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J. Pathol. 189: 265272 (1999)

IMMUNOHISTOCHEMICAL ANALYSIS OF THE
ACTIVATION OF NF-B AND EXPRESSION OF
ASSOCIATED CYTOKINES AND ADHESION
MOLECULES IN HUMAN MODELS OF ALLERGIC
INFLAMMATION
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1
University Medicine, University of Southampton, U.K.
2
Pharmacia & Upjohn Inc., Kalamazoo, U.S.A.
3
Signal Pharmaceuticals, San Diego, CA, U.S.A.
SUMMARY
To investigate the role of NF-B in regulating allergic inammation, a monoclonal antibody directed to the activated form of NF-B
has been developed and immunohistochemistry has been employed to study the pro-inammatory transcriptive function of NF-B and
the adhesion molecules and cytokines that it regulates. Human umbilical vein endothelial cells (HUVECs) exposed to physiological levels
of TNF demonstrated dose- and time-dependent cytoplasmic and nuclear activation of NF-B, followed by up-regulation of ICAM-1.
This was suppressed by the selective inhibitors of NF-B activation, calpain and gliotoxin. Using monoclonal antibodies directed to
NF-B and associated cytokines and adhesion molecules, immunohistochemistry was applied to bronchial explants stimulated ex vivo
with TNF, and to nasal polyp tissue, embedded in glycol methacrylate. Stimulation of the bronchial explants increased expression of
NF-B, IL-8, and GM-CSF in the epithelium and endothelium and ICAM-1 in the endothelium. In nasal polyp, expression of NF-B
was in the epithelium, the endothelium and in submucosal mast cells, eosinophils, T and B lymphocytes, and macrophages. Thus,
immunohistochemistry can be used to determine the cellular provenance of NF-B and its activation status in single cell and complex
tissue systems, in parallel with appropriate inammatory markers. Copyright 1999 John Wiley & Sons, Ltd.
KEY WORDSallergic inammation; adhesion molecules; cytokines; gene regulation; NF-B
INTRODUCTION
The transcription facotor NF-B is a key regulator in
allergic inammation, mediating the transcription of
genes for the adhesion molecules ICAM-1, VCAM-1,
and E selectin, and the cytokines GM-CSF, TNF, IL-6,
and IL-8.
1,2
NF-B is usually a heterodimer comprising
the subunits p50 and p65. Each subunit contains an
NF-B/rel/dorsal domain, consisting of an N-terminal
DNA binding site and C-terminal nuclear location sig-
nal (NLS), necessary for translocation across the nuclear
membrane.
3
NF-B exists in the cytoplasm of cells in an
inactive form due to reversible binding of an inhibitory
protein (IB), which prevents nuclear translocation
by obscuring the NLS.
4,5
Activation of NF-B and
subsequent nuclear translocation occur when IB is
phosphorylated, dissociates from the complex, and is
degraded.
6,7
These events may be initiated by a range of
inammatory mediators.
79
NF-B regulatory elements
have been shown to be expressed in the endothelium,
epithelium, T and B lymphocytes, and macrophages.
8
Traditionally, the electrophoretic mobility shift assay
has been used to investigate the activation of NF-B.
10
This technique can be applied to isolated cells and
tissues and provides information about the overall
activation status of NF-B; however, because nuclear
protein preparations are required for the assay, it fails
to provide information about the cellular provenance,
the number of cells expressing activated NF-B, the
architectural relationship of the cells to their surround-
ing tissue, or the intracellular distribution of p65/p50.
To overcome these disadvantages, we have developed
a monoclonal antibody (MAb 2C7) to the active dimeric
form of NF-B, which is directed to an epitope overlap-
ping the NLS on p65. We have used this MAb and MAb
G96 directed to an epitope near the C terminus of p65 of
NF-B, which is immunoreactive with both the inactive
NF-BIB trimer and the active dimer of NF-B, and
MAbs to inammatory cells, adhesion molecules, and
cytokines to study the role of NF-B in human models
of allergic inammation.
MATERIALS AND METHODS
Monoclonal antibody development
Balb/C mice were immunized and boosted three times
with a p65 NLS-derived peptide (CDTDDRHRIEEKR
KRKT)-KLH conjugate,
11
hybridomas prepared,
12
and
the supernatants screened by ELISA with the p65 NLS
peptide and recombinant p65 protein.
11
Selected sub-
clones were assessed by western blotting with recom-
binant p65 and nuclear and cytoplasmic fractions of
*Correspondence to: Dr Susan J. Wilson, University Medicine,
Centre Block, Level D, Southampton General Hospital, Southampton
SO16 6YD, U.K. E-mail: sjw1@soton.ac.uk
CCC 00223417/99/11026508$17.50
Copyright 1999 John Wiley & Sons, Ltd.
