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.
ABSTRACT ___________________________________________________________ 152
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
CAMP. .............................................................................................................................................. 110
6:IL10
ION/PMA,ANTICD3ANTICD3/ANTICD28RP/rhIL2. .......... 112
7:IL4
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
,
,
1.1.1.

.
,
i)
, , ii) ,

, iii)
,
,
, ,
.

.
, ,
( 1-2) (Abbas &
Lichtman, 2001) (Goldsby RA, 2004).
.
1-2. :
:
.
:.
, .
14
:

,

( 1-3),
1-3 : (scanning
electron microscope SEM). Copyright 2007 Dennis Kunkel Microscopy, Inc.
1.1.1.1.

, .

(CD4, CD8 ..).

,
, (nave) .
-


(TCR: T Cell Receptor)
.
15
.
,
( 1-4). ,

G0 .
G1, DNA ( S).
G2 .
,
, ,
RNA .
, 24
,
1000 ( ).

,
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
(Abbas & Lichtman, 2001) (Goldsby

RA, 2004).
,
,
.
(V, Variable)
.
(C, Constant)
,

TCR ,

CD3 ( 1-5). CD3
, , .
TCR CD3, ,
, ITAM (Immunoreceptor
Tyrosine Based Activation Motifs).

(Reth et al., 2004).
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
,
1.1.1.3.

MHC
. MHC ,
, I (MHC I) II (MHC II):
MHC ,
8-9
, CD8 .

, ( 1-7).
MHC ,
, 13-18
, CD4 .
,
1-7. APCs, MHC I MHC II ,

.
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.


CTLA-4 CD152. CTLA-4 CD28
, CD80
CD86,
(Alegre et al., 2001; Bashian et al., 1997; Frauwirth et al., 2002).
, ,
,
,
23
1.1.1.4.
.

MHC I (T C : T
cytotoxic cells), CD8


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
(Tr1 cells) CD4+

IL-10 IL-10 .
T H 1 T H 2
,
IL-10.
TGF-, IL-5
IFN-, IL-4 IL-10,
4
12 24 (O'Garra et al., 2004a; Wu et al., 2007).
(nregT CD4+CD25+Foxp3+ T cells)

IL-2 (IL-2R) (CD25)
CD4+CD25+.
,
.
(cell to cell contact)

Foxp3 (forkhead box transcription factor). , Tr1
Foxp3 CD4
(O'Garra et al., 2004a; Rouse, 2007). Foxp3

(Jonsson et al., 2005; O'Garra et al., 2004a).

,

,
-
(Horwitz et al., 2003).
, ,

,
, .

26
-MHC- .

IL-2
,

(Chandok et al., 2004).

CD45.
,

. CD45
.
CD45RO,
CD45RA () CD45RB
() .
(Chandok et
al., 2004).
1.1.2.

,
. (cytokines) (<30 kDa)
.
(lemphokines),
(monokines),
(interleukins)
.
,
24 ( 1-26, IL-1IL-26).
(interferones),
(tumor necrosis factor).

27


,
. / ,
,
.
,
,

( 1-12) (Abbas & Lichtman, 2001).
1-12.
.
(autocrine),

28
, (paracrine)
,
(endocrine)
, ,
( 1-13).
..

1-13. .

/
,
1-14.
/ .
29
,
,
,
.
,
( 1-11) (Abbas & Lichtman, 2001).
1.1.2.1. -10 (IL-10)

-10 (IL-10)
.
.

,
T H 1- . IL-10
(Mege et al.,
2006). T H 1 T H 2 IL10
-
(Tregs) (O'Garra et al., 2004b; Rouse, 2007; Taylor et al., 2006; Wu et al., 2007).
IL-10 ,
IL-10 -.
IL-10

Epstein-Barr IL-10.
Epstein-Barr IL-10
IL-10 80 %. IL-10 160 ,
18.5 kDa 37 kDa (Mege et al.,
2006; Moore et al., 1993; Pestka et al., 2004).
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
( )
-
-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-.
1-21. ATP cAMP .
..
&
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).
1-23. cAMP- , PKA.

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
/ .. CREB, CREM, ATF1, NFB (Fimia et al.,

2001; Sands et al., 2008; Sassone-Corsi, 1998; Torgersen et al., 2002).

,
,
.
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

PKA
(Protein Kinase Inhibitors, PKI).
PKI (, )
.
(chaperones) C PKA
45
cAMP
1.2.2.5.
,
cAMP 5-AMP
cAMP ( 1-25).
PKA,
(Lynch et al., 2005).
1-25. cAMP 5-AMP cAMP.
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
(lipid rafts). PKA,

PKA (Torgersen et al., 2002).
PKA 2-4 Csk ,
364,
Lck
(Tasken et al.,
2006).
70-80 % PKA
PKA 20-25 % PKA
Golgi . PKA

(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
4 mef2 , mef2a, -b, -c, -d,

,
. mef2d
(Liu, 2005). MEF2
, - (Black et al.,
1998).
MEF2 ( 1-26) N
, MADS-box ( 57 )
MEF2 domain ( 29 ) ( 1-24). MADS-box,
MEF2
MADS-box ,
,
DNA CTA(A/T) 4 TAG/A. MEF2 domain
MEF2
MEF2 . MADS-box MEF2
domain
DNA,
, C (McKinsey
et al., 2002).
1-26. MEF2. MEF2 ,

MADS N- , -
MEF2 DNA CTA(A/T) 4 TAG/A. MADS
MEF2- DNA
. MEF2
C- . MEF2-
. MEF2 ()
(). .
, MEF2
, Cabin1 -
52
(Histone deacetylases class II-HDACs) HDAC4,

5, 7 9 ( 1-27a). Cabin1 cain,
-
( -
),
- . HDACs 3
(Liu, 2005; McKinsey et al., 2002).
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
(basicdomain-leucine-zipper, bZip). , CREB,

-
CRE
.
CREB,
(transactivators)
(De Cesare et al., 1999).
N CREB (Activation
domain), 2 .
(Phosphorylation box-P box)
60 /
, 133
PKA. , 2
, Q1 Q2 ,

( 1-28). P box
Q1 Q2 , Q2
, CREB (De Cesare et al.,
1999; Mayr et al., 2001). C CREB
, (basic-domain-leucine-zipper,
bZip) (Mayr et al., 2001).
1-28. CREB. (Q1

Q2), DNA, (basic domain)
(Leucine zipper), (P-box).
133, PKA.
CREB
.. p38 - ,
.. (Mayr
et al., 2001).
55
1.3.3. cAMP
NFB/Re1/NFAT,
AP-1
CREB/ATF, PKA (Sands et al., 2008; Torgersen et al., 2002).

PKA CREB 133
cAMP ( 1-29)
(De Cesare et al., 1999; Fimia et al., 2001; Sands et al., 2008; Sassone-Corsi, 1998;
Torgersen et al., 2002).
CREB, CBP p300,
CREB/CBP/p300
CRE (Fimia et al., 2001; Mayr et al., 2001; Sands et al., 2008;
Sassone-Corsi, 1998; Torgersen et al., 2002),

(Torgersen et al., 2002).
1-29. cAMP- CREB.

CREB
/ PP-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
-32P ATP Amersham Pharmacia Biotech

SCHERING PLOUGH
SIGMA
DNA GIBCO BRL

// (125:24:1) SIGMA
KODAK
- SIGMA
62

Virion/Serion D
MERCK
(Trypan blue) BIOCHROM
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
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
Trypan blue ( 0.5 % (/) PBS)

:
25 ml
Falcon 50 ml 50 ml
, 250 x g, 9 min, 4 C.
Pasteur 25 ml,
,
50 ml.
2 . 3
200 ml, AET

-.
, Pasteur
20 ml
AET/, 1:5 (:),
20 37C ().
50 ml
250 x g 9 min 4C.
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 ,
.
2.1.5. L-- (Leucine-methyl-ester).

L-Leucine-methyl-ester

.
:
(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),
(Ionomycin) (SIGMA) PMA

(Phorbol 12-Myristate 13-Acetate),
(TCR) -,
anti-CD3 .
:
0,05 %
, 1-10 x 106 / ml RPMI 1640 10 %
FBS 40 g/ml
24 falcon
106 M Forskolin, 106 M PGE2, 106 M 8-Br-cAMP
79
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
PKA IL-10 cAMP,

10, 25 50 RP-8-Br-cAMP (BioLog).
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.
(RT-PCR Reverse Transcription Polymerase chain reaction)

:
RNA cDNA
(reverse transcriptase).
RNA cDNA,
dT
.
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.
First Strand Buffer

DTTs
ddH 2 O
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.
(RTPCR, Real Time PCR)

RT-PCR PCR,
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
Maxim Biotech Inc.

:
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
, .
E.coli DH5 (competent cells),

Promega.
E.coli
.
:
DH5 (20l competent cells),
10-50ng DNA (
). 30 , 2
42C 2 . 1
ml SOC L-Broth 1-2
37C
. 1200 x g 5

L-broth 1.5 % ,
DNA, 16
37 C.
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, firefly luciferase

10
[1037bp Z30175] (Platzer et al., 1995) ( . ).
pRL-TK Renilla luciferase
(Herpes simplex virus
thymidine kinase promoter) (Promega Corporation, Madison, WI).
SecI, p3782, pVUII, BSU DraI
firefly luciferase 135, 235, 310, 500 1010 bp,
TATA IL-10 ( .
).
SRa-PKA
PKA-. ( Muramatsu et al., 1989)
MEF2/luc ( Han et al., 1995) CREB/luc
(STRATAGENE) firefly luciferase
MEF2 CREB .
PKA SRa.
(Muramatsu et al., 1989),
95
IL-10/Luc, SecI,
p3782, pVUII, BSU DraI pGL3 (INVIVOGEN).
2.4.1.7.
BRADFORD.
BIORAD Protein Assay (5x) ,

Bradford (Bradford, 1976)
.
:

. Coomassie Brilliant
Blue G-250 450 nm.

595 nm. Coomassie
Brilliant Blue G-250 .
:
5 .. BSA
.
1.2g/ml
10g/ml .
96 50 l 2
l 48 l . 250 l
BIORAD (1x)
.
5
.

,
.
(
g/ml) .
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.
, (Turner Designs DT20/20)

.
,
562nm.
98
,
562 nm
(RLU: Relative Light Units).
mg ,
(standards),

Renilla .
2.4.2. In vitro
5
2.4.2.1.
DNA (5 end labeling)

:

DNA (EMSA),
DNA
.
4 (PNK),
Promega. PNK
[-32P] ATP (Amersham Pharmacia Biotech) 5-
DNA. DNA

.
:
eppendorf :
6.
[-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
4.1.2. cAMP IL-2

-2 T H 1 ,

,
. ,
T H 1 cAMP
IL-2
( 5). ,

, .
5: IL-2
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
Rp / rhIL-2.
4.1.4. cAMP IL-4

IL-10 cAMP,
T H 2 ,
IL-4 ( 7).

, .
7: IL-4
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
DNA (transfection by electroporation)

.
IL-10
PGE2 Rolipram I/PMA ( 10).
, anti-CD3/anti-CD28 PGE2
IL-10 50 %
, Rolipram
60 %.
anti-CD3 ,
Luciferase Renilla
.
115
10: cAMP
IL-10 .
PKA, IL-10
PKA ( 11).
11: PKA IL-10

.
4.2.2. cAMP
mRNA IL-10
cAMP mRNA IL-10

mRNA
-10 ,
.
116
Rolipram, mRNA IL-10

I/PMA. ,
anti-CD3/anti-CD28, Rolipram
mRNA IL-10 80 % ( 12).
12: cAMP mRNA IL10 .
4.2.3. mRNA IL-10

, cAMP,

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 %.
13: cAMP mRNA IL10 .
CD3/CD28
Rolipram mRNA IL-10 220

CD3/CD28
4 , ( 13).
anti-CD3/anti-CD28 Rolipram
mRNA IL-10 30 120
, anti-CD3/anti-CD28
Rolipram mRNA
240 . (240
) mRNA IL-10 30 % Rolipram,
Rolipram 70 %.

I/PMA cAMP
mRNA IL-10 antiCD3/anti-CD28 cAMP .
118
4.2.4. cAMP 500

IL-10
IL-10

(CR/CD28).

IL-10, cAMP.

IL-10

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 ).
14: cAMP IL-10

.
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
Conservation profile of Human_IL10
Conservation profile of Mouse_IL10
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
MEF2, PGE2 Rolipram
( 17). MEF2
,

.
17: cAMP
MEF2.
123
4.4.2.
cAMP
CREB in vivo
CRB
/ cAMP-
.
firefly
Luciferase CREB
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),

. 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.

, 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. IL-2 IL-4
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

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-
5.2.1. cAMP- IL10

,
,
cAMP IL-10,
. , cAMP
in vitro
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

, 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
(Skalhegg et al., 1998),

,
PKA I,

(Skalhegg et al., 1997).
IL-10,
PKA.
PKA
PKA (PKA-C) (Muramatsu et al., 1989).
cAMP
PKA . ,
3 ,
PKA R C , C
PKA (Skalhegg et al., 1997).
PKA,
C ,
cAMP- IL-10
.
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-.
5.2.2. cAMP- IL-2 IL-4

cAMP-
IL-10,
T H 2 ,
T H 2
. ,
IL-2, T H 1
IL-4, T H 2

cAMP.
cAMP
IL-2
IL-4
(Anastassiou et al., 1992; Van der Pouw-Kraan et al., 1992;
Zidek, 1999). ,
cAMP IL-2

cAMP (Anastassiou et al.,
1992). IL-2

cAMP,
139
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
5.3.1. cAMP- IL-10
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
,

. -
.
5.3.2. cAMP- mRNA IL-10

cAMP-
IL-10, cAMP-
mRNA .
RT-PCR
, mRNA IL-10
cAMP ,
. ,
142

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
et al., 1998), MEF2

cAMP-
IL-10 . MEF2
, NFAT
, MyoD
..
CREB
(Berdeaux et al., 2007; Potthoff et al., 2007).
,
MEF2 ,

Mef2- (Cripps et al., 2004;
Potthoff et al., 2007). , MEF2 RNAs
(miRNAs), -
3 UTR mRNA
, MEF2C
HDAC9,
(He et al., 2004; Potthoff et al., 2007).
MEF2 ,

, ,
,
/ -
. cAMP-
IL-10 ,
.
5.4.2. CREB
CREB
cAMP- /,
PKA Ser133.
CREB, CBP
p300, CREB/CBP/p300
148
CRE (Fimia et al., 2001; Mayr et

al., 2001; Sands et al., 2008; Sassone-Corsi, 1998; Torgersen et al., 2002).

CREB Ser133
, (Ca2+)
MAP (p38) (Yu et al., 2001)
, DNA

cAMP, cAMP IL-10. ,
cAMP
IL-10 (Platzer
et al., 1999), IL-10 ,
,
, IL-10
.

cAMP IL-10,
CREB .

CREB IL-10, CREB
IL-10,

, , /

.
, cAMP
CREB DNA (Montminy, 1997a; Montminy, 1997b),
cAMP
CREB DNA .
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
which controls the immune response, while IL-10 is considered to be an important

regulator of this response. Elevation of intracellular concentration of cAMP has been
shown to increase IL-10 production by monocytes. The aim of this study was the
elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a
mechanism that remains unclear.
Fresh Human -lymphocytes derived from PBMC of healthy donors where
stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence
of cAMP elevating agents (10-6 Forskolin, 10-6 PGE2, 5x10-6 Rolipram and
10-6 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the
production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was
determined by the use of Actinomycin D (10 M). The activity of IL-10 promoter was
measured by luciferase reporter assay, after transfection of cells with plasmids
carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010,
-500, -310, -235, -135bp). PKA role was examined either by cotransfection
experiments with a plasmid carrying a constitutively active mutant of the catalytic
subunit of PKA- isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP
(10-50 M). The presence of binding sites of transcription factors in the first 500bp of
the IL-10 promoter, was validated using the web-based program CONSITE. Binding
of the transcription factors MEF2 and CREB was investigated in nuclear extracts of
stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB
was investigated independently with transfection experiments using plasmids
containing the lusiferase reporter under the control of the transcription factors.
Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%,
when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation
with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP
reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the
same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP
elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the
anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly
transcriptional. IL-10 promoter activity is reduced up to 60% when cells are
stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is
152
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.
KEYWