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Κ. ΛΙΟΠΕΤΑ ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ 2008
Κ. ΛΙΟΠΕΤΑ ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ 2008
cAMP
2008
cAMP
, A , .
, , .
, , .
, A , .
, , .
, , .
, , .
, , .
, , .
, , .
&
.
...
-
, ...
.. ,
... ,
..- !
. , - -
, , , , , ..
, , ...
, ...
, ,
, .
, , , , ,
..... - - , .
.
. .
, .
, .
, . ,
, .
.
. , . ,
. , . , . , .
, .
.
. .
. (. ), . (.
) . (),
,
.
.
, . .
.
. . ,
, -
- ,
.
, .
, ,
,
. , ,
, , , ,
, , &
.
,
, ,
..
cAMP
. cAMP
IL-10 .
cAMP IL-10 -
.
- antiCD3/anti-CD28, anti-CD3 Ionomycin/PMA
cAMP (10-6 Forskolin, 10-6 PGE2, 5x10-6
Rolipram 10-6 8-Br-cAMP). IL-10
ELISA mRNA IL-10 Real Time PCR.
IL-10
(1327 bp) (-1010, -500, -310, 235, -135 bp). MEF-2 CREB
EMSA,
MEF-2 CREB.
cAMP IL-10
50-60% Ion/PMA, 80-90% anti-CD3
anti-CD3/anti-CD28. IL-10 antiCD3, IL-10,
(IL-2 & IL-4). IL-10
mRNA 50%
. IL-10
cAMP
. ,
cAMP .
IL-10 cAMP (50 %
) 500 bp TATA box,
MEF-2 CREB,
Consite. MEF-2
- 70% cAMP
. , cAMP
(x 2,5) CREB (x 2).
cAMP IL-10 , .
- . cAMP
IL-10 -
MEF-2 CREB.
/ MEF-2 CREB
.
-: , cAMP, IL-10,
, , -, mRNA, IL10, , MEF2, CREB.
_________________________________________________________________ 4
_____________________________________________________________ 6
_________________________________________________________ 10
______________________________________________________12
1.
_____________________________________________________________ 12
1.1
______________________________________________________12
1.1.1
__________________________________________14
1.1.1.1
_________________________________________________________15
1.1.1.2
___________________________________________________17
1.1.1.3
____________________________________________20
1.1.1.4
______________________________________________24
1.1.2
________________________________________________________________27
1.1.2.1
10(IL10) ____________________________________________________30
1.1.2.2
2(IL2) ______________________________________________________33
1.1.2.3
4(IL4)_______________________________________________________34
1.2
__________________________________________35
1.2.1
G ___________________37
1.2.1.1
G_____________________________________________________________37
1.2.2
cAMP _______________________________________________________________________39
1.2.2.1
cAMP _________________________________________________________40
1.2.2.2
_______________________________________________________41
1.2.2.3
cAMP _______________________________________________41
1.2.2.4
(PKA) _________________________________________________43
1.2.2.5
cAMP ________________________________________________________46
1.2.2.6
______________________________________________________46
1.2.2.7
cAMP _________________________________________________47
1.2.2.8
cAMP ____________________________________________________48
1.3
_______________________________________________________50
1.3.1
MEF2 ______________________________________________51
1.3.2
CREB _______________________________________________54
1.3.3
cAMP ___________________56
1.3.4
IL10
_________________________________________________________________58
2.
_____________________________________________________ 60
______________________________________________________62
1.
2.
________________________________________________________________ 62
____________________________________________________________ 68
2.1
_________________________68
2.1.1
___________________________________________________________________68
2.1.2
___________________________________________________________68
2.1.3
________________________________71
2.1.4
__________________________________________________72
2.1.4.1
__________________________________72
2.1.4.2
Erosetting___________________________________________73
2.1.5
L
(Leucinemethylester). _________________________________________________________75
2.1.6
MACS____________________________________________75
2.1.7
_______________________________________________________________________ 77
2.2
__79
2.2.1
____________________________________79
2.2.1.1
________________________________________________________80
2.2.1.2
rhIL2 __________________________________80
2.2.1.3
AI(PKAI) ________________________________________________________80
2.2.2
(10,2
4)
. ____________________________________________________________________81
2.3
IL10cAMP
____________________________________________________83
2.3.1
RNA __________________83
2.3.2
____________84
2.3.3
mRNA10
__________________________________________85
2.3.3.1
(RTPCRReverse
TranscriptionPolymerasechainreaction)______________________________________86
2.3.3.2
(RTPCR,
RealTimePCR)___________________________________________________________87
2.3.3.3
mRNA10
,D. ___________89
2.4
_________________________________________90
2.4.1
Invivo _________________90
2.4.1.1
E.coli
(transformation) _________________________________________________________90
2.4.1.2
/ ___________________90
2.4.1.3
________________________________91
2.4.1.4
DNA _______________________92
2.4.1.5
DNA(transfectionbyelectroporation) _________________93
2.4.1.6
______________________________________________________________95
2.4.1.7
BRADFORD. __________________________________________________96
2.4.1.8
______________________________97
2.4.2
Invitro _______________________________99
2.4.2.1
5DNA(5end
labeling) ________________________________________________________________99
2.4.2.2
____________________________100
2.4.2.3
DNA
_____________________________101
2.4.2.4
DNAconsensus ________________________________________________103
2.4.3
IL10 ____________________________103
3.
____________________________________________________________ 104
4.
_____________________________________________________ 106
4.1
4.1.1
4.1.2
4.1.3
4.1.4
4.1.5
4.2
&
. __________________________________________106
cAMPIL10
. _____________________________________________________________106
cAMPIL2
. _____________________________________________________________110
cAMPIL10
IL2.______________________________111
cAMPIL4
. _____________________________________________________________112
cAMPIL10,TA
,PKA. _________________________________113
IL10CAMP
. _______________________________________115
4.2.1
4.2.2
4.2.3
4.2.4
4.3
4.4
4.4.1
4.4.2
4.4.3
4.4.4
cAMP
IL10.________________________________________115
cAMPmRNAIL10
.________________________________________________116
mRNAIL10,
cAMP,. ___________________________117
cAMP500
IL10. _________________________________________________________119
500IL10CONSITE. ______________120
Invivoinvitro. _____________________________123
cAMP
MEF2invivo. ______________________________________________________123
cAMP
CREBinvivo. _________________________________________________________________124
cAMP
MEF2invitro. ________________________________________________________________125
CREB
cAMPinvitro. _____________________________________________________126
5.
___________________________________________________________ 128
5.1
__129
5.1.1
__________________________129
5.1.2
IL10 __________________________________129
5.1.3
IL2IL4 __________131
5.1.3.1
IL2__________________________________________________________131
5.1.3.2
IL4__________________________________________________________132
CAMP
5.2
________________________________________________________________134
5.2.1
cAMPIL10
___________________________________________________134
5.2.1.1
PKAcAMPIL10
_____________________________________________137
5.2.1.2
cAMPIL10
IL2 ____________________________________138
5.2.2
cAMPIL2IL4
__________________________________________________________139
5.3
IL10CAMP
________________________________________________________________141
5.3.1
cAMPIL10
_______________________________________________________________141
5.3.2
cAMPmRNAIL10
_________________________________142
5.3.2.1
cAMPIL10
__________________________________________________________144
5.4
MEF2CREBIL10
CAMP____________________________________146
5.4.1
MEF2 ______________________________________146
5.4.2
CREB_______________________________________148
6.
_____________________________________________________ 150
7.
8.
_______________________________________________________ 154
9.
_______________________________________________________ 160
1.1.,
. .................................................................................................................................. 12
1.2.. .................................................................................................................................... 14
1.3.. ..................................................................................................................... 15
1.4.
. ................................................................................................................................................ 16
1.5.T/CD3/. .............................................................. 18
1.6.).)
...................................................................... 19
1.7.APCS,MHCIMHCII,. .......... 20
1.8.IL2.. .................................................................................................. 21
1.9.IL2
.
..................................................................... 22
1.10....................................................... 22
1.11.
. ........................................................................................................................................................ 25
1.12.. ....... 28
1.13.. ....................................................................................................... 29
1.14./. ........................................................................ 29
1.15.,
10. ........................................................................................................ 31
1.16.,
..10,...................................................................................................... 32
1.17.................... 36
1.18.G........................................................... 37
1.19.G. .................. 38
1.20.AMP. .................................................................................................................. 39
1.21.ATPCAMP. ............................... 40
1.22.
GSGi. ....................................................... 41
1.23.CAMP,PKA. ................ 42
1.24.PKACAMP.... 44
1.25.CAMP5AMPCAMP........................................... 46
1.26.MEF2.. .......................................................................................................................................... 52
1.27.MEF2.. ..................................... 53
1.28.CREB.. ........................................................................................................................................... 55
1.29.CAMPCREB.. ............................................ 56
2.1........................................69
2.2.MM(NEUBAUER)............................................................71
2.3.
MACS.................................................. 76
2.4.(ELISA). ....................................... 81
2.5.DNA ............................ 91
2.6.DUALLUCIFERASE. .... 98
1 ................................................................................................................................................................................ 71
2 ................................................................................................................................................................................ 71
1............................................................................................................................................................................... 87
2............................................................................................................................................................................... 87
3............................................................................................................................................................................... 87
4............................................................................................................................................................................... 88
5............................................................................................................................................................................... 89
6............................................................................................................................................................................... 99
7............................................................................................................................................................................. 100
8............................................................................................................................................................................. 102
10
1:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28. ........................................... 107
2:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28. ....................................................................... 107
3:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28................... 108
4:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28
CAMP. .............................................................................................................................................. 109
5:IL2
ION/PMA,ANTICD3ANTICD3/ANTICD28
CAMP. .............................................................................................................................................. 110
6:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28RP/rhIL2. .......... 112
7:IL4
ION/PMA,ANTICD3ANTICD3/ANTICD28
CAMP. .............................................................................................................................................. 112
8:IL10
Rp8BrCAMP. .............................................................................................. 114
9:IL10
50MRp8BrCAMP/CAMP. ............................................................ 114
10:CAMP
IL10. ......................................... 116
11:PKAIL10
.................................................................................................................................... 116
12:CAMPmRNAIL10
. ............................................................................................................ 117
13:CAMPmRNAIL10
. ............................................................................................................ 118
14:CAMPIL10
.................................................................................................................................... 119
15:CONSITE. ..................................... 120121
16:500IL10
CONSITE.............................................................................................................................. 122
17:CAMPMEF2.......... 123
18:CAMPCREB........... 124
19:CAMPMEF2
.A)EMSAGEL.)%
..................................................................................... 125
20:CAMPCREB
11
.A)EMSAGEL.)%
..................................................................................... 126
1.
1.1.
,
.
, ,
,
,
( 1-1) (Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1-1.
,
.
,
.
,
, 20 ,
12
. ,
,
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
,
, ,
(Abbas & Lichtman, 2001).
,
,
.
.
,
(autoimmune reactions)
(Abbas & Lichtman,
2001) (Goldsby RA, 2004).
,
,
.
, , ,
(Abbas
& Lichtman, 2001) (Goldsby RA, 2004).
,
. , ,
.
,
,
.
,
13
,
,
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1.1.1.
.
,
i)
, , ii) ,
, iii)
,
,
, ,
.
.
, ,
( 1-2) (Abbas &
Lichtman, 2001) (Goldsby RA, 2004).
.
1-2. :
:
.
:.
, .
14
:
,
( 1-3),
(Abbas & Lichtman, 2001).
1-3 : (scanning
electron microscope SEM). Copyright 2007 Dennis Kunkel Microscopy, Inc.
1.1.1.1.
, .
(CD4, CD8 ..).
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
,
, (nave) .
-
(Abbas & Lichtman, 2001).
(TCR: T Cell Receptor)
.
15
.
,
( 1-4). ,
G0 .
G1, DNA ( S).
G2 .
,
, ,
RNA .
, 24
,
1000 ( ).
,
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1-4.
.
-
,
(memory T cells),
,
(Abbas & Lichtman, 2001) (Goldsby
RA, 2004).
16
,
. :
(MHC-major histocompatibility
complex) ,
( T -self-reactive T cells)
. 3%
.
(Romagnani, 2006).
(central self-tolerance).
,
(peripheral self-tolerance).
( )
(Romagnani, 2006).
,
(Abbas & Lichtman,
2001).
1.1.1.2.
-
TCR MHC ,
-..
17
1-5. T /CD3 / .
18
, ,
.
(protein tyrosin kinases-PTK)
. ,
Src, Lck, ITAM
CD3
( 1-6). ZAP-70
.
,
lipid rafts
(Reth et al., 2004).
1-6. ) . )
.
TCR ,
,
19
. / TCR /,
.
/ TCR,
/ TCR
,
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1.1.1.3.
MHC
. MHC ,
, I (MHC I) II (MHC II):
MHC ,
8-9
, CD8 .
, ( 1-7).
MHC ,
, 13-18
, CD4 .
,
( 1-7) (Abbas & Lichtman, 2001) (Goldsby RA, 2004).
20
,
,
.
(Romagnani, 2006).
G0 G1
-2 (IL-2 Receptor-IL-2R)
.. IL-2, IL-3, IL-4.
IL-2 ,
. -
IL-2 ( 1-8).
1-8. IL-2. 3 , , ,
.
, IL-2, ,
( )
(Cantrell et al., 1983). IL-2
G1 S
,
. IL-2,
21
G0
(Cantrell et al., 1984) ( 1-9).
1-9. IL-2
.
( 1-10).
MHC/
. , ,
(Alegre et al., 2001; Frauwirth et al., 2002;
Weaver et al., 2008).
1-10. . )
1 . ) 2
, .
22
CD28
, T44 1987 CD28.
44 kDa 90 % CD4+ 50 % CD8+ - .
CD28
CD80 (B7.1) CD86 (B7.2),
(APCs)
. , CD28 1
, ,
(Weaver et al., 2008).
,
. 1,
, ,
CD28.
(Weaver et al., 2008).
CTLA-4 CD152. CTLA-4 CD28
, CD80
CD86,
(Alegre et al., 2001; Bashian et al., 1997; Frauwirth et al., 2002).
, ,
,
,
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
23
1.1.1.4.
.
MHC I (T C : T
cytotoxic cells), CD8
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
MHC (T H : T helper
cells) .
CD4
,
.
, T H 1 T H 2,
( 1-11).
T H 17 (Romagnani, 2006).
TH1
-2
. T H 2
-4 (IL-4), -5 (IL-5), -6 (IL-6), -10
(IL-10) -13 (IL-13),
IL-10.
-12 T H 1 T H 2
, IL-4 T H 2
T H 1 (Romagnani, 2006).
24
1-11.
.
T H 17 IL-23
IL-4
T H 2 IFN- T H 1 .
T H 17 IL-17 IL-22
,
(Romagnani, 2006).
.
CD4+ ,
,
, .
,
(Tr1) (nTreg:
naturally occurring Treg),
,
(O'Garra et al., 2004a; Rouse,
2007; Wu et al., 2007).
25
-MHC- .
IL-2
,
(Chandok et al., 2004).
CD45.
,
. CD45
.
CD45RO,
CD45RA () CD45RB
() .
(Chandok et
al., 2004).
1.1.2.
,
. (cytokines) (<30 kDa)
.
(lemphokines),
(monokines),
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
(interleukins)
.
,
24 ( 1-26, IL-1IL-26).
(interferones),
(tumor necrosis factor).
27
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
,
. / ,
,
.
,
,
( 1-12) (Abbas & Lichtman, 2001).
1-12.
.
(autocrine),
28
, (paracrine)
,
(endocrine)
, ,
( 1-13).
..
(Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1-13. .
/
,
( 1-14) (Abbas & Lichtman, 2001) (Goldsby RA, 2004).
1-14.
/ .
29
,
,
,
.
,
IL-10
IL-10.
IL-10 IL-10
, IL-19, IL-20, IL-22, IL-24 IL-26. IL10 , ,
30
.
IL-10 -,
, (Mege et al., 2006; O'Garra et al.,
2004b; Pestka et al., 2004).
, , ,
IL-10 -.
IL-10
. IL-10
IL-10 Epstein-Barr DNA
(.. ) (Moore et al., 1993).
IL-10
IL-10 , ..
, , ... IL-10,
-
( 1-15) (Ozaki et al., 2002).
,
,
. IL-10 (IL10R) , IL-10R1 IL-10R2.
IL-10R1 IL-10 IL-10R2
IL-10 (Mege et al., 2006; Moore et al.,
2001; Pestka et al., 2004).
1-15.
-10.
31
IL-10 ,
,
.
IL-10,
JAK .
STAT ,
STAT1, STAT3 STAT5,
,
- ( 116). STAT3 IL-10,
. IL-10
SOCS-1 SOCS-3. SOCS-1
IFN-, IL-10 IL-4 (Mege et al., 2006; Moore et al., 2001; Pestka et
al., 2004).
1-16.
, .. -10, .
/.
-10 :
(.. IL-12, IL-18)
CD80, CD86
MHC II .
32
IL-2, (TNF),
IL-5, CD4+ .
, IL-2, TNF-, IFN- IL4.
, .
TNF IL-1 .
Gram(+)
LPS Gram(-) , in vivo in vitro
.
. ..
NFB AP-1 CD8+
CD4+ .
IL-10
,
(O'Garra et al.,
2004a; O'Garra et al., 2007; O'Garra et al., 2004b).
1.1.2.2.
-2 (IL-2)
IL-2 ,
.
(Hidaka, et al., 1996) (Gomperts,
Kramer, & Tatham, 2002).
IL-2
(IL-2 receptor, IL-2R)
-
. IL-2
G1 S .
, .. -
IL-2,
33
. IL-2
(Gaffen et al., 2004).
IL-2
( )
-
(Gaffen et al., 2004).
-4(IL-4)
1.1.2.1.
IL-4 T H 2 CD4+
, ,
, . ,
IL-4 CD4+
T 2,
T 1 IFN-.
. ,
,
MHC , . -4
(IL-4)
, . IL-4 :
-.
MHC- ,
.
,
.
- .
IgE.
34
1.2.
( -first messengers),
.
(signal transduction pathway signaling
cascade)
, . ,
,
(Hancock, 2005; Gomperts, Kramer, & Tatham, 2002; Hidaka, et al.,
1996).
.
,
.
, :
, /
,
,
/,
.
(second
messengers) -AMP (cAMP) (Ca2+).
..
.
35
,
,
..
( 1-17).
1-17. .
.
.
, ,
, ,
, .
,
.
.
36
1.2.1.
G-
,
.
:
,
,
G-.
G-
, ,
. ,
( 1-18) ,
, (Hancock, 2005;
Gomperts, Kramer, & Tatham, 2002).
1-18. G-.
G-
1.2.1.1.
G-
, , .
, G-
G-
, (Malbon,
2005).
G- . G s -
- (G stimulatory -proteins),
G i - (G inhibitory -proteins) 37
. - G s - s -
G i - i .
G-,
G-,
G- ( 1-19b).
G- GDP G-
GTP. G , .
G-
. - ,
.. cAMP- ,
.
- GTP GDP,
G- ( 119c)(Malbon, 2005).
1-19.
G . a)
,
. b)
G . c)
G
.
38
1.2.2. cAMP
AMP (cAMP, 3'-5'-
- 1-20)
, 1957,
(1971) Earl W. Sutherland (Hancock, 2005; Gomperts, Kramer, &
Tatham, 2002).
1-20. AMP.
,
. cAMP
,
,
,
(Hancock, 2005).
cAMP
(Adenylate
cyclase, AC) cAMP
(Phosphodiesterases, PDEs)(Barritt, 1992). cAMP
/ (), Epacs
(Epac1 Epac2) guanine nucleotide exchange factors (GEFs),
, cyclic
nucleotide-gated ion channels (CNGs).
cAMP ,
, ,
(Dremier et al., 2003).
39
cAMP
.
- , Ca2+
cAMP
Ca2+
1, cAMP (Barritt, 1992).
(Cooper, 2003).
cAMP
1.2.2.1.
cAMP ATP,
(5-10 mmol/l). ATP cAMP
( 1-21)
G-.
..
&
cAMP
,
(Hancock, 2005; Gomperts, Kramer, & Tatham, 2002; Barritt, 1992).
40
1.2.2.2.
.
9 (I-IX) ,
.
, ,
, ,
cAMP .
s G s -, i
G i - ( 1-22) (Klinger et
al., 2002).
1-22. Gs Gi- .
Forskolin ,
(Ca2+) (Cooper, 2003).
1.2.2.3.
cAMP-
cAMP-
G s -,
G s - ( 1-23). G s -
, G s G s , . G s -
G s - ,
41
.
cAMP
cAMP ,
PKA
(Hancock, 2005; Gomperts, Kramer, & Tatham, 2002).
G s -,
,
cAMP, .
- (,
,
(Fimia
et
al.,
2001).
, PP-1, -2A, -2B
-2C (Barritt, 1992).
,
42
. cAMP ,
.
G , ,
ATP cAMP
PKA.
10
104 (Barritt, 1992).
(PKA)
1.2.2.4.
PKA
(0.2-2mol/l).
, ..
MAP ,
cAMP
(Fimia et al., 2001).
PKA - AKAPs (A Kinase Anchoring Proteins)
(Malbon et al., 2004). AKAPs
, PKA,
, ..
cAMP.
AKAPs ,
(Tengholm, 2007). , AKAPs
(Colledge et al., 1999).
cAMP, PKA
(R)
(C) ( 1-24).
43
1-24. PKA
cAMP.
,
, (35% identity) cAMP.
cAMP
PKA, cAMP
. cAMP,
R 2 C 2 104-105,
.
PKA
/ . ,
cAMP PKA
cAMP 5-AMP
,
(Barritt, 1992; Hancock, 2005).
cAMP-
PKA
,
,
.
44
PKA ,
15-20
. 4-6
.
cAMP ,
, ,
(Tengholm, 2007).
PKA , PKA PKA
. PKA
PKA , - AKAPs. , PKA
PKA , RI
RII . RI RII
cAMP, cAMP (Taylor et
al., 2004).
PKA
.
(RI, RI, RII, RII)
(C, C, C)
24
(Fimia et al., 2001).
PKA
(Protein Kinase Inhibitors, PKI).
PKI (, )
.
(chaperones) C PKA
(Fimia et al., 2001).
45
cAMP
1.2.2.5.
,
cAMP 5-AMP
cAMP ( 1-25).
PKA,
(Lynch et al., 2005).
1.2.2.6.
(PDEs)
cAMP 3 , 5 14
PDEs, 1 14
(PDE1-14), 25 ,
50 ,
,
(Conti et al., 2007; Essayan, 2001).
PDEs, 4 7
cAMP. , PDE4
cAMP .
46
4
(Conti et al., 2007; Lynch et al., 2005).
PDE4
4 (PDE4A, B,
C, D). c,
, , , , .
PDE4 rolipram
PDE4H , roflumilast cilomilast
PDE4H PDE4L
(Conti et al., 2007;
Essayan, 1999; Essayan, 2001).
1.2.2.7.
cAMP
cAMP- .
cAMP. , 2 (PGE2)
cAMP-
cAMP ,
G-.
(Tasken et al., 2006; Torgersen et al., 2002).
cAMP-
PKA
cAMP, Epac
CNGs, (Tasken et al., 2006),
PKA
(Torgersen et al., 2002).
,
cAMP PKA
47
(Tasken et al., 2006).
cAMP
PDEs 4 7 (PDE4, PDE7),
.
,
7 (PDE7) (Li et al., 1999)
-2 (IL-2) (Torgersen et al., 2002),
PDE4 ,
cAMP (Bopp et al., 2007).
- cAMP/PKA
AIDS (HIV ) CVI
(Tasken et al., 2006; Torgersen et al., 2002), cAMP (Tasken
et al., 2006).
1.2.2.8.
cAMP
cAMP
, cAMP-
(Hancock, 2005).
48
cAMP
IL-2 -,
, mRNA
,
IL-2 (Anastassiou et al., 1992; Jimenez
et al., 2001; Johnson et al., 1988; Tasken et al., 2006; Torgersen et al., 2002; Zidek,
1999).
cAMP TNF-
(Zidek, 1999)
(Jimenez et al., 2001). IL-3
GM-SF
2 (Zidek, 1999)
IL-2- IL-5
(Heijink et al.,
2003; Jimenez et al., 2001) IL-10
,
cAMP (Jimenez et al., 2004).
cAMP IL-6 IL-10
IL-4
2 (Platzer et al., 1999; Zidek, 1999).
49
1.3.
,
,
.
. ,
.
,
. :
,
,
.
,
(Papavassiliou, 1995a; Papavassiliou, 1995b).
,
DNA.
DNA ,
,
DNA.
, DNA,
: () -- (helix-turn-helix), ()
(zinc fingers), () (leucine zipper) () --
(helix-loop-helix).
, DNA .
,
,
. (Papavassiliou, 1995b).
50
.
.
.
. , ,
.
,
,
(Papavassiliou, 1995a; Papavassiliou,
1995b) .
1.3.1. MEF2
MEF2 (Myocyte Enhancer Factor-2) ,
A/T DNA
- (Brand, 1997). ,
,
(McKinsey et al., 2002).
MEF2
MADS (MADS-box family of transcription factors),
-
(MCM1, Agamous, Deficiens, Serum response factor).
, MEF2
(protein-protein interaction)
,
MADS-box (Black et al., 1998; Brand, 1997).
51
, MEF2
, Cabin1 -
52
1-27. MEF2. a)
MEF2 ,
, Cabin1 HDACs,
. b) , MEF2
.
, MEF2
( 127b). .. - , --
(CaMK) HDACs, Cabin1,
- 14-3-3,
MEF2. MEF2
53
,
, .. - p300,
(Liu, 2005; McKinsey et al., 2002).
MEF2
(Liu, 2005; McKinsey et al., 2002) MEF2
(Black et al., 1998; Liu, 2005; McKinsey et al., 2002; Shin et al., 1999;
Wang et al., 2005a; Yang et al., 1999) (Belfield et al., 2006). ,
MEF2
(Belfield et al., 2006; Pan et
al., 2005; Pan et al., 2004),
(Pan et al., 2005; Shin
et al., 1999) .. -2 (Pan
et al., 2004).
1.3.2. CREB
CREB (cAMP-Responsive element-binding
protein) cAMP-
CRE (cAMP-Responsive element). CRE,
- somatostatin,
TGACGTCA
cAMP-
(De Cesare et al., 1999; Fimia et al., 2001; Sands et al., 2008; SassoneCorsi, 1998; Torgersen et al., 2002), CREB
,
(De Cesare et al., 1999).
3 , CREB,
CREM ATF-1,
CREB, CREM ATF-1. CREB ATF-1
, CREM
- (neuroendocrine) (Mayr et al., 2001).
54
CREB
.. p38 - ,
.. (Mayr
et al., 2001).
55
1.3.3. cAMP
NFB/Re1/NFAT,
AP-1
-
cAMP NFB.
56
Rel ,
PKA. NFB
IB.
IB IB NFB
.
PKA IB NFB/
IB IB
NFB PKA(C).
PKA NFB
CBP/p300 (Sands et al., 2008; Torgersen et al., 2002), PKA
CBP p300
(Vo et al., 2001).
MEF2
IL-2 Jurkat
(Pan et al., 2004), cAMP-
,
(Wang et al., 2005a). , Wang et al.,
MEF2
cAMP- cAMP- PKA
20,
DNA (Wang et al., 2005a).
cAMP
- MEF2 ,
CREB (Belfield et al., 2006).
cAMP PKA lipid rafts
-,
.
57
1.3.4. IL-10
cAMP- IL-10
. Platzer et al.
cAMP
IL-10 (Platzer et al.,
1999; Platzer et al., 1995). , cAMP-
IL-10
CREB ATF-1,
IL-10 TATA box (Platzer et al., 1999).
IL-10
,
SP1 c-JUN ,
3-UTR (Powell et al., 2000; Wang et al.,
2005b).
cAMP
,
IL-10,
cAMP,
, AIDS
.
58
59
2.
cAMP IL-10
,
.
60
61
1.
DIFCO
GIBCO BRL
MERCK
MERCK
SIGMA
SHOERING PLOUGH
- MERCK
SIGMA
62
Virion/Serion D
MERCK
MERCK
LS MACS
KODAK
AppliChem
SIGMA
8-Br-cAMP SIGMA
A
Actinomycin D PROMEGA
AET SIGMA
Anti-goat SANTA-CRUZ BIOTECHNOLOGY
Anti-MEF2D SANTA-CRUZ BIOTECHNOLOGY
63
B
Bactopeptone DIFCO (BECTON DICKINSON)
BIORAD Protein Assay BIORAD
Bromophenol blue SERVA
BSA SIGMA
C
CD3-FITC IMMUNOTECH Beckman Coulter
CD45RA-PE IMMUNOTECH Beckman Coulter
CD45RO-PE IMMUNOTECH Beckman Coulter
Coomassie Brilliant Blue R250 MERCK
D
DEPC SIGMA
DH5a Competent Cell Kit INVITROGEN
DNA Labeling System INVITROGEN
DTT SIGMA
Dual Luciferase Reporter Assay System PROMEGA
E
EDTA (-- ) SIGMA
EGTA GIBCO BRL
F
FBS GIBCO BRL
Ficol-Hypaque-1077 SIGMA
FORSKOLIN SIGMA
Freezing Medium GIBCO BRL
64
H
HCl MERCK
HEPES (-2---2- ) SIGMA
I
IgG-FITC IMMUNOTECH Beckman Coulter
IgG-PE IMMUNOTECH Beckman Coulter
IL-10 ELISA kit BIOSOURCE
IL-2 ELISA kit BIOSOURCE
IL-4 ELISA kit BIOSOURCE
Ionomycin SIGMA
K
KCl
KCl MERCK
KH 2 PO 4 SIGMA
L
L-Leucine-methyl-ester SIGMA
M
Memory CD4+ T cell Isolation Kit Miltenyi Biotech
MgCl2 MERCK
N
NaCl MERCK
NaOH MERCK
Nonidet P-40 (NP-40) IGEPAL CA630 SIGMA
O
Oligo-DTs INVITROGEN
65
P
PBS GIBCO BRL
pCREB-Luc STRATAGENE
PGE2 SIGMA
pGL3 PROMEGA
PMA SIGMA
PMSF SIGMA
Poly dIdC ROCHE
pRL-TK PROMEGA
R
rhIL-2 R&D SYSTEMS
ROLIPRAM SIGMA
Rp-8-Br-cAMP BioLog
RPMI 1640 GIBCO BRL
RT-PCR Kit IL-4 MAXIM Biotech Inc.
RT-PCR Kit 18S MAXIM Biotech Inc.
RT-PCR Kit IL-10 MAXIM Biotech Inc.
S
SDS MERCK
Sodium acetate SIGMA
SuperScriptII Reverse Transcriptase (SSIIRT) INVITROGEN
T
TBE INVITROGEN
TBS GIBCO
TEMED SIGMA
Tris-HCl pH 7.4 GIBCO BRL
TRIZOL INVITROGEN
66
Y
Yeast extract DIFCO
15, 50ml, ,
96 / ,
eppendorf 2ml
CORNING COSTAR.
67
2.
2.1.
2.1.1.
, ,
RPMI 1640 (+ HEPES, + Glutamine GIBCO BRL), 10%
(Fetal Bovine Serum) 40 g/ml .
2.1.2.
:
Histopaque-1077 1.077
0.001 g/ml
(Ficoll) (Hypaque).
( 2-1).
68
2-1. .
, ,
o (Ficoll-Hypaque).
,
. ,
,
4 C
. ,
,
, ,
.
(Peripheral Blood Mononuclear Cells, PBMC).
69
:
Ficoll-Isopaque ( Ficoll-Hypaque, Lymphoprep, Histopaque)
FALCON 50 ml,
SORVAL
RPMI 1640 + 10% FBS + 40g/ml
Pasteur, Gilson tips
( Neubauer)
Trypan blue ( 0.5 % (w/v) PBS)
:
, (1:1)
, (PBS Mg+2/Ca+2)
1:2 o 15 ml
Histopaque-1077, .
400 x g, 30 min, 20 oC .
,
.
Pasteur,
, FALCON
50 ml PBS. 250 x g, 10 min,
4C . 50
ml PBS 2
(250 x g, 10 min, 4C).
10 ml RPMI 1640 PBS
Neubauer .
4C PBS RPMI 1640
.
70
2.1.3.
10 ml
RPMI 1640 PBS, 1:10
0.05 %, .
5 , (~10l)
Neubauer ( 2-2) ,
.
2-2. ( Neubauer).
9 (.. 6, 7, 8 9)
0.1 mm3 . ,
(.. 6, 7, 8 9)
4
. ,
10 1 mm3:
1.
mm
10
, ,
:
2.
71
100
2.1.4.
:
. ,
,
Fc IgG.
(rosettes) ()
CD2,
2-(2--)
(AET) .
. ,
.
2.1.4.1.
:
37C
AET :
6.4 gr 2-(2--) (AET)
3.2 ml 10 N NAOH
156.8 ml dd H 2 O.
FALCON 50 ml,
(0.9 % )
SORVAL
RPMI 1640 + 10% FBS + 40g/ml
Pasteur
,
( Neubauer)
72
Pasteur
5 . 6
, RPMI 1460
(+FBS+) 50 ml .
4 C 1 ,
.
,
.
2.1.4.2.
E-rosetting
:
FALCON 50 ml,
SORVAL
RPMI 1640 + 10% FBS + 40g/ml
PBS
73
Pasteur
,
( Neubauer)
Trypan blue ( 0.5 % (w/v) PBS)
8.3 gr/lt
M Tris-HCl pH 7.5
:
,
,
Falcon, 200 x 106
/falcon 25 ml PBS 10 ml
.
10 , 5 250 x g
4C 4C 1 12 .
. 15
ml 300 x g
30 . 3
. ,
.
,
(- )
Falcon .
,
.
,
3 5 ml
. 10
7 250 x g
4C.
, , 4 ml
, 10 7
74
250 x g 4C.
. 3 ml , 10
.
, 10 ml
PBS ,
.
.
:
(Thiele et al., 1983). , 5 x
106 /ml - FBS - 2.5
mM L--- 40 .
3 (250 x g 4C 7 )
50ml - FBS -.
, 10 ml
FBS ,
.
2.1.6.
MACS
:
(CD45RO+CD4+) (CD45RA+
CD4+)
CD4+ ,
Miltenyi Biotech.
,
(
75
CD8, CD14, CD16, CD19, CD36, CD45RA, CD56, CD123, TCR/ Glycophorin
A - 1 )
(anti-biotin - 2
). MACS ( LS)
( 2-3),
( ).
.
2-3.
MACS.
:
MACS:
PBS pH 7.2
2 mM EDTA
0.5 % BSA
LS MACS
MidiMACS
76
:
, 300 x g 10 4
C 107 40 l MACS.
10 l Biotin-Antibody Coctail
Gilson 10 4 C 30 l
MACS 20 l Anti-Biotin Microbeads.
15 4 C.
1-2 ml MACS 300 x g
10 4 C.
