
Lk)
False positive rate (%)

varying definitions of test positivity. The cut-off values for test positivity in table 5.2
correspond to individual points on the ROC curves.
5.5 Discussion
In Chapter 4 it was established that SAg-dependent T cell VP-restricted adherence and
activation on the surface o f HUVEC monolayers results in activation of the HUVEC. The
in vitro studies outlined in this chapter now demonstrate that a further consequence of
this interaction is EMP generation, (both increased E-selectin and CD 105 EMP
formation). This effect occurred earlier, and was comparable (and on the whole greater)
than the HUVEC response to stimulation with TNF-a, emphasising the potential for
vascular injury in response to SAgs.
It is currently still unclear in this in vitro model whether this microparticle formation
occurs as a result o f apoptosis alone, or is the result o f cellular activation in the absence
of apoptosis. Moreover, the functional significance of endothelial microparticle formation
in response to SAg dependent T cell activation remains to be elucidated, although it is
known that microparticles rich in PS can participate in the dissemination o f pro-adhesive
and pro-coagulant activities in thrombotic disorders (Boulanger, Scoazec and others,
2001; Combes, Simon and others, 1999; Jimenez, Jy and others, 2001; loop, Berckmans
and others, 2001; Mallat, Hugel and others, 1999; Mallat, Benamer and others, 2000;
Nieuwland, Berckmans and others, 2000), thus potentially amplifying the vascular injury.
294
It was the opinion of the author that the generation of endothelial microparticles in
response to many injurious stimuli including TNF-a and, (as described for the first time
in this chapter) in response to SAg-dependent T cell mediated endothelial injury,
constituted a fascinating and possibly important observation, with potential direct
relevance to the study of patients with vasculitis. This result therefore prompted the
studies of this phenomenon in children with vasculitis syndromes described in this
chapter.
There is an emerging concept that endothelial cell dysfunction is central to the
pathogenesis of many diseases including atherosclerosis (Mallat and Tedgui, 2001;
Tedgui and Mallat, 2001), multiple sclerosis (Minagar, Jy and others, 2001b), and
autoimmune diseases which are typified by thrombosis and vascular injury (TTP
(Jimenez, Jy and others, 2001) and SLE with antiphospholipid syndrome (Combes,
Simon and others, 1999)). In support of this concept, previous studies have been able to
demonstrate an elevated number o f microparticles o f endothelial origin in these disorders,
as a consequence of increased endothelial cell activation or apoptosis. The data presented
in this study would now suggest that the same is true o f the systemic vasculitides of
childhood, and it was possible to demonstrate increased levels o f circulating endothelial
microparticles expressing E-selectin and CD 105 in the active stage of the disease.
It could be argued that childhood vasculitides would be the “prototypic” disease states in
which to study this phenomenon since endothelial dysfunction occurs in most vasculitis
295
syndromes, and children do not have atherosclerosis, which may be a confounding factor
when studying endothelial microparticles in adult disease states. Thus it is now apparent
that increased endothelial microparticle formation is not specific to any single disease
entity, but rather is the endpoint of a variety of immunological and inflammatory
pathways, which culminate in endothelial injury.
A number of endothelial surface markers of varying specificity were utilised to
characterise the microparticles as being o f endothelial or platelet origin. CD 105
(endoglin) is constitutively expressed by endothelial cells, but is also expressed on some
activated monocytes and leukaemic cells (Barclay AN, Brown MH and others, 1997b).
As such, it is relatively specific for the endothelium. E-selectin is only expressed on
activated endothelial cells in vivo (Barclay AN, Brown MH and others, 1997c), although
very recently has been demonstrated to be upregulated on CD4 T cells in vitro stimulated
by a novel interaction between membrane-bound TNF-a and rabbit anti-human TNF-a
antibody (Harashima, Horiuchi and others, 2001). Thus, at least in vivo, E-selectin is an
entirely specific marker for endothelial cells. ICAM-1 is constitutively expressed on
endothelial cells, although it is upregulated during endothelial cell activation (Barclay
AN, Brown MH and others, 1997a). It has a much wider tissue expression, however, and
is also expressed on activated leucocytes (Barclay AN, Brown MH and others, 1997a).
As such, it is a less specific endothelial cell marker. Similarly, although VCAM-1 is
predominantly expressed on vascular endothelium, it also has been identified on dendritic
cells, macrophages, and non-vascular cell populations within several organs including the
joints and kidney (Barclay AN, Brown MH and others, 1997d). CD42a is restricted to
296
and constitutively expressed on platelets and megakaryocytes and is therefore a relatively
specific platelet marker (Barclay AN, Brown MH and others, 1997e). P-selectin is
present on activated platelets and activated endothelial cells, and is therefore a poor
discriminator between activated platelets and endothelial cells (Barclay AN, Brown MH
and others, 1997f).
The main finding in the in vivo study was an increased level o f circulating microparticles
expressing E-selectin and CD 105 in the children with active vasculitis. It is suggested
that these microparticles are truly of endothelial cell origin, although one potential
confounder which cannot be excluded is that soluble E-selectin (known to be increased in
many vasculitis syndromes) (Nash, Shah, and Dillon, 1995) could bind
phosphatidylserine-rich microparticles released from platelets. We suggest that this is
unlikely however, because although it is known that L-selectin can bind to negatively
charged phospholipids such as phosphatidylserine and cardiolipin, E-selectin has been
shown unequivocally not to have any binding capacity to phospholipid (Malhotra, Taylor,
and Bird, 1996). Moreover, the fact that both E-selectin and CD 105 microparticles were
observed to correlate with vasculitic disease activity, whereas microparticles expressing
less specific endothelial markers failed to do so indicates that the E-selectin and CD 105
microparticles are truly of endothelial origin. It should be stated, however, that 3-colour
flow cytometry was not performed (because o f time constraints) to examine for CD42a
and E-selectin “double-positives” to unequivocally exclude the possibility o f soluble E-
selectin (which is known to be increased in vasculitis syndromes, including KD) binding
297
to platelet-derived MP and thus potentially confounding the observations described in
this study.
An increased number of microparticles of platelet origin expressing CD42a in the active
vasculitis group was also observed, although the size o f this difference between the
groups was less dramatic than that observed for EMP. This finding is not entirely
surprising since most vasculitis syndromes are characterised by high platelet counts and
by micro and macroscopic thrombus formation; indeed anti-platelet therapy is
recommended for most forms of childhood vasculitic illness (Brogan and Dillon, 2000b).
Interestingly, however, there was no correlation between platelet microparticles and
absolute platelet count in the vasculitis patients, or the disease controls.
Importantly, this study did not address the functional significance of increased
microparticle numbers (of all types) in active vasculitis, although it is possible that
microparticles could contribute to the pathogenesis of vasculitis via several mechanisms.
Firstly, the prothrombotic potential of cellular and platelet microparticles is well-
established, and has been demonstrated in acute coronary syndromes, atherosclerosis,
anti-phospholipid syndrome, and meningococcal sepsis. This occurs mainly as a result of
the rich PS content of the microparticles, which acts as the one o f the essential lipid co
factors for clotting and supports thrombin generation via the tissue factor-factor VII-
mediated pathway. Since a prothrombotic tendency occurs in vasculitis (Kiraz, Ertenli
and others, 2002; Savage and Cooke, 1993), it is entirely conceivable that the increased
circulating microparticles observed during active vasculitis may contribute to this.
298
Secondly, it has been demonstrated in vitro that microparticles of neutrophil origin can
activate endothelial cells in tissue culture (Mesri and Altieri, 1998). This is interesting
since an important sub-group of small vessel vasculitides (Wegener's granulomatosis,
microscopic polyangiitis, and Churg-Strauss syndrome) are associated with the presence
of anti-neutrophil cytoplasmic antibodies (ANCA) which are thought to activate
neutrophils and endothelial cells, causing vascular injury. It is not yet known if neutrophil
microparticles are released from neutrophils following activation by ANCA, or indeed
whether microparticles derived from other leucocytes could further contribute to
endothelial activation in vasculitis, but clearly this is an important area worthy of further
study.
The test characteristics of endothelial microparticles for the diagnosis of active vasculitis
based on a limited number of children with active vasculitis are encouraging, and
certainly compare favourably with invasive tests such as visceral angiography (Brogan,
Davies and others, 2002), and tissue biopsy (Albert, Rimon, and Silvers te in, 1988). It
must be emphasised, however, that increased circulating EMP are not specific to the
vasculitides, but rather are a feature of a number of diseases characterised by endothelial
cell dysfunction. Used in the right clinical context, however, they may provide important
diagnostic information. Furthermore, we have shown that they correlate with clinical
parameters of vasculitic disease activity such as the BVAS, and may be useful when
measured longitudinally in individual patients to monitor disease activity.
299
Lastly, the observation that endothelial microparticles correlated with conventional acute
phase reactants in the context of vasculitic disease activity, but less so in the disease
control group suggests that they are not behaving like a conventional acute phase
reactant, and may be able to discriminate between sepsis and the inflammatory response
associated with active vasculitis. In accordance with this result, a study of children with
meningococcal sepsis failed to show any difference in the levels o f circulating EMPs in
this disease compared with controls, although patient numbers were limited (Nieuwland,
Berckmans and others, 2000).
In conclusion, the consequence of SAg-dependent T cell mediated HUVEC interactions
is HUVEC activation and EMP formation, potentially a marker of endothelial injury. In
addition, this study has demonstrated increased levels of circulating EMP in children with
active vasculitis, and it is proposed that this novel finding may provide a "window" to the
activated endothelium. The functional significance of this observation, however, remains
to be elucidated.
300
Chapter 6: General discussion and future directions
301
6.1 General discussion
This thesis has attempted to examine one aspect of the relationship between infections
and vasculitis in the young and specifically asks the question whether or not S Ags could
be involved in the aetiopathogenesis of vascular injury associated with vasculitis
syndromes in the paediatric population. Throughout this thesis a general theme has been
the role that the endothelium plays in vasculitis- both as a target for injury and as a
potential amplifier of the inflammatory response. Chapter 5 expands on this concept, and
for the first time demonstrates increased endothelial microparticles in children with active
vasculitis.
There are important limitations to the data presented in this thesis many o f which have
been discussed in the relevant chapters, and in addition many unanswered questions. This
chapter will highlight these limitations and proposes areas of further research that may
address some of the unresolved issues. In addition, limited preliminary data will be
presented to illustrate the sort of approaches that may be used in the future to further the
data presented in this thesis.
6.1.1 What is true skewing o f the V p repertoire?
In chapter 1, evidence for peripheral blood T cell activation (CD69 upregulation) in
patients with active vasculitis was found, and there was some evidence o f T cell Vp
repertoire skewing in the vasculitis patients over and above that observed in the disease
control children despite comparable degrees o f T cell activation. As discussed, this
302
provides evidence for a role of T cells in vasculitis but does not provide definitive
evidence that SAgs are involved in all forms of vasculitis. Despite attempts to control for
potential confounding factors, this observation may still be artefactual.
It is useful in this context to consider what is actually meant by “skewing” o f the Vp
repertoire to highlight the complexity of this concept, particularly when considering
diseases with a chronicity that can span months to years (such as PAN or WG). The
typical response of a T cell (CD4+ or CD8+) following SAg activation has been
described: activation, followed by proliferation (expansion), and finally deletion. Thus,
depending on the timing of the blood sampling in relation to disease onset, particular
peripheral blood Vp family percentages may be normal, increased, or decreased. Even
when considering Kawasaki disease, which in contrast to other vasculitic syndromes such
as PAN has a well-defined and relatively acute onset, previous studies have suggested
that the timing of the blood sample is crucial to detect expansion o f Vp2 T cells (Curtis,
Zheng and others, 1995). This point is summarised in figure 6.1 which considers a
hypothetical study examining the percentage of Vp2 T cells in the peripheral blood of
healthy controls, KD at the acute (day 10) and convalescent (week 5) phases o f the
disease, and PAN with disease onset anywhere between 3 and 12 months fi*om the time of
sampling. As illustrated in this figure, studies which only examined changes in the mean
percentage of Vp families and ignored changes in the variance (spread) may miss true
“skewing”. This could explain some of the negative studies relating to KD, and illustrates
the importance of considering both expansions and deletions when looking for evidence
within the Vp repertoire of an effect of SAgs.
303
Figure 6.1: What is T cell VP skewing? A hypothetical study examining controls, KD
and PAN (n=3 in each group)
Higher mean Same mean

same variance bigger variance
25 ♦ controls
■ KD day 10
20 A KD week 5
• PAN 3-12 months?
15
10
I
A
0 vp2
w
Lower mean
g same variance
In fact the concept of skewing is even more complex than this, purely as a result of
simple arithmetic: when an individual responds to a SAg (e.g. TSST-1) and increases the
percentage of circulating Vp2 T cells, by definition there must be a concomitant decrease
in the percentage of other Vp families. Conversely, if an individual has a deletion of a
particular Vp family in response to a SAg, there will always be a concomitant increase in
the percentage of other VP femilies. In other words, true skewing also dictates that SAgs
will cause specific changes in the responding Vp family, and non-specific changes shared
over the whole T cell Vp repertoire. Again, this could account for some o f the
inconsistencies regarding changes in the Vp repertoire observed in previous studies
relating to KD.
6.1.2 Vp-specific activation in the absence o f Vp skewing
Another issue relating to the previously described model of the response o f a T cell to a
SAg which may explain some of the negative data relating to KD is that Vp-specific
activation may occur in the absence o f proliferation early on in the response. This
concept is highlighted by the in vitro experiments described in chapter 4, which showed
Vp-specific CD69 upregulation on T cells incubated for 4 hours with SAg, but with
relatively little change in Vp percentages. Theoretically if a patient with vasculitis which
was triggered by a SAg was sampled early in the disease process, there may be Vp-
specific activation without Vp expansion. The 3-colour flow cytometry protocol for
examining specific Vp family CD 69 upregulation within the CD4 or CDS T cell subsets
305
(which effectively provides data relating to both Vp-specific activation and
expansion/deletion) would be the obvious way to address this potential confounding
factor, and it is with some regret that this approach was not utilised from the outset in the
studies leading to this thesis.
Preliminary results using this approach were obtained from one infant aged 3-months
with KD who was included in the study described in chapter 3. This infant was of
particular interest since he was the identical twin brother o f a case of infantile KD
(complicated by coronary and axillary arterial aneurysm formation) who had presented
10 days previously (again included in chapter 3). Since the parents had been warned
regarding an increased risk of KD in siblings of index cases they presented relatively
early with the second twin, allowing blood sampling on day 6 o f the illness. Analysis of
the peripheral blood CD4 and CDS v p repertoire (by percentages) did not reveal any
major expansions or deletions. Specific VP expression o f CD69 was measured for Vp2
and V p l4 (limited blood volume was obtained from this small infant thus precluding
analysis of CD69 expression on other Vp families). These preliminary results are shown
in figure 6.2. As illustrated, although only two Vp femilies were analysed within the CD4
and CDS populations and cell numbers were limited, there was CD69 upregulation within
the Vp2 subset (true for both CD4 and CDS), which was not observed for V p i4, and with
comparatively little perturbation of the percentage o f cells expressing Vp2 as compared
with healthy control children. Polymerase chain reaction for toxin genes from nasal
isolates of Staphylococcus aureus from this infant revealed genes encoding for the toxins
SEA, SEG, SEI and TSST-1 (analysed by the Central Public Health Laboratory,
306
Colindale). The presence of TSST-1 and the observed specific activation o f CD4 and
CDS T ceils expressing Vp2 is of considerable interest in this context, and emphasises
that analysis of Vp repertoire skewing by consideration o f percentages is a relatively
insensitive means of studying this phenomenon.
307
Figure 6.2: VP-specific expression of CD69 on peripheral blood CD4 and CD8 T cells in
a 3-month old infant with KD (day 6 of illness).
CD69 MFI 144

11% 28% CD69 "high
Double gate
10* 10*
CD69PE
Vb2F
FSC
CD69 MFI 1.14
4.3% CD69 "high"
Double gate
c
u
10’ l O* 10 * 10*
CD69PE
Vbl4F
CD69 MFI 46
8.9 «/( 32.2% CD69 "high’
Double gate
Û
u
10' 10*
CD69PE
Vb2F
CD69 MFI 2.7
3.9'% 6.9% CD69 "high’
oo Double gate
Q
U
w
o
00
10*
C069PE
Vbl4F
A further extension of this argument would be to correlate such Vp-specific activation
profiles in patients with KD (or other vasculitides) with toxins isolated from individual
patients. For example, bacterial culture supernatants could be incubated with PBMCs
from a healthy adult donor, and the specific Vp activation profile obtained (CD69
upregulation across the Vp repertoire) could be compared with that actually observed in
the patient. Such an approach has been proposed in KD and sample collection is
underway as part o f a collaborative study undertaken by the London Kawasaki Disease
Research Group (KDRG) (Brogan, Bose and others, 2002).
6.1.3 V^-restricted binding o f the T cells to the H U VEC monolayer:
compartmentalisation o f the immune response in vasculitis
In chapter 4 it was demonstrated that in vitro MHC class E-expressing HUVEC can
present SAg to T cells, and one consequence of this is Vp-restricted binding of the T cells
to the HUVEC monolayer. This observation may provide insight into the
compartmentalisation of the immune response in vasculitis, and could also explain some
o f the conflicting data relating to studies of peripheral blood T cell VP repertoire skewing
in KD. For example, if this mechanism was operational in vasculitic syndromes such as
KD it could be anticipated that sampling the peripheral blood compartment may not be
revealing in terms of T cell activation since the majority o f the activated T cells would be
adherent to the endothelium of lesional tissue. As discussed in chapter 1 there are data
suggesting that lesional sequestration of T cells expressing Vp2 occurs in KD which
would be compatible with this theory (Leung, Giomo and others, 1995; Yamashiro Y,
309
N agata S and others, 1996). However it should be acknowledged that the mechanism
proposed in chapter 4 is not the only possible way that T cells expressing Vp2 may be
sequestered into lesional tissue in KD. For example, T cells may become activated at sites
away from the endothelial surface (for example, within the respiratory tract, or gut-
associated lymphoid tissue) and subsequently migrate to cardiovascular tissue under the
influence o f endothelial cell adhesion molecules. This latter mechanism may also utilize
homing receptors and lL-12 (Leung, Gately and others, 1995). The contribution o f
homing receptors is currently being examined in KD as part o f the London KDRG
collaborative study (Brogan, Bose and others, 2002).
It was anticipated that a study o f lesional T cell Vp expression in childhood PAN and
MPA would have been undertaken as part o f this thesis. Indeed, ethical committee
approval for this was obtained, and preliminary work in establishing antibody binding
using T cells embedded in wax blocks was undertaken. The idea had been to examine
tissue from patients in whom peripheral blood T cell Vp profiles were known, and
calculate in vivo “ Vp ratios” (in other words, the ratio o f the percentage o f particular Vp
families o f T cells in lesional tissue to peripheral blood) in order to further examine the
hypothesis that there may be specific Vp T cell sequestration in vasculitic lesional tissue
in PAN. Time constraints precluded such a study, however.
6.1,4 Clonal versus poly dona! Vp restricted expansion
Another limitation o f the T cell study presented in chapter 3 was that it did not address
the issue o f clonal versus polyclonal expansion. Generally speaking SAg stimulation
310
produces a Vp restricted expansion of both CD4 and CDS T cells that is polyclonal since
stimulation is induced independent of the TCR junctional regions. Conventional antigen
stimulation typically leads to a clonal expansion of T cells (with consequently limited
TCR junctional diversity) that is not Vp restricted. However, there have been reports
suggesting that conventional antigen exposure can lead to a clonal expansion of cells with
restricted Vp usage (Deckhut, Allan and others, 1993; Wedderbum, O'Hehir and others,
1993). In addition, the expansion o f a CD8+ Vp4+ clone was reported in an individual
following hepatitis B vaccination (Abbott, Geursen and others, 1996). Therefore, to be
certain that an expansion of T cells belonging to a particular Vp family is caused by SAg
exposure, it is preferable to confirm polyclonality by analysis o f the CDR3 region. That
said, this paradigm can arguably now be challenged since it has been established the
Mycoplasma SAg MAM binds both to human V p l7 segments and the CDR3 region (see
chapter 1, section 1.3.2.1). Whether this is true of other SAgs remains to be established,
but from a mechanistic point of view it is no longer true to say that SAgs always induce
polyclonal CDR3 VP restricted expansion, or conversely that clonal expansion as
determined by CDR3 analysis precludes a SAg-mediated response.
6.1.5 Flow cytometry versus polymerase chain reaction fo r the study o f T cell Vp
repertoires
Flow cytometry was used in the studies described in this thesis to analyse changes in the
peripheral blood T cell Vp repertoire although some comment regarding semi-
quantitative polymerase chain reaction (PCR), and the advantages and disadvantages of
311
these two alternative means of examining the Vp repertoire is required. One of the
principal advantages of the use of monoclonal antibodies and flow cytometry is that this
technique detects TCR proteins rather than measuring RNA levels. PCR analysis may
detect non-functioning transcripts including pseudogene transcripts, transcripts of non
functional rearrangements, and transcripts that are not expressed as functional TCR
proteins due to poor pairing with the TCR alpha chain. Secondly, the use o f monoclonal
antibodies and flow cytometry allows accurate measurement o f the number o f cells
expressing a particular Vp gene product. Direct comparison o f the results o f analysing
specific T cell Vp families with well-characterised monoclonal antibodies with that
performed by semi-quantitative PCR has demonstrated how PCR analysis can give
misleading quantitative results (Diu, Romagne and others, 1993). This is because RNA
levels are not necessarily equivalent to actual cell numbers or percentages since the
amount of TCR p chain mRNA expressed by individual T cell clones may differ up to
100 fold. Thirdly, the use of multicolour staining with monoclonal antibodies allows the
T cell Vp repertoire to be analysed with respect to other phenotypic characteristics such
as CD4 or CDS positivity, or expression of activation markers such as CD69. Such
analysis is more difficult using PCR and requires the T cell population to be separated,
for example by magnetic beads (see chapter 2), before mRNA isolation.
PCR analysis has two main advantages over FACS analysis. Firstly, oligonucleotide
primers can be designed to probe the complete T cell Vp repertoire. In contrast the
availability of monoclonal antibodies against many o f the TCR Vp families has
historically been limited, although recently much improved. The second advantage of
312
PCR techniques is that less sample is required, a feature which is of particular relevance
to the study of children, where the volume of blood available for study is usually limited.
Lastly, PCR techniques allow Vp expansions to be classified as polyclonal or clonal by
probing the CDR3 region (see table 1.3.2, and text above).
Taking into account these considerations flow cytometry was overall considered to be a
suitable technique to answer the main question being asked regarding T cell activation
and T cell VP-repertoire skewing in the studies described in this thesis, although the
limitations of this technique need to be acknowledged.
6,1.6 Colonisation o f study subjects with SAg-producing organisms
A final limitation of the study described in chapter 3 is that it did not provide data
relating to colonisation of vasculitis patients with SAg-producing organisms. Clearly this
would have been a useful adjunct to the argument and indeed it was anticipated that
swabs (nasal, pharyngeal, and rectal or stool) would have been obtained from all the
vasculitis patients, controls, and disease control children described in chapter 3. This
would have allowed comparison of the toxin profiles (analysed using PCR for known
common staphylococcal and streptococcal SAgs) and the peripheral blood T cell VP
repertoire. In fact, none of the children (or their parents) included in the studies described
consented to rectal swabs, and only a minority of vasculitis patients consented to nasal
swabs (it was of some surprise to this author that children would rather have a blood test
than a nasal swab!). That said, nasal colonisation with staphylococcus and/or p-
haemolytic streptococci was detected in 5/16 children with PAN, a rate comparable to
313
previously published nasal carriage rates within the healthy paediatric population as a
whole (Burgner, Curtis and others, 2001). Again, because o f time constraints, these
isolates were not analysed for toxin production. In any case such an approach would only
have identified known or common staphylococcal or streptococcal SAgs, and novel SAgs
would not have been detected. Moreover, the chronicity of the vasculitides studied
(mainly PAN) and the fact that many of these children were hospitalised for considerable
periods of time and received several courses of antibiotics would make such data very
difficult to interpret, and indeed control for.
6.1.7 HLA-mismatch between T cells and HUVEC in co^culture experiments
In chapter 4 data were presented suggesting that endothelial cells can function as
competent SAg presenting cells for T cells. It was previously discussed that the major
limitation of those data was that no attempt was made to control for HLA-mismatch, and
any observations made are occurring on the background o f a mixed lymphocyte-
endothelial culture. There are two potential ways to overcome this problem. Firstly, the
experiments could be repeated using an endothelial cell line with known tissue type, and
T cells donated from an adult with a compatible match. The practical limitation o f this
approach is that it would place a considerable burden on that individual donor
(throughout the course of 2 years, over 1000 mis of blood was collected from healthy
adult donors in order to obtain T cells or PBMC). In addition, the use o f immortalised cell
lines as opposed to first or second passage HUVEC may introduce another confounding
factor relating to cell adhesion molecule expression, or response to cytokine stimulation
(Haraldsen, Kvale and others, 1996). The second way of overcoming the HLA mismatch
314
problem would be to use HUVEC from a single umbilical cord and T cells extracted from
cord blood from the same individual. From a practical point o f view this is simpler, but
the main disadvantage is that naive T cells from the foetus may not respond to SAgs in
the same way as T cells from more mature donors. That said, KD has been described in
neonates (Stanley and Grimwood, 2002), and such studies may therefore be valid in their
own right.
6.1.8 EM P generation from SAg-dependent T cell mediated activation o f HUVEC
Another intriguing result presented in chapter 5 related to the generation of EMP in
response to SAg-dependent T cell mediated activation. As shown in figure 5.1, there was
an initial increase in EMP in the co-culture supernatant maximal by 2-4 hours, followed
by a return to just above normal by 24 hours. This was most obvious for E selectin EMP
(figure 5.Id). The obvious question relating to this observation is: where are the EMP
going? One clue may be provided by the study by Brezinschek et a l (Brezinschek,
Oppenheimer-Marks, and Lipsky, 1999). Using an in vitro model in which human
peripheral blood CD4+ T cells migrated through confluent monolayers o f HUVEC, the
authors demonstrated that activated T cells acquired a variety of endothelial cell surface
determinants including CD31, CD49d, CD54, CD61 and CD62E (E selectin).
Importantly, the acquisition of these endothelial markers occurred as a result of
endothelial cell membrane transfer as documented using endothelial cells whose cell
membrane had been pre-labelled with the lipophilic dye, DiOC-16. Another relevant
observation that the authors made was that membrane blebs occurred on the sur&ce of
the endothelial cells during their interaction with T cells. It is intriguing to speculate that
315
this endothelial cell membrane transfer to T cells occurred as a result o f endothelial
microparticle formation, and certainly this observation would explain the results
illustrated in figure 5.1. To examine the hypothesis that the EMP were binding to the T
cells present in the co-culture (and thus were effectively being removed from the
supernatant), preliminary experiments were performed examining surface E selectin
expression on T cells in the co-culture model described in chapter 4, and the results of a
single experiment are shown in figure 6.3. At 2 hours, there was increased surface
expression of E selectin on CD3+ T cells co-cultured with MHC class n+ HUVEC and
TSST-1 (10 ng/ml) which would be compatible with the hypothesis that E selectin EMP
released from activated HUVEC are binding to the T cells present in the co-culture. To
confirm this, however, it would be important to examine mRNA expression for E selectin
in these T cells to ensure that the T cells are not synthesising E selectin de novo
(Harashima, Horiuchi and others, 2001). Additionally, it would be important to examine
this phenomenon over a longer time frame (0-24 hours). Nonetheless, these preliminary
data do suggest that uptake of EMP by T cells may be a valid hypothesis to account for
the decrease in EMP in the supernatant of the co-culture experiments described in chapter
5. Moreover, transfer of E selectin to the T cell may have functional implications, for
example by conferring an increased tendency for T cells to adhere to other leukocytes or
endothelial cells.
316
Figure 6.3: Preliminary experiment demonstrating surface expression of E selectin
(CD62E) on T cells (CD3+) from a healthy adult donor following co-culture with MHC
class 11+ HUVEC and TSST-1 (10 ng/ml).
1% 0.4%
u
o
0
............. 10* 10^ 10^

CD3F CD3F CD3F
0 hours 1 hour 2 hours

U)
6.1.9 A proposed modelfo r SAg-induced vascular injury
The exact mechanism by which SAgs cause disease and in particular vascular injury
remains unclear. Intense cytokine release, especially TNF-a, is believed to be particularly
important in the pathogenesis of the capillary leak underlying the toxic shock syndrome.
Based on the data described in chapter 4 it is possible to propose a model whereby SAgs
may cause vascular injury (figure 6.4). Central to this model is the concept that the
endothelial cell can operate as a SAg presenting cell. SAg may initially meet and be
presented to T cells at sites such as the upper respiratory tract or gut-associated lymphoid
tissue. It is well established that such an interaction results in the production of a plethora
of pro-inflammatory cytokines including IFN-y, TNF-a, IL-1, and IL-2. This in itself
could upregulate MHC class II and cell adhesion molecule expression on endothelial cells
locally and/or systemically. Systemic absorption of SAg on the background o f this
endothelial activation could result in further VP-restricted adherence to the endothelium,
with further massive T cell activation directly on the luminal endothelial surface of blood
vessels. This could result in further endothelial activation and injury mediated by several
potential downstream events including recruitment of other inflammatory cells. In some
vasculitic syndromes (e.g. KD) this may be the initial and predominant triggering event,
whereas in others (such as PAN or WG) such SAg-mediated endothelial injury may be
operating as an additional risk factor on the background of several other genetic (e.g.
FcyR polymorphisms) and environmental (e.g. silica exposure) predisposing factors. This
318
Figure 6.4: Proposed model for SAg-induced vascular injury
KEY
T lymphocyte
(CD4 or CDS)
Activated endothelial
cell
I I M HC class II
Vascular
injury , SAg
APC = antigen presenting cell

T Ly = T lymphocyte
INF-Y
TNF-a
U)
VO C
proposed model does not, however, preclude other mechanisms of vascular injury and
may operate in conjunction with endothelial and vascular injury mediated by
conventional antigens, autoantibodies, complement, or immune complexes.
6.1.10 Future work: SAg peptide antagonists
The fascinating experiments relating to SAg peptide antagonists published by Arad et al
(Arad G, Levy R and others, 2000) open up another line of future experimental work. It
would be interesting to see whether the p i2 SAg antagonist could block Vp restricted T
cell endothelial adherence and activation. In September 2000 contact was made with the
senior author of this paper who unfortunately declined any collaboration. This may have
been because their group is developing this technology as protection against potential
germ warfare with superantigenic toxins (Kaempfer, Arad and others, 2002). SAg
antagonists may prove to be important blockers of SAg-dependent T cell mediated
endothelial injury, however, and this would be an area worthy of considerable future
research.
6.1.11 Cellular microparticles: Potential novel mechanisms o f vascular injury
The observations made in chapter 5 relating to EMP generation from stimulated HUVEC
in vitro led to the in vivo study of EMP in children with vasculitis. In some respects this
was a “sideways” step, but (in the opinion of the author) an important step to take since
the results provide important insight into the endothelial injury associated with vasculitis
in the young. It should be emphasised that the observation o f increased EMP in children
320
with active vasculitis in no way implies that SAgs are necessarily involved in the
pathogenesis of these vasculitic syndromes (although SAg-dependent T cell mediated
endothelial injury clearly is associated with increased EMP formation in vitro), and it is
likely that EMP formation is the endpoint of a number o f different immunological
pathways culminating in endothelial injury.
It is conceivable that microparticles (endothelial, platelet, or other) could be functionally
important in the vascular injury and prothrombotic state associated with vasculitis, and
future work examining this area may provide insight into novel pathogenetic
mechanisms. For example, the prothrombotic potential of PS-rich cellular and platelet
microparticles is likely to be relevant to the vasculitides. An obvious future experiment
would be to compare the fibrin-generating potential o f microparticles derived from either
activated or resting endothelial cells in vitro. Furthermore, it is interesting that
microparticles of neutrophil origin can activate endothelial cells in vitro (Mesri and
Altieri, 1998). Another set of experiments could examine the potential for neutrophil
microparticle generation in response to ANC A stimulation (following TNF-a priming).
In terms of SAgs, it might also be interesting to study the generation of Vp specific
microparticles fi*om T cells in vasculitis patients. Given the inherent problems in
considering T cell VP repertoire skewing such an approach may cast further light on this
complex issue by providing evidence of Vp-specific lymphocyte activation and/or
apoptosis, which would be compatible with an effect of SAgs.
321
It would also be fascinating to investigate if MHC class H positive endothelial cells can
generate MHC class n positive microparticles. One implication o f this hypothesis would
be that such MHC class II positive microparticles might be able to present SAg to T cells
raising the possibility of a mechanism which could massively amplify the immune
response to SAgs and profoundly contribute to the systemic inflammatory response
associated with SAg-mediated diseases.
6.1.12 Organ-specific endothelial microparticles?
Another intriguing question relating to EMP would be to ask whether it was possible to
identify different phenotypic subclasses of EMP that correlated with either the size o f the
blood vessel involved or the organs involved in the vasculitic process. For example,
identification of a specific glomerular endothelial marker may allow the study of specific
glomerular EMP profiles, which may then cast light on the extent of specific renal injury
associated with vasculitis. Another obvious example would be in KD where the
identification of a coronary arterial endothelial marker expressed on EMP may have
prognostic implications in terms of the risk of developing coronary arterial aneurysms. At
the time of writing this thesis the author is not aware of any organ-specific endothelial
markers that would allow such a study, although clearly this approach would be of
considerable interest.
322
6.1.13 Concluding remarks
In conclusion the data presented in this thesis would suggest that T cells are involved in
the aetiopathogenesis of vasculitis syndromes of the young. Moreover, it was possible to
demonstrate peripheral blood T cell VP skewing over and above that observed in disease
controls. Whilst these observations would be compatible with a superantigenic effect,
these data do not unequivocally resolve this contentious issue. One way that SAgs could
contribute to vascular injury may involve the endothelial cell as a SAg presenting cell,
resulting in VP specific CD4 and CDS T cell adherence and activation directly on the
luminal endothelial surface. The consequence of such an interaction is upregulation of
endothelial cell adhesion molecules and the generation o f endothelial microparticles.
Lastly, endothelial microparticles are increased in the circulation o f children with active
vasculitis, and correlate with clinical and laboratory parameters o f vasculitic disease
activity. This observation provides further evidence of endothelial injury in vasculitis,
and it is anticipated that it may provide insight into novel pathogenetic mechanisms of
vascular injury.
323
Appendix 1: Classification of vasculitic syndromes
1. Chapel Hill Consensus (Jennette, Falk and others, 1994)
Karnes and definitions o f vasculkides adopted by the Chapel Hill Consensus Conference on
the Nomenclature o f Systemic Vasculitis____________________________________________
Large vessel vasculitis
G iant cell (tem poral) arteritis Granulomatous arteritis of the aorta and its m ajor branches, w ith a predilection for the extracranial
branches o f the carotid artery. Often involves the tem poral artery. Usually occurs in patients older
than 50 and often is associated w ith polymyalgia rheumatica.
Takayasu arteritis Granulomatous inflammation o f the aorta and its m ajor branches. Usually occurs in patients younger
than 50.
Medium-sized vessel
vasculitis
Polyarteritis no d o sa| (classic N ecrotizing inflammation o f medium-sized or small arteries w ithout glomerulonephritis or vasculitis
polyarteritis nodosa) in arterioles, capillaries, or venules.
Kawasaki disease Arteritis involving large, medium-sized, and small arteries, and associated with muco-cutaneous
lymph node syndrome. Coronary arteries are often involved. Aorta and veins may be involved.
Usually occurs in children.
Small vessel vasculitis
Granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting
W egener’s granulomatosisj
small to medium-sized vessels (e.g., capillaries, venules, arterioles, and arteries). N ecrotizing
glomerulonephritis is common.
Churg-Strauss syndrome^ Eosinophil-rich and granulomatous inllammation involving the respiratory tract, and necrotizing
vasculitis affecting small to medium-sized vessels, and associated w ith asthma and eosinophilia.
M icroscopic polyangiitisf N ecrotizing vasculitis, with few or no immune deposits, affecting small vessels (i.e., capillaries,
(m icroscopic polyarteritis)): venules, or arterioles). Necrotizing arteritis involving small and medium- sized arteries may be
present. Necrotizing glomerulonephritis is very common. Pulmonary capillaritis often occurs.
Henoch-Schonlein purpura Vasculitis, with IgA-dominant immune deposits, affecting small vessels (i.e., capillaries, venules, or
arterioles). Typically involves skin, gut, and glomeruli, and is associated with arthralgias or artliritis.
Essential cryoglobulinémie vasculitis Vasculitis, w ith cryoglobulin immune deposits, affecting small vessels (i.e., capillaries, venules, or
arterioles), and associated w ith cryoglobulins in serum. Skin and glomeruli are often involved.
Cutaneous leukocytoclastic angiitis Isolated cutaneous leukocytoclastic angiitis w ithout systemic vasculitis or glomerulonephritis.
♦Large vessel refers to the aorta and the largest branches directed toward major body regions (e.g., to the
extremities and the head and neck); medium-sized vessel refers to the main visceral arteries (e.g., renal,
hepatic, coronary, and mesenteric arteries); small vessel refers to venules, capillaries, arterioles, and the
intraparenchymal distal arterial radicals that connect with arterioles. Some small and large vessel
vasculitides may involve medium-sized arteries, but large and medium-sized vessel vasculitides do not
involve vessels smaller than arteries. Essential components are represented by normal type; italicised type
represents usual, but not essential, components.
tPreferred term. J Strongly associated with antineutrophil cytoplasmic autoantibodies (ANCA).
324
2. American College of Rheumatology (ACR) Criteria (for polyarteritis nodosa
(Lightfoot, Jr., Michel and others, 1990) modified for children (Brogan, Davies and
others, 2002).
CRTTEIUON DEFINITION
1. Weight loss o f 5% or more, O R failure to thrive Loss o f 5% or more o f body weight since illness began, not
caused by dieting or other factors, O R fall off in weight from the
child's normal centile
2. Livedo reticularis M ottled reticular pattern over the skin o f portions o f the
extremities or torso
3. Testicular pain or tenderness Pain or tenderness o f the testicles, not caused by infection,
trauma, o r other causes
4. M yalgias, weakness, o r leg tenderness Diffuse myalgias (excluding shoulder and hip girdle) or
weakness of muscles or tenderness o f leg muscles
5. M ononeuropathy o r polyneuropathy Developm ent o f mononeuropathy, multiple mononeuropathies, or

polyneuropathy
6. Systemic hypertension Systolic or diastolic B P greater than age-related reference range
7. Elevated blood urea nitrogen or creatinine Elevaticm o f B U N or creatinine above age related reference
range, not due to dehydration or obstruction
8. H epatitis B virus Presence of hepatitis B surface antigen or antibody in serum
9. Arteriograpihic abnormality Arteriogram showing aneurysms or occlusions o f the visceral

arteries, not caused by arteriosclerosis, fibromuscular dysplasia,
or other non-inflammatory causes
10. Biopsy of small- or medium-sized artery containing Histological changes showing the presaice o f granulocytes or
polymorphs granulocytes and m ononuclear leukocytes in the artery wall
The presence o f 3 or more o f the criteria w ill define a pwtient as having PAN.
3. ACR criteria for classification of Henoch-Schonlein purpura (Mills, Michel and

others, 1990)
Critci4n» ^
Palpiable purpura Slightly raised “pialpable” hemorrhagic skin lesions not related to
thrombocytopenia
Age </=20 yr at onset Patient </=20 yr old at onset o f first symptcms
Bow el angina Diffuse abdominal pjain, w orse after meals, or the diagnosis o f
bowel ischemia, usually including bloody diarrhoea
Wall granulocytes on biopsy Histologic changes showing granulocytes in he w alls o f arterioles
or venules
For purposes of classification, a patient shall be said to have Henoch- Schonlein purpura if at least two of
these criteria are present. The presence of any two or more criteria yields a sensitivity of 87.1% and
specificity of 87.7%.
325
4. ACR criteria for the classification of hypersensitivity vasculitis (Calabrese,
Michel and others, 1990)
................. ...............................................................
Age at onset >16 yr Developm ent o f symptoms after age 16 yr
M edication at disease onset M edication that may have been a precipitating factor was taken at
the onset o f symptoms
Palpable purpura Slightly elevated purpuric rash over one or more areas o f skin;
does not blanch w ith pressure and is not related to
thrombocytopenia
M aculopapular rash Flat and raised lesions o f various sizes over one or m ore areas o f
the skin
B iopsy (including arteriole and venule) H istologic changes showing granulocytes in a perivascular or
exlravascular location
For purposes of classification, a patient shall be said to have hypersensitivity vasculitis if at least three of
these criteria are present. The presence of any three or more criteria yields a sensitivity of 71.0% and
specificity of 83.9%. The age criterion is not applicable for children.
5. ACR criteria for the classification of Wegener’s Granulomatosis (Leavitt, Fauci

and others, 1990)
CHtcrîMi Defîaitfttn: :
Nasal or oral inflammaiton Painful or painless oral ulcers or purulent or bloody nasal
discharge
Abnormal-apDpwaring chest radiograph Nodules, fixed infiltrates or cavities
■Abnormal urinary sediment M icrohem aturia (>5 R BC /hpf) or R B C casts
Granulomatous inflammation Granulomatous inflammation within the w all of an artery or in
the perivM culy or extrayascular area o f m artery or arteriole
Diagnosis of Wegener’s granulomatosis requires the presence of two of the four criteria. The presence of
any two or more criteria has a sensitivity of 88.2% and a specificity of 92.0%. RBC, red blood cells.
326
Appendix 2: The Birmingham Vasculitis Activity Score
(EVAS) (Luqmani, Bacon and others, 1994)
Tick box only if abnormality is newly present or worsening within the previous 4 weeks
and ascribable to vasculitis
1. SYSTEMIC 3 (maximum total)

none 0
malaise 1
myalgia 1
arthralgia/arthritis 1
fever (<38.5°C) 1
fever (>38.5®C) 2
wt loss (1 -2 kg) within past month 2
wt loss (>2 kg) within past month 3
2. CUTANEOUS 6 (maximum total)

none 0
infarct 2
purpura 2
other skin vasculitis 2
ulcer 4
gangrene 6
multiple digit gangrene 6
3. MUCOUS MEMBRANES/EYES 6 (maximum total)

none 0
mouth ulcers 1
genital ulcers 1
conjunctivitis 1
episcleritis 2
uveitis 6
retinal exudates 6
retinal haemorrhage 6
4. ENT 6 (maximum total)

nil 0
nasal discharge/obstruction 2
sinusitis 2
epistaxis 4
crusting 4
aural discharge 4
otitis media 4
new deafiiess 6
hoarseness/laiyngitis 2
subglottic involvement 6
5. CHEST 6 (maximum total)

none 0
dyspnoea or wheeze 2
nodules or fibrosis 2
327
pleural efiüsion/pleurisy 4
infiltrate 4
haemoptysis/haemorrhage 4
massive haemoptysis 6
6. CARDIOVASCULAR 6 (maximum total)

none 0
bruits 2
new loss of pulses 4
aortic incompetence 4
pericarditis 4
new myocardial infarct 6
CCF/cardiomyopathy 6
7. ABDOMINAL 9 (maximum total)

none 0
abdominal pain 3
bloody diarrhoea 6
gall bladder perforation 9
gut infarction 9
pancreatitis 9
8. RENAL 12 (maximum total)

none 0
hypertension (diastolic >90) 4
proteinuria (>1 + or >0.2 g/24 h) 4
haematuria (> 1 + or >10 rbc/ml) 8
creatinine 125-249 pmol/1 8
creatinine 250-499 pmol/1 10
creatinine >500 pmol/1 12
rise in creatinine > 10% 12
9. NERVOUS SYSTEM 9 (maximum total)

none 0
organic confusion/dementia 3
seizures (not hypertensive) 9
stroke 9
cord lesion 9
peripheral neuropathy 6
motor mononeuritis multiplex 9
MAXIMUM SCORE 63
328
Appendix 3: 96-well Antibody plate plan for determination of
the T cell Vp repertoire
Cells CD3F Vp8F VP8F & CD3PE CD69PE cx> m ^ CD4^QR tD S Q R

alone isotype Isotype ^ PE ^ lsot)pe
.
control control control
/'
CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE
CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR
V plF Vp2F \p 3 F VPS.IF VP5.2F VP7.IF VP8F V P IIF V pl2F VP13.1F \ p l3 .6 F V p l4 F
CD3PE CD3PE CD3PE CD3PE CD3PE

CD4QR CD4QR CD4QR CD4QR CD4QR
VPI6F V pl7F Vp20F VP2I.3F VP22F
CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE CD3PE
CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR CD8QR
V plF Vp2F Vp3F VPS.IF VP5.2F VP7.1F VP8F V p llF V pI2F VP13.1F VP13.6F VI 4P F
CD3PE CD3PE CD3PE CD3PE CD3PE

CD8QR CD8QR CD8QR CD8QR CD8QR
VpI6F V17PF Vp20F V P2I.3F VP22F
CD3F CD3F
CD69PE CD69PE
CD8QR CD4QR
CD3F CD3F
CD25 PE CD25PE
CD4QR CD8QR
3 29
Appendix 4: 96-well antibody plate plan for determination of T
cell VP-specific CD69 expression
Cells Vp8F Vp8F % W 9PE CD4 QR ;

alone Isotjpc ■ Isotype
control control
CD69PE CD69PE CD69PE CD69PE CD69PE CD69PE

CD4QR CD4QR CD4QR CD4QR CD4QR CD4QR
VplF VP2F \P 3 F VpS.lF Vp8F VP12F
CD69PE CD69PE CD69PE CD69PE CD69PE CD69PE

CD8QR CD 80R CD8QR CD8QR CD8QR CD8QR
VplF VP2F \p 3 F VP5.1F VP8F V pi2F
330
Appendix 5: Flow cytometry instrument settings for PBMC
and T cells, HUVEC and microparticles
PBM C/f ceils