Received 3 August 1998
Revised 22 January 1999
Accepted 5 May 1999
266 S. J. WILSON ET AL.
Copyright 1999 John Wiley & Sons, Ltd. J. Pathol. 189: 265272 (1999)
TNF-stimulated HUVECs. Antibody specicity was
conrmed by pretreating HUVECs with the inhibitors
of NF-B activation, calpain
13
or gliotoxin,
14
prior to
stimulation and immunouorescent staining. Clone 2C7
was selected as suitable for immunohistochemistry.
Immunohistochemical studies
Endothelial cellsHUVECs (BioWhittaker, Walkers-
ville, MD, U.S.A.) were grown to conuency and stimu-
lated with rTNF (R&D Systems Europe, Abingdon,
U.K.). Optimal conditions were determined in time (0,
05, 3, 6, 9, and 24 h with 5 ng/ml rTNF) and dose
response studies including physiological concentrations
of TNF (0, 25, 50, 100, 500, and 5000 pg/ml).
15,16
Following stimulation, HUVECs were xed in parafor-
maldehyde and stained using an indirect FITCavidin
method using MAbs to NF-B (2C7) and to ICAM-1
(clone RR1, Boehringer-Ingleheim, Ridgeeld,
U.S.A.).
17
The stained cells were examined using a Leica
TCS4D confocal laser scanning microscope and the
mean intensity uorescence was recorded.
Lung explantPieces of bronchial tissue (2 mm
3
) were
dissected from normal tissue of lobectomy samples and
maintained in tissue culture medium (LHC9, Gibco Life
Technologies, Paisley, U.K.) overnight. The explants
were stimulated with rTNF (5 ng/ml) for 6 h or main-
tained in culture medium alone, xed in acetone,
and processed into glycol methacrylate resin (GMA).
18
Sections of 2 m were stained immunohistochemically
using the Strept-avidin biotin-peroxidase detection
system and MAbs to NF-B (clone 2C7, MAb G96,
Pharmigen, San Diego, U.S.A.), IL-8 (BioWhittaker,
Wokingham, U.K.), GM-CSF (R&D Systems,
Abingdon, U.K.), TNF (Celltech, Slough, U.K.),
ICAM-1, and endothelium (EN4, Bradsure Biologicals,
Loughborough, U.K.). Control sections were incubated
with isotype-matched immunoglobulins. The number of
positive cells per mm
2
of submucosa, the percentage
expression of NF-B and cytokines in the epithelium,
and the proportion of stained vessels were enumerated.
Nasal polypNasal polyp (2 mm
3
) was processed into
GMA, stained, and analysed as described above. In
addition, double IHC was used to identify the cellular
provenance of NF-B using cell-specic markers:
tryptase for mast cells (AA1, Dako, High Wycombe,
U.K.), cationic protein for eosinophils (EG2,
Pharmacia and Upjohn, Milton Keynes, U.K.), CD3 for
T lymphocytes (Dako), CD14 for macrophages (Dako),
and CD20 for B lymphocytes (Dako), followed by
staining for NF-B.
RESULTS
Monoclonal antibody development
On initial ELISA screening, 300 supernatants were
positive with the p65 NLS peptide and ve were also
positive with the p65 recombinant peptide. Following
subcloning, two clones (2C7 and 9B8) maintained
immunoreactivity (Fig. 1A). By western blotting
(Fig. 1B), the subclones of 2C7 (lanes 1, 2, and 48)
exhibited reactivity with recombinant p65, whereas 9B8
(lane 9) did not. There was no reactivity with the
negative control antibody.
The 2C7 MAb was shown to recognize a 65 kD
protein in cytosolic lysates of HUVECs and was un-
aected by TNF stimulation (Fig. 1C, lanes 1 and 2).
In unstimulated cells, there was only weak reactivity
with the 65 kD protein in nuclear lysates, but strong
reactivity following TNF stimulation. Following TNF
activation, immunouorescent staining of HUVECs
with MAb 2C7 (subclone SD8) was observed in the
nuclei and perinuclear area (Fig. 1D). Pretreatment
of the cells with the selective inhibitors of NF-B
activation, calpain and gliotoxin, almost completely
abolished staining with MAb 2C7 (Fig. 1D), conrm-
ing antibody specicity for activated NF-B. MAb 2C7,
clone SD8 was employed for further immunohisto-
chemical studies.
Immunohistochemical studies
Endothelial cellsThere was low constitutive expres-
sion of the active form of NF-B and of ICAM-1.
NF-B immunoreactivity was localized to the cyto-
plasm, while ICAM-1 was intracellular. After TNF
exposure, expression of activated NF-B was evident
after 05 h as weak cytoplasmic and nuclear staining
accompanied by stronger perinuclear staining. NF-B
activation peaked at 6 h, with strong staining of cyto-
plasm and nuclei. ICAM-1 staining lagged behind that
of NF-B, occurring rst at 3 h, peaking at 6 h, and
being maintained for up to 24 h (Figs 2A2C). When
studied at 6 h, both activated NF-B and ICAM-1
immunoreactivity showed dose-dependent increases to
physiological concentrations (25100 pg/ml) of TNF
(Figs 2D2F).
Lung explantIn lung explant tissue, a variety of cells
within the submucosa expressed inactive and active
NF-B, IL-8, GM-CSF, and TNF. Immunoreactivity
with MAbs 2C7 and G96 was predominantly cyto-
plasmic. Following stimulation with rTNF, there were
no changes in the number of positively stained sub-
Fig. 1Characteristics of NF-B antibodies. Analysis of p65 directed MAb by ELISA (A) showed that the subclones 9B8 and 2C7 were reactive
with both the NLS peptide and the recombinant p65 peptide. Analysis of p65 antibodies (B) by western blotting of recombinant p65 indicated that
the 2C7-derived clones were reactive (lanes 1, 2, and 48). The 9B8 clone (lane 9) and the negative control MAb (lane 3) were non-reactive. Analysis
of 2C7 MAb by western blotting of HUVEC fractions (C) indicated positive reactivity with a 65 kD protein in the cytoplasmic fractions, pre- and
post-TNF stimulation (lanes 1 and 2). Weak reactivity is observed with the 65 kD protein in the nuclear fractions of untreated HUVECs, and strong
reactivity after TNF stimulation (lanes 3 and 4). In TNF-treated HUVECs, immunouorescent staining with MAb 2C7 (activated NF-B) resulted
in nuclear and strong perinuclear immunoreactivity (Di and Dii). Positive staining is green. When pretreated with the inhibitors of NF-B activation,
calpain (10 m) (Diii) and gliotoxin (05 g/ml) (Div), minimal staining is observed. Scale bar=20 m
267 EXPRESSION OF NF-B IN HUMAN MODELS OF ALLERGIC INFLAMMATION
Copyright 1999 John Wiley & Sons, Ltd. J. Pathol. 189: 265272 (1999)
Fig. 2Time and dose responses for NF-B and ICAM-1 expression following TNF stimulation of HUVECs. At time
zero, expression of activated NF-B (MAb 2C7) and ICAM-1 is minimal (AC). Nuclear and cytoplasmic expression of
activated NF-B, seen as green immunouorescence (B), is evident at 05 h and peaks at 6 h. Expression of ICAM-1, seen
as green immunoreactivity with orange nuclear counterstaining (C), lags behind that of activated NF-B, being rst evident
at 3 h and then increasing in a time-dependent manner. The expression of activated NF-B and ICAM-1 increases in a
dose-dependent manner (DF). Scale bar=20 m (n=3)
268 S. J. WILSON ET AL.
Copyright 1999 John Wiley & Sons, Ltd. J. Pathol. 189: 265272 (1999)
Fig. 3Expression of NF-B, cytokines, and adhesion molecules in bronchial explants. The percentage expression of NF-B and
associated cytokines and adhesion molecules is increased in the endothelium (A) and epithelium (B) of bronchial explants following
TNF stimulation. This increased expression can be observed in GMA sections stained with MAb directed to activated and unactivated
NF-B (G96) (C), activated NF-B (D), IL-8 (E), GM-CSF (F), and ICAM-1 (G). In all photographs, positive staining is red, control
sections are shown on the left, and TNF-treated on the right of each pair. In CG, the scale bar equals 40 m
269 EXPRESSION OF NF-B IN HUMAN MODELS OF ALLERGIC INFLAMMATION
Copyright 1999 John Wiley & Sons, Ltd. J. Pathol. 189: 265272 (1999)
Fig. 4Immunohistochemical staining for NF-B in GMA-embedded nasal polyp. Immunoreactivity with MAb G96
directed to activated and inactivated NF-B is observed in the epithelium, endothelium, and submucosal cells (A). MAb
2C7 immunoreactivity is seen in the endothelium and cells within the submucosa (B). Double immunohistochemical
staining with MAb 2C7 (blue) for activated NF-B is seen in the nuclei and co-localizes with the numerous cells (stained
brown). Strongest reactivity is seen in mast cells (C), eosinophils (D), and B lymphocytes (G); weaker activity is observed
in T lymphocytes (E) and macrophages (F). Scale bar=20 m
270 S. J. WILSON ET AL.
Copyright 1999 John Wiley & Sons, Ltd. J. Pathol. 189: 265272 (1999)
mucosal cells. In vascular endothelium, TNF increased
cytoplasmic staining of vessels using either MAb to
NF-B (2C7 or G96) and this was accompanied by
increased immunostaining for IL-8, GM-CSF, and
ICAM-1. In the epithelium, TNF increased staining for
both forms of NF-B with some nuclear but predomi-
nantly perinuclear staining; IL-8 and GM-CSF were
also increased (Figs 3A3G). Isotype-matched control
sections were all negative.
Nasal polypStaining with both MAb 2C7 and MAb
G96 was observed in the basal and lateral surfaces of
epithelial cells and in the cytoplasm of endothelial and
submucosal cells (Figs 4A and 4B). Double IHC
revealed strong nuclear staining for both forms of
NF-B in mast cells, eosinophils, and B cells; weaker
nuclear staining in macrophages; and cytoplasmic
staining in T lymphocytes (Figs 4C4G).
DISCUSSION
We have developed a MAb directed to activated
NF-B and have demonstrated its use in human cell and
tissue models of inammation. Using this MAb, we have
shown that NF-B activation occurs in several key cell
types involved in allergic inammation, including mast
cells, eosinophils, lymphocytes, endothelial cells, and
epithelial cells. The expression and activation of NF-B
in mast cells and eosinophils have not been previously
reported.
MAb 2C7 developed in this study was selected speci-
cally to recognize an epitope overlapping the p65 NLS
which is exposed once IB has dissociated from the
p65/p50/IB complex. MAb 2C7 immunoreactivity was
not conned to cell nuclei, as under several experimental
conditions, cytoplasmic and perinuclear staining of
distinct cell types was also observed. The absence of
staining in the presence of calpain, a proteosome
inhibitor,
13
and gliotoxin, which inhibits IB degrada-
tion,
14
conrmed immunoreactivity towards activated
NF-B. The staining pattern observed with MAb 2C7
demonstrates incomplete nuclear translocation of p65/
p50, suggesting that continued exposure to an activating
stimulus exceeds that required to induce gene transcrip-
tion, resulting in an accumulation of active NF-B in the
cell cytoplasm and perinuclear region. This would also
account for the diculty in visualizing nuclear expres-
sion of activated NF-B in sections of lung explant and
nasal polyp when nuclei have been counterstained with
haematoxylin. Nuclear expression of activated NF-B
with MAb 2C7 is evident in the tissue sections stained in
Figs 4C4G, where nuclear counterstaining has been
omitted. This pattern of staining with MAb 2C7 is
similar to that reported by Kaltschmidt et al.,
19
who
developed a commercially available MAb (Boehringer
Mannheim) directed to the activated form of NF-B. In
our systems, this commercial antibody was less sensitive
than MAb 2C7.
In HUVECs, there was minimal constitutive acti-
vation of NF-B and ICAM-1 expression. Following
TNF stimulation, expression of activated NF-B was
observed within 30 min and reached maximum at 6 h.
ICAM-1 expression was seen at 3 h and also peaked at
6 h. This time course for in vitro up-regulation of
ICAM-1 is similar to that reported in vivo in the
bronchial mucosa of asthmatic subjects after segmental
allergen challenge,
20
consequent upon TNF release
from activated mast cells.
2123
Increased expression of
activated NF-B and ICAM-1 was observed at TNF
concentrations as low as 25 pg/ml, levels that have been
measured in nasal and bronchial lavage of patients with
rhinitis
15
and asthma,
16
respectively. Comparable
responses have also been observed in passively sensitized
and challenged bronchial explants.
23
Lung explants and nasal polyps provide an integrated
model
21,23,24
to investigate NF-B-driven pro-
inammatory events. We employed immunohistochem-
istry applied to GMA sections, which oers a number of
distinct advantages over conventional wax or frozen
sections
18
and in the lung explant we were able to
extrapolate the ndings observed in single cell systems.
In the endothelium, there was up-regulation of ICAM-1,
IL-8, and GM-CSF which was accompanied by an
increased expression of both the active and the inactive
forms of NF-B. In the epithelium, there was
up-regulation of IL-8 and GM-CSF.
Using nasal polyp as a model for studying allergic
inammation, we suggest an important role for NF-B
activation in the endothelium, the epithelium, and a
variety of cells within the submucosa. Double IHC
demonstrated that the mucosal cells expressing high
levels of NF-B were mast cells, eosinophils, T and B
lymphocytes, and macrophages. Expression of activated
NF-B within mast cells and eosinophils implies that
these cells have an important regulatory function.
Taken together, our observations show the advantage
of using immunohistochemistry to study the importance
of NF-B as a regulator of the initiation and mainten-
ance of the allergic inammatory response. By providing
detail of the cellular provenance and co-localization of
NF-B-regulated genes such as adhesion molecules and
cytokines, this approach is an important adjunct to
conventional gel shift assay.
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