500 l MACS.
MACS
, 3 ml MACS. ,
Falcon.
3 ml MACS 3
. 5 ml
MACS, ,
Falcon, 300 x g
10 4 C, 2 ml PBS,
.
2.1.7.
.
:
20 l CD45RO-PE
20 l CD45RA-PE
20 l IgG-PE
20 l CD3-FITC
20 l IgG-FITC
:
Ficoll
, 2 PBS,
NeuBaeur Eppendrof
1x 105 - 1x 106 /100 l PBS/ Eppendorf.
77
2 .
20 l
(FITC) (PE) 20 l
.
4 C. , 1 ml PBS/
4 C 7 1500 x g.
, 500 l PBS/,
FACS 1 .
78
2.2.
2.2.1.
:
-cAMP
cAMP.
:
Forskolin (SIGMA),
cAMP .
2 (PGE2) (SIGMA)
cAMP EP2 ,
G-.
4, Rolipram (SIGMA),
cAMP
8-Br-cAMP (SIGMA),
cAMP.
- (anti-CD3) (CALTAG)
CD28 (anti-CD28) (CALTAG),
5 x 106 M Rolipram .
1 g/ml 2.5 ng/ml PMA,
2 g/ml anti-CD3 1 g/ml anti-CD28,
2 g/ml anti-CD3 , 16 - 36
.
,
250 x g, 10 , 4 C.
, o
,
.
2.2.1.1.
anti-CD3
,
RPMI 1640 FBS
1 4o C).
,
FBS ,
(Geppert et al., 1986).
2.2.1.2.
rhIL-2
IL-2 IL-10
,
20 g/ml (ng/l) human IL-2,
R&D SYSTEMS.
2.2.1.3.
A I (PKA I)
(Protein Kinase A PKA)
RP-8-Br-cAMP,
,
cAMP (Gjertsen et al., 1995; Holm et al., 2003). H
80
2.2.2.
-2
-4)
(-10,
.
:
-10 (
-2, -4)
. IL-10 ( IL-2, IL-4)
, ,
IL-10 ( IL-2, IL-4) .
,
, IL-10 ( IL-2, IL-4)
1 ,
--10
( IL-2, IL-4)- - ( 2-4).
2-4.
(ELISA).
81
,
IL-10 ( IL2, IL-4) .
H 2 O 2
490 nm.
-10 ( IL-2, IL-4)
.
:
-10 (IL-10) -2
(IL-2)
-4
(IL-4)
(ELISA)
BIOSOURCE.
.
82
2.3.
IL-10
cAMP
2.3.1. RNA
:
RNA
,
.
H TRIZOL
, RNA ,
RNA Chomczynski Sacchi
(Chomczynski et al., 1987).
TRIZOL RNA,
.
,
. RNA
, 75%
TE .
:
TRIZOL (Invitrogen)
Eppendorf 1.5 ml,
SORVAL Eppendorf
RPMI 1640 + 10% FCS + 40g/ml
Gilson tips
H 2 O RNA
75% (DEPC-treated H 2 O)
:
5-10 x 106 700 l TRIZOL
.
83
eppendorf 5 ..
140 l
15 sec.
2-3 12000 g
4C 15 .
eppendorf 350
l . ,
10 12000 g,
4C 10 . ,
700 l 75% /
7500 g, 4C 5 .
30 l TE RNA.
2.3.2.
:
DNA/RNA.
,
.
0,6-2% ./..
.
:
1%
1 x TBE.
.
RNA
(gel loading buffer) 50% , 1mM EDTA, 6.25%
0.25% .
84
1
120 Volt.
U.V.
RNA . RNA
2 28S 18S
DNA
.
2.3.3. mRNA
-10
cAMP mRNA IL-10
-
(RT-PCR).
(PCR polymerase chain reaction)
DNA.
, ,
DNA
DNA.
DNA .
PCR :
1) DNA (denaturation),
90 C.
2) DNA (annealing),
DNA 50 60 C.
3) (extension) ,
, 72 C.
, 30-40 ,
DNA .
85
PCR DNA
. RNA
, DNA (RTPCR reverse transcription PCR).
2.3.3.1.
Invitrogen.
.
:
2 g RNA
1 l DNTPs (10 mM)
1 l oligoDTs
4 l First Strand Buffer (5x)
2 l DTT (0.1 M)
1 l SSIIRT
ddH 2 O
PCR 0.2 ml
PCR
:
PCR 0,2 ml ,
:
86
1.
stock
(l) 20 l
2 g
10 mM
20 g
x l
1 l
1 l
12 l
RNA
dNTPs
-dTs (15)
ddH 2 O
65
C 5 . :
2.
stock
(l) 20 l
5x
0.1 M
4 l
2 l
1 l
,
42 C 2 , :
3.
SuperScript II RT
stock
(l) 20 l
200 U/l
1 l
42 C 50 70 C 15
.
cDNA
.
2.3.3.2.
PCR
, RT-PCR
(Peters et al., 2004).
87
RT-PCR
Rotor-Gene RG-3000 CORBETT Research.
,
,
(Rotor gene, 6
Windows).
:
H mRNA IL-10
Maxim Biotech Inc.
.
:
, .
IL-10
18S. RT-PCR
, 0.1 ml. ,
, :
4.
RT-PCR
probes
Taq DNA
cDNA (0.01-0.1g)
ddH 2 O
stock
(l) 25 l
2x
12.5 l
10x
10x
5 U/l
2.5 l
2.5 l
0.25 l
2.5 l
4.75 l
88
5.
PCR
IL-10
45
95C
10 sec
60C
30 sec
72C
30 sec
72C
10 min
IL-4
45
95C
10 sec
60C
30 sec
72C
30 sec
72C
10 min
18S
45
95C
10 sec
60C
30 sec
72C
30 sec
72C
10 min
RT-PCR.
2.3.3.3.
mRNA -10
,
D.
D mRNA
mRNA.
:
10 x 106 /ml RPMI 5 x 106
M Rolipram 30 1 g/ml
2.5 ng/ml PMA, 2 g/ml antiCD3, 1 g/ml anti-CD28, 2 g/ml anti-CD3
, 3 . 4 .
, ,
250 x g 10 4 C, 5 g/ml
D 3
, 2 4 37 C 5.0 %
CO 2 . ,
250 x g 10 4 C
RNA, cDNA RT-PCR.
89
2.4.
2.4.1. In vivo
E.coli
2.4.1.1.
(transformation)
:
DNA , DNA
, .
2.4.1.2.
1 5ml LB
DNA. 8 37o
C 1 ml 8-
90
250 ml L-broth . 24
37 C
Falcon 50 ml, 7 2500 x g 4 C
DNA .
2.4.1.3.
IL-10/Luc, pRL-TK
E.coli
NucleoBond Xtra Maxi Macherey-Nagel
.
:
DNA (200-50,000bp)
DNA
pH.
,
DNA, . DNA
, , RNA,
DNA, ,
( 2-5). ~90% 0.55g DNA.
2-5.
DNA
91
:
DNA 12 ml
RNA (resuspension buffer RES
+ RNAse A), falcon 50 ml. ,
25 ml (equilibration buffer
EQU). 12 ml
(lysis buffer LYS) , 5-6 ,
5 . 12 ml
(neutralization buffer NEU) 10-15
.
,
15 ml (equilibration buffer EQU).
25 ml
(wash buffer WASH) . 15 ml
(elution buffer ELU),
falcon.
10,5 ml
1 7500g 4C.
, 2 ml 70 %
/ DNA
eppendorf. 15000 g 5
,
Gilson, 10-15
. DNA 200 l
TE, DNA
.
2.4.1.4.
DNA
.
DNA DNA
(Ready LoadTM 100bp DNA Ladder, Gibco BRL).
92
2.4.1.5.
DNA
(transfection
by
electroporation)
:
DNA - -
()
.
DNA
.
DNA .
DNA 12-72
.
DNA
. ,
.
:
) :
,
DNA.
.
250V/cm. 30 50 %
.
) :
, .
DNA .
20-50sec.
93
) :
, ,
.
4oC.
) DNA:
DNA
DNA
1-40 g/ml.
) :
(HEPES buffered saline) .
- :
,
DNA .
stress .
DNA:
DNA
, RNA . DNA
.. milli-Q H 2 O
0.2 1 mg/ml. DNA
260nm 280nm,
1.8 .
:
25 x 106 /
1100 x g 10 4C. ,
(0.4cm) 50 g DNA.
10% (FCS),
94
, 25 x
106 /0,3 ml .
Pasteur (25x106/50g
/0,3 ml RPMI 1640+10% FCS/).
(360Volt) gene pulser (ECM 399,BTX)
falcon 5 ml
.
2 ,
16 .
1100 x g 10 4C
. ,
-70C
.
2.4.1.6.
IL-10/Luc, SecI,
p3782, pVUII, BSU DraI pGL3 (INVIVOGEN).
2.4.1.7.
BRADFORD.
96
2.4.1.8.
,
Promega Dual-Luciferase Reporter Assay System
.
:
Dual reporter
.
.
Luc (
)
, Luc .
(firefly luciferase
Photinus pyralis).
, DNA
.
ATP.
562nm . kit
Promega
.
Luc, pRL/TK
Renilla (Renilla luciferase Renilla reniformis)
.
,
.
,
.
97
.
10fg-10ng .
(peak 0.3-0.5sec)
.
Promega (AcetylcoA),
.
:
40 l ( 40 g)
eppendorf
, .
, firefly
Renilla, .
80 l
firefly 80 l
Renilla,
firefly
Renilla ( 2-6).
2-6.
Dual
Luciferase.
,
562 nm
(RLU: Relative Light Units).
mg ,
(standards),
Renilla .
2.4.2. In vitro
5
2.4.2.1.
[-32P]
DNA
4 PNK buffer
T 4
ddH 2 O
stock
(l) 20 l
(3000 Ci/mmole)
2 g/ l
10x
10 U/l
2 l
1 l
2 l
2.5 l
12.5 l
30 37 C
DNA
99
95 C
. DNA
(ROCHE Diagnostics Co,
Indianapolis, IN, USA)
DNA (-counter).
(cpm/min).
2.4.2.2.
:
(Dignam et al., 1983).
( )
(
C).
:
7.
A
10 mM HEPES pH 7.9
10 mM KCl
0.1 mM EDTA
0.1 mM EGTA
1 mM DTT
0.5 mM PMSF
C
20 mM HEPES
0.4 M KCl
1 mM EDTA
20 % Glycerol
1 mM DTT
0.5 mM PMSF
:
20 x 106 10 ml TBS
250 x g 4C 7 .
1 ml TBS
Eppendorf. 250 x g 4C 7
. , 400 l A
100
15 . 25 l 10 % / NP-40
vortex 10
250 x g 4C 4 .
, Gilson
30 l C
15 4 C.
250 x g 4 C 10 ,
Gilson
Eppendorf -70 C (-196 C).
,
Biorad
Protein Assay Biorad ( Bradford).
DNA
2.4.2.3.
32P DNA
,
(Ausubel F.M., et al, 1989 in: Current Protocols in
Molecular Biology).
:
-DNA
DNA . , (
),
32
DNA ( 5 )
(.. MEF2).
. DNA
-
DNA (probe).
DNA
. ,
DNA,
101
.
( ),
(
).
:
5 x MEF2
10 mM Tris HCl pH 7.5
50 mM NaCl
1 mM DDT
5% glycerol
4 g BSA
4 g PolyDIDC
:
32P DNA
,
eppendorf :
8.
BSA
PolyDIDC
ddH 2 O
stock
(l) 18 l
10 g
2 g/ l
2 g/ l
5x
x l
2 l
2 l
4 l
x l
10 .
DNA 2 pmol DNA probe (2-5 x 104
cpm) . 30
3 l (gel loading buffer)
(5 % ), 1 35 mA.
, 35 mA
2 . ,
102
70C 12
.
PhopshorImager FUJIFILM FLA
3000 (FUJIFILM Europe) AIDA Windows.
DNA consensus
2.4.2.4.
consensus
EMSA:
) MEF2 ( IL-10):
5-GTAGCCCATCCTAAAAATAGCTGTAATGC-3
) CREB:
5- AGAGATTGCCTGACGTCAGAGAGCTAG -3
2.4.3.
IL-10
500
IL-10
CONSITE (Sandelin et al., 2004). CONSITE
online (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite)
DNA.
CONSITE :
,
/
,
,
,
.
103
3.
Sorvall RC5C
Beckmann
Mini Trans Blot Electrophoretic Transfer Cell
BioRad
DNA/RNA Horizon 11-14 Gibco BRL
VORTEX Genie 2 Scientific Industries
37oC BioLine
37C Forma Scientific 3250
() Holten HB 2448
Power/PAC 300 BioRad
Turner Designs DT20/20
eppendorf 1.5 ml Eppendorf
5417R
()
96- Sorin Biomedica
Behkman
Hettich Universal 32R
- Swip KL-2 alpha scientific
Rotor-Gene RG-3000 CORBETT Research
.
PhopshorImager FUJIFILM FLA 3000 (FUJIFILM Europe)
104
105
4.
4.1.
&
4.1.1. cAMP IL-10
in
vitro
,
, .
(Ionomycin-Ion) PMA.
CD3 (anti-CD3)
CD28
(anti-CD28).
,
(in vivo)
.
CD3
(anti-CD3).
, CD28 ,
.
24, 48
72 ,
10 (IL-10). IL-10
48
IL-10 (peak) 48
.
( 1),
106
24 .
1: IL-10
Ion/PMA, anti-CD3 anti-CD3/anti-CD28.
,
24 , IL-10 30
( 2).
IL-10 Ion/PMA, CD3/CD28 CD3
.
2: IL-10
Ion/PMA, anti-CD3 anti-CD3/anti-CD28.
IL10
(Memory T cells CD45RO+)
(Nave T cells CD45RA+) .
24 , 3
107
IL-10
.
3: IL-10
Ion/PMA, anti-CD3 antiCD3/anti-CD28.
cAMP-
IL-10 ,
cAMP
.
:
P2 (PGE2)
cAMP E2 ,
G-. PGEs,
PGE2,
.
,
(immune
complexes).
Forskolin (FS),
cAMP
.
4, Rolipram (RP),
cAMP. Rolipram
(antidepressant drug) in
vitro
108
8-Br-cAMP,
cAMP.
,
cAMP IL-10
( 4). , I/PMA PGE2,
Rolipram Forskolin IL-10 75, 75 60 % ,
Ion/PMA.
anti-CD3/anti-CD28 PGE2, Rolipram Forskolin,
IL-10 82, 91 89 % .
anti-CD3 IL-10
, PGE2, Rolipram Forskolin
IL-10 76, 82 74 % .
IL-10
cAMP Rolipram, PGE2
Forskolin .
4: IL-10
Ion/PMA, anti-CD3 anti-CD3/anti-CD28
cAMP.
109
5: IL-2
Ion/PMA, anti-CD3 anti-CD3/anti-CD28
cAMP.
IL-2
Ion/PMA anti-CD3/anti-CD28, anti-CD3
IL-2
. PGE2, Rolipram Forskolin
IL-2 45,
50 35 % Ion/PMA, 66, 50 60 %
110
anti-CD3/anti-CD28 .
Ion/PMA,
IL-2 PGE2 .
IL-2
Ion/PMA anti-CD3/anti-CD28, cAMP,
IL-10.
4.1.3. cAMP- IL-10
IL-2
IL-2
. - IL-2
IL-10 cAMP,
IL-2
IL-10. ,
IL-10 IL-2,
IL-2
,
rh-IL-2,
.
( 6), rh-IL-2
I/PMA, anti-CD3/anti-CD28 anti-CD3
IL-10, rh-IL-2
Rolipram I/PMA, anti-CD3/anti-CD28 anti-CD3
IL-10 ( I/PMA, anti-CD3/antiCD28 anti-CD3 ).
111
6: IL-10
Ion/PMA, anti-CD3 anti-CD3/anti-CD28
Rp / rhIL-2.
IL-4 ( 7).
, .
7: IL-4
Ion/PMA, anti-CD3 anti-CD3/anti-CD28
cAMP.
112
IL-4
Ion/PMA,
anti-CD3/anti-CD28 anti-CD3 .
cAMP, IL-4
.
4.1.5. cAMP- IL-10,
TA ,
PKA
PKA cAMP-
, cAMP-
, .
PKA cAMP- IL10, PKA, RP-8-Br-cAMP.
PKA (PKAI),
cAMP.
PKA I (PKA I), .
Rp-8-Br-cAMP
I/PMA, anti-CD3 anti-CD3/anti-CD28
IL-10
Rp-8-Br-cAMP
(50 ) ( 8). ,
50 Rp-8-Br-cAMP.
113
8: IL-10
Rp-8-Br-cAMP.
50 Rp-8-Br-cAMP I/PMA,
anti-CD3 anti-CD3/anti-CD28
cAMP ( 9),
IL-10
( I/PMA, anti-CD3 antiCD3/anti-CD28) 50 Rp-8-Br-cAMP. ,
50 Rp-8-Br-cAMP.
9: IL-10
50M Rp-8-Br-cAMP / cAMP.
114
4.2.
-
IL-10 cAMP
4.2.1. cAMP
IL-10
cAMP-
IL-10
- .
IL-10 cAMP,
(1327 )
IL-10 IL-10. ,
DNA
Firefly
IL-10
115
10: cAMP
IL-10 .
PKA, IL-10
PKA ( 11).
4.2.2. cAMP
mRNA IL-10
cAMP mRNA IL-10
mRNA
-10 ,
.
116
mRNA , cAMP
IL-10
IL-10 mRNA. mRNA,
Rolipram,
mRNA
-10 ,
D .
( 13)
mRNA 50 %
(half life ) 100
I/PMA Rolipram,
I/PMA (180 ). (
I/PMA I/PMA Rolipram), mRNA IL117
10 30 240 (
). (240 )
mRNA IL-10 10 % Rolipram, Rolipram
30 %.
CD3/CD28
118
DNA
(transfection
by
electroporation)
CD3/CD28 PGE2 Rolipram.
, cAMP
1010 bp (DraI) ( 14). , PGE2
Rolipram 500,
310, 235 135 IL-10 (BSU, PVUII, p3782, SecI
) 50 % (
CD3/CD28 ).
119
4.3.
500
IL-10
CONSITE
CONSITE
500
IL-10 (Human IL-10 promoter GeneBank: Z30175-1327bp)
IL-10 (Mouse IL-10 gene
GeneBank: M84340).
cAMP.
14.
IL-10 (Human IL-10
promoter GeneBank: Z30175) IL10 (Mouse IL-10 gene GeneBank: M84340). ,
(conserved) 2
.
,
MEF2 CREB,
15.
Legend
Human_IL10
Mouse_IL10
Coding region
Non-displayed
Detected
region
site (clickable)
binding Conservation
cutoff
Percent identity
120
15. CONSITE.
121
A)
Score
16.302
16.302
ID
Name
Class
Species
Position
1037
1350
Sequence
Human_IL10
Mouse_IL10
MA0052
MEF2
MADS
Homo sapiens
Sequence
A
C
G
T
Lit ref
Medline
[ 1 0 57 2 9 6 37 2 56 6 ]
[50 0 1 1 0 0 0 0 0 0 ]
[ 0 0 0 0 0 0 0 0 2 50 ]
[ 7 58 0 55 49 52 21 56 0 2 ]
B)
Score
9.531
10.116
Position
899
1207
ID
Name
Class
Species
Sequence
Human_IL10
Mouse_IL10
MA0018
CREB
bZIP
Homo sapiens
Sequence
Lit ref
Medline
A
C
G
T
[0
[7
[5
[4
3
5
4
4
0 2 5 0 0 16 0 0 1 5 ]
3 3 1 0 0 0 16 0 5 6 ]
6 11 7 0 15 0 0 16 0 3 ]
7 0 3 16 1 0 0 0 10 2 ]
16. 500
IL-10 CONSITE.
122
4.4.
In
vivo
in
vitro
4.4.1. cAMP
MEF2 in vivo
MEF2 Ca2+-
,
Jurkat,
cAMP-
.
firefly Luciferase
MEF2
DNA
(transfection by electroporation)
CD3/CD28 PGE2 Rolipram.
CD3/CD28
MEF2, PGE2 Rolipram
( 17). MEF2
,
.
17: cAMP
MEF2.
123
4.4.2.
cAMP
CREB in vivo
CRB
/ cAMP-
.
firefly
Luciferase CREB
CD3/CD28 PGE2 Rolipram.
CD3/CD28
CREB,
( 18). PGE2 Rolipram 2.5
CREB , PGE2
.
cAMP-
CREB IL-10.
IL-10
cAMP /
CREB.
18: cAMP
CREB.
124
4.4.3.
cAMP
MEF2 in vitro
MEF2
,
In vitro
.
MEF2
90% CD3/CD28 ( 19).
PGE2 Rolipram
, MEF2
.
A)
B)
RESTING
CD3/CD28
PGE2
RP
MEF2D COLD
PROBE
+
-
+
-
+
+
-
+
+
+
-
19: cAMP
MEF2 . A) EMSA gel. )
% .
125
4.4.4.
CREB
cAMP In vitro
CREB
, in vitro
In vitro
.
( 20)
CREB 65% CD3/CD28.
PGE2 Rolipram
2.5 , CREB
.
)
B)
RESTING
CD3/CD28
PGE2
RP
Unrelated PROBE
CREB COLD PROBE
+
-
+
-
+
+
-
+
+
-
+
+
+
-
+
+
+
20: cAMP
CREB . A) EMSA gel. )
% .
126
127
5.
cAMP. , 2 (PGE2)
cAMP ,
G- (Abbas & Lichtman, 2001; Barritt,
1992).
cAMP-
(Tasken et al., 2006; Torgersen et
al., 2002). cAMP
HIV CVI
(Tasken et al., 2006; Torgersen et al., 2002),
(Tasken et al., 2006).
. cAMP
, cAMP-
(Hancock, 2005).
cAMP
IL-2 (Anastassiou et al.,
1992; Jimenez et al., 2001; Johnson et al., 1988; Tasken et al., 2006; Torgersen et al.,
2002; Zidek, 1999), TNF-
(Jimenez et al., 2001), IL-3 GM-SF
(Zidek, 1999) IL-5
(Heijink et al., 2003; Jimenez et al., 2001) IL-10
(Jimenez et al., 2004).
IL-10 .
128
5.1.
5.1.1.
.
. ,
, IL-10, IL-2 IL4, .
5.1.2. IL-10
, .
.
in vitro
(Ion/PMA, CD3/CD28, CD3)
in vitro
.
PMA
C (PKC) (Barritt, 1992; Abbas & Lichtman, 2001).
,
,
(Gaffen et al., 2004), .
CD3
CD28
anti-CD3 CD3 ,
(Geppert et al., 1986; Manger et al., 1987).
129
IL-10
, .
IL-10
PMA,
CD3
CD28
CD3 .
IL-10 Ion/PMA ,
/ ,
TCR/CD28. ,
,
IL-10 .
CD3 ,
IL-10.
CD3
,
(Alegre et al., 2001; Frauwirth et
al., 2002; Rudd et al., 2003). (Gallo et al., 2006; Weaver et
al., 2008)
. 1,
,
, CD28.
(Weaver et al., 2008).
, IL10 , anti-CD3 (Wang et
al., 2005).
130
,
.
CD3
IL-10.
IL-10 ,
IL-2 IL-4
.
,
, ,
IL-10. ,
, IL-10
,
.
IL-10
. IL-10
background
.
IL-10 ,
,
.
5.1.3.1.
IL-2
L-10 T H 2 ,
,
T H 1 .
cAMP- L-10
T H 1 ,
IL-2. IL-2 T H 1 ,
131
,
(Cantrell et al., 1983; Gaffen et al., 2004).
IL-2
( ).
IL-2
PMA CD3/CD28 (
PMA),
IL-2
(Gaffen et al., 2004).
CD3
IL-2
IL-2
(Cantrell et al., 1984; Cantrell et al., 1983; Gaffen et al., 2004; Romagnani, 2006),
(Abbas & Lichtman, 2001) (Goldsby RA, 2004)
anti-CD3
IL-2 (Jimenez et al., 2001).
Jimenez et al.,
IL-2
.
,
24 ,
IL10. 24
IL-2
.
5.1.3.2.
IL-4
cAMP-
IL-10
132
T H 2 , IL-4.
PMA
IL-4 ,
, IL-4
.
IL-4
,
.
anti-CD3 antiCD3/antiCD28, IL-4
(Tokoyoda et al., 2004).
IL-4 anti-CD3 (He et al., 1999).
.
.
PKC
IL-4,
anti-CD3 (Van der Pouw-Kraan et al., 1992).
IL-4
anti-CD3/anti-CD28.
3 ,
IL-4 24
,
.
133
5.2.
cAMP-
.
cAMP
IL-10 ,
,
(Jimenez et al., 2001).
IL-10 ,
,
. ,
cAMP IL-10
IL-10
,
cAMP
IL-10 (Harizi et al., 2006; Platzer et al., 1999). ,
IL-10 cAMP ,
,
IL-10
cAMP .
IL-10, cAMP-
134
IL-10 . ,
IL-10
PMA, anti-CD3,
antiCD3/antiCD28. IL-10
,
IL-10
Ion/PMA.
Ion/PMA
Ca2+
C (PKC),
(Barritt, 1992).
cAMP
Ca2+ (Wayman et al., 1997). Ca2+
Ca , /
,
(Cooper, 2003a; Cooper, 2003b)
PDE1 (Cooper et al., 2004).
,
, , cAMP
cAMP. PDEs
cAMP,
, ,
cAMP.
, IL-10
PMA,
.
IL-10
cAMP,
, .. STAT
135
,
.
PMA Ca2+
cAMP
PDEs. cAMP-
IL-10
Ion/PMA
.
cAMP- IL-10
,
cAMP- IL-4 (Tokoyoda et al., 2004).
cAMP,
. cAMP
PGE2,
EP
10 (Zidek,
1999). PGE2-
anti-CD3 (Choudhry et al., 1999),
cAMP. ,
cAMP PKA-
Csk , Lck
ITAMs
CD3 (Vang et al., 2001). PGE2, Forskolin
cAMP,
,
.
cAMP,
cAMP
,
cAMP. , ,
cAMP-
. ,
136
cAMP Forskolin
- (Choudhry et al., 1999;
Paliogianni et al., 1993). , cAMP
CAM (Paliogianni et al., 1993; Wayman et al., 1997)
IL-2.
cAMP- -
,
,
.
IL-10
Rolipram,
cAMP PDE4. PDE4
cAMP
(Tasken et al., 2006).
, cAMP
.
PDE4, cAMP-
IL-10 cAMP.
5.2.1.1.
PKA cAMP-
IL-10
cAMP-
PKA
cAMP, Epac CNGs,
(Tasken et al., 2006). ,
cAMP-
IL-10 PKA-. Rp-8BrcAMP, PKA,
PKA I. PKA I
137
5.2.1.2.
cAMP- IL-10
IL-2-
IL-2
. IL-2
,
.
cAMP IL-2
(Anastassiou et al., 1992; Jimenez et al., 2001; Johnson et al., 1988;
Paliogianni et al., 1993; Tasken et al., 2006; Torgersen et al., 2002; Zidek, 1999),
.
IL-2 PI3-,
PDE4, cAMP (Heijink et al., 2003).
138
IL-10
IL-2,
. IL-2-
IL-5 (Heijink et al., 2003).
IL-2 IL-10 IL-2
.
cAMP- IL-10,
IL-10 cAMP
IL-2-.
T H 1 T H 2
cAMP (Munoz et al., 1990; Stam et al., 1993).
cAMP
T H 2 T H 1 (Zidek, 1999).
cAMP IL-10 ,
,
T H 2 , .
cAMP- IL-10
.
,
.
IL-10
IL-10,
. , cAMP- IL-10
140
5.3.
IL-10
TH
cAMP
IL-10 cAMP,
cAMP-
IL-10.
, firefly
Luciferase 10 (1327
) [Z30175], Platzer et
al. (Platzer et al., 1995).
, IL-10
cAMP
.
,
, IL-10
- .
IL10 Ion/PMA (Wang
et al., 2005).
IL-10 Ion/PMA,
cAMP. IL-10
cAMP,
,
IL-10
, -
.
Wang et al.
141
.
, Wang
et al .
.
, IL-10
50 % .
(80%)
IL10 , , - .
cAMP-
IL-10
IL-10, :
,
-.
,
IL-10
,
. -
.
cAMP mRNA
IL-10.
, IL-2, IL-6 IL-3,
mRNA (Gaffen et al., 2004).
, cAMP
mRNA
mRNA IL-2,
-
(Anastassiou et al., 1992).
mRNA IL-10,
D, - mRNA.
, mRNA IL-10
cAMP . ,
mRNA IL-10,
. IL-10 mRNA ,
IL-10
. cAMP
mRNA IL-10 , cAMP
RNA mRNA IL10 .
T H 1
T H 2 , T H 1 IL-10
IL-10 mRNA (Im et al., 2004;
Wang et al., 2005). T H 2 IL-10
mRNA ,
(Im et al., 2004; Wang et al., 2005).
Powell et al. EL-4
, 3-UTR IL-10
mRNA, (mRNAdestabilizing motifs) (Powell et al., 2000). , IL143
10 mRNA
,
. mRNA IL-10
PMA response elements, ,
Ion/PMA,
3-UTR
(Powell et al., 2000). cAMP- ,
,
mRNA
IL-10 ,
.
5.3.2.1.
cAMP- IL-10
IL-10
cAMP
,
cAMP.
-
cAMP
IL-10.
(5 deletion mutants
),
IL-10 ( 135, 235, 310, 500 1010 bp TATA box)
Luciferase firefly,
.
IL-10
cAMP (50 %), 500 .
IL-10
cAMP, IL-10
.
144
Luciferase
cAMP, ,
.
500 ,
IL-10 cAMP,
CONSITE, ,
cAMP IL-10. MEF2
CREB.
145
5.4.
MEF2
CREB IL-10 cAMP
5.4.1. MEF2
MEF2
, ,
,
. 4 , MEF2D
(Brand, 1997; Liu, 2005).
MEF2
/ -
. ,
MEF2 - IL-2
,
TATA (Pan et al., 2004) consensus
MEF2
IL4, IL-9, IL-13 IFN- (Esau et al., 2001).
MEF2 IL-10
.
,
MEF2
IL-10
,
cAMP ,
cAMP MEF2 cAMP IL-10.
, .. NFAT,
,
MEF2
,
(McKinsey et al., 2002).
146
,
- (Class II Histone Deacetylases-HDACs)
Cabin1, MEF2,
(Lu et al.,
2000).
,
, , cAMP-
, MAP
- (Esau et al., 2001; McKinsey et al., 2002).
MAP
MEF2
(Esau et al., 2001; McKinsey et al., 2002; Savignac et al., 2007)
- CAM K , MEF2
p300 (Brand, 1997; Liu, 2005; McKinsey et al.,
2002; Savignac et al., 2007).
cAMP Ca2+
,
. cAMP Ca2+
MEF2 Ca2+ -
- (Belfield et al., 2006).
,
cAMP-
MEF2.
, ,
cAMP
EF2.
MEF2
. ,
MEF2
DNA (Black
147
5.4.2. CREB
CREB
cAMP- /,
PKA Ser133.
CREB, CBP
p300, CREB/CBP/p300
148
149
6.
cAMP
IL-10 ,
IL-10. , IL-10 cAMP
.
IL-10 cAMP
,
,
.
IL-10 cAMP
PKA .
TCR/ ,
, cAMP
IL-10, mRNA. ,
IL-10 cAMP
.
T
/ , cAMP
IL-10.
cAMP - 500 bp
IL-10.
cAMP-
IL-10
MEF2, CREB
IL-10.
MEF2 CREB ,
.
IL-10 ,
cAMP
150
151
7.
ABSTRACT
cAMP is a second messenger playing a crucial role in the signal transduction
not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with
constitutively active PKA mutant. Transfection assays with the different IL-10
promoter fragments revealed that the most responsible part of IL-10 promoter to
cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains
binding sites for the transcription factors MEF-2 and CREB, as validated by the webbased program Consite. Increased intracellular cAMP reduces the binding of MEF2 to
nuclear extracts of stimulated T cells by 70 %, however its activity is not affected
significantly. On the contrary, both the binding and the activity of CREB are
increased in the presence of elevated cAMP.
cAMP mediated inhibition of IL-10 production is PKA mediated and specific
for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent
regulation of IL-10 production is controlled by transcriptional and/or posttranscriptional mechanisms depending on the nature of stimulus. Transcriptional
mechanisms involve the transcription factors MEF2 and CREB, however the exact
mechanisms of action of these factors deserves further elucidation. Cell and stimulus
specific mechanism of regulation of IL-10 production is necessary for its
immunoregulatory function.
153
8.
Abbas AK, & Lichtman AH (2001). Basic Immunology:Functions and
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Goldsby RA, Kindt TJ, & Osborne BA. (2004). Kuby Immunology. New
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Gomperts BD, Kramer IM, & Tatham PE. (2002). Signal Transduction. USA:
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Hancock JT (2005). Cell Signalling. New York: Oxford University Press.
Hidaka H, Nairn AC, Krebs EG, Cohen P, Yamakawa T, Takaku F, .
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cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at
multiple levels. J Immunol 1992; 148 (9):2845-52.
Bashian GG, Braun CM, Huang SK et al. Differential regulation of human,
antigen-specific Th1 and Th2 responses by the B-7 homologues, CD80 and CD86. Am
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155
Lynch MJ, Baillie GS, Mohamed A et al. RNA silencing identifies PDE4D5
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AcetylcoA: Acetyl coenzyme A
AC: Adenylate Cyclase
Ag: Antigen
AIDS:Acquired Immunodeficiency Syndrome
AKAPs: A Kinase Anchoring Proteins
AP: Alkaline Phosphatase
APCs: Antigen Presenting Cells
AP-1: Activator Protein-1
ATP: Adenosine Triphosphate
ATF-1: Activation of Transcription Factor-1
BCA: Bicichoninic Acid
BSA: Bovine Serum Albumin
bZip: Basic-domain-leucine-zipper
cAMP:Cyclic adenosine monophosphate
Ca2+: Calcium
CaMK: Ca2+/calmodulin-dependent protein kinases
CBP: CREB Binding Protein
CD3: Cluster of Differentiation 3
CD4: Cluster of Differentiation 4
CD8: Cluster of Differentiation 8
CD28: Cluster of Differentiation 28
CD45: Cluster of Differentiation 45
CNGs:Cyclic Nucleotide-Gated ion channels
CRE: Cyclic AMP Response Element
CREB: Cyclic AMP Response Element Binding Protein
CREM:Cyclic AMP Responsive Element Modulator
CTLA-4 (CD152): Cytotoxic T-Lymphocyte Antigen 4
CVI: Common Variable Immunodeficiency
DNA: Deoxyribonucleic acid
DNTPs:deoxyribonucleotide triphosphate
DTT:Dithiothreitol
ELISA: Enzyme-Linked Immunosorbent Assay
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