Detector Voltage Amp gain Mode Threshold Compensation
Forward EOO 1.00 Linear 105 FL1-0.5%FL2
scatter FL2-36% FL l
Side scatter 440 2.3 Linear 52 FL2-37%FL3
(for CD4)
FLl (FITC) 582 - Logarithmic 52 FL2-3.7% FL3
(for CD8)
FL2 (PE) 645 - Logarithmic 52 FL3-22.4%FL2
FL3 (QR) 715 - Logarithmic 52
HUVEC
Forward El - Logarithmic 80 Non-apphcable
scatter since only single
Side scatter 359 1.00 Linear 52 colour FACS
analysis
FLl (FITC) 401 - Logarithmic 52 performed
FL2(PE) 408 - Logarithmic 52
FL3 (CYC) 159 - Logarithmic 52
Microi>;9»ticles
Forward EOl - Logarithmic 80 FL1-4.8%FL2
scatter FL2-13.7% FLl
Side scatter 377 - Logarithmic 52 FL2-0%FL3
FL3-18.1%FL2
FLl (FITC) 782 - Logarithmic 136
FL2 (PE) 749 - Logarithmic 52
FL3 (CYC) 803 - Logarithmic 52
3 31
Appendix 6: Equation used to derive the number of 3 pm latex
beads added for the analysis of microparticles (Sigma product
information)
OR N«'1.828x10**
« X P sX d ’
Wh^re:
number of beads per ml
S« % solids (wAv)
D - diameter (pM)
Fs^ density of bulk polymer (g/ml)
Pi “ density of latex (g/ml)
When;
S» îo% solids
S - L05 g/tn, (polystyrene).
PL- U005 g/ml
332
Appendix 7: Plate plan for T cell and HUVEC co-culture experiments
(1000W #)#T :
r' ' cells)
Z'/ZiA '''
iW"'
^»''A//'A"
iV-V,
S]
333
Appendix 8: Plate plan for integrin blockade of SAg-mediated T cell adhesion to HUVEC
' "m
' c' c ^ 'm
' „ÿ, ,
„
##&
a
« U 0 »
' i 'i i
# F
M M m
f
I
334
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Publications from this thesis:
Brogan PA, Bose A, Burgner D, Shingadia D, Tulloh R, Klein N, Michie C, Levin M,
Booy R, Dillon MJ. Kawasaki disease; an evidence based approach to diagnosis and
treatment and proposals for future research. Arch Dis Child 2002; 86: 286-290.
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2000; 2:411-416.
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vasculitides. Clin Exp Immunol 2003;131:517-527.
Papers submitted and pending peer review:
Brogan PA, Brachet C, Shah V, Hamden A, Mant D, Klein N, Dillon MJ. Endothelial
microparticles: just blood dust or a must for the diagnosis o f childhood vasculitides?
Arthr Rheum Mar 2003 (original article).
Brogan PA, Shah V, Klein N, Dillon MJ. Superantigenic activation o f T lymphocytes and
endothelial cells: a mechanism for superantigen mediated vascular injury. Arthr Rheum
Mar 2003 (original article).
Brogan PA. Recent advances in Kawasaki disease: In: Recent advances in paediatrics
(David TJ, Ed; book chapter submitted April 2003).
402

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University o f London

Superantigens, endothelial injury and

Paul Anthony Brogan BSc (Hon) MBChB (Hon), MRCP, MSc.

Submitted for the degree o f Doctor o f Philosophy.

Department o f Nephro-Urology, Institute o f Child Health,

All rights reserved

INFORMATION TO ALL USERS

Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author.

All rights reserved.

I would like to acknowledge my supervisors. Professor Michael Dillon and Dr Nigel

studies described in this thesis.

Paul Brogan, October 2002.

Superantigens, endothelial injury, and vasculitis of the young.

CHAPTER 1; INTRODUCTION AND THESIS AIMS______________________________________ 15

1*1 ^^S^%UIjl 1 IS 'I'lHi

L2 THE IMMUNO PATHOGENESIS OF VASCULITIS_______________________________ 58

C.TJI^n'IS •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••« 1OS

CHAPTER 2. MATERIALS AND

2.1 INTRODUCTION_________________________________________________________________ 132

2.2 SUBJECTS, CLASSIFICATION OF VASCULITIC SYNDROMES, AND ASSESSMENT OF

2.3 MATERIALS_____________________________________________________________________ 135

2.5 ISOLATION AND ANALYSIS OF WHOLE BLOOD CELLULAR AND PLATELET

2.5.1 P repa ra tio n o f platelet poor pla sm a fo r m ic r o pa rticl e a n a l y s is .........................................158

CHAPTER 3; T CELL ACTIVATION AND V p REPERTOIRES IN CHILDHOOD

3.2 INTRODUCTION_________________________________________________________________ 177

3.3 lEN 1S IMETH^IDS .....m..m..m.........m..m.......m..m.....m..m......m.......m..m.....m..m..m.......................179

CHAPTER 4: Vp-RESTRICTED T CELL ADHERENCE TO ENDOTHELIAL CELLS: A

4.3 MATERIALS AND METHODS_____________________________________________________ 218

4.5 ^)ISC«%ISSI^)N.....w #.........M..w*. ......w......M.....w#..... . . . . . . . . . . . . . .............M.. 251

CHAPTER 5: ENDOTHELIAL AND PLATELET MICROPARTICLES IN CHILDHOOD

5.2 JL^i l lt^)DU(.x 1 I^)N . .. . .M . .. . .. M . .. . .. . .. . .. . M . . .. . .. M .. . .. . M .. . . . . . . . . . . . M . . . M . . M . . M . . M . . . . . . . . . . . . . M . . . M . . . . . . . . . . M . M . . M . . M . . . . . M M . . 26 I

5.4 RESULTS________________________ 270

CHAPTER 6: GENERAL DISCUSSION AND FUTURE DIRECTIONS_____________________ 301

6.1 CrENFIj^l^^Ij ^HSCfUSSI^IN.m*.m..................... m.m............... m. . . . . . . . . . . . . . 3 0 2

APPENDIX 1; CLASSIFICATION OF VASCULITIC SYNDROMES_______________________ 324

APPENDIX 2: THE BIRMINGHAM VASCULITIS ACTIVITY SCORE (BVAS) (LUQMANI,

APPENDIX 3: 96-WELL ANTIBODY PLATE PLAN FOR DETERMINATION OF THE T CELL

APPENDIX 4; 96-WELL ANTIBODY PLATE PLAN FOR DETERMINATION OF T CELL VP-

APPENDIX 5: FLOW CYTOMETRY INSTRUMENT SETTINGS FOR PBMC AND T CELLS,

APPENDIX 6: EQUATION USED TO DERIVE THE NUMBER OF 3 ^M LATEX BEADS ADDED

APPENDIX 8; PLATE PLAN FOR INTEGRIN BLOCKADE OF SAG-MEDIATED T CELL

REFERENCE LIST__________ 335

]PUIfIjIC^^^lT^)^fS 'I'HlS 'H lESIS .............................M.. . . . . . . . . . . . . . . . . . . . . . . . M......M.M......M..M..... 402

1.1 Vasculitis of the young

1.2 The immuno-pathogenesis of vasculitis

1.3 Superantigens and vasculitis

1.4 Thesis aims

1.1.1 Introduction- definition o f vasculitis

The vasculitides are a group of disorders characterised by the presence o f inflammatory

infiltrates (which may be predominantly neutrophilic, eosinophilic, or mononuclear) in

Vasculopathy is a broader term encompassing abnormalities o f blood vessels that may be

inflammatory, but may also be developmental or degenerative. More recently, the

distinction between certain degenerative vasculopathies and inflammatory vasculopathy

atherosclerosis (previously considered to be a degenerative vasculopathy) involves an

systemic vasculitis e.g. microscopic polyangiitis), or one aspect of a more widespread

tissue diseases) (Flores-Suarez and Alarcon-Segovia, 2000).

aetiology o f most vasculitis syndromes. Moreover, as in adult vasculitic syndromes,

features o f the various individual diseases that will be discussed below.

process: Wegener’s granulomatosis, for instance, is classified as a small vessel disease

the renal artery and aorta.

comparison with previously published studies. The limitations of such classification

but not to diagnose vasculitis in the first instance (Hunder, 1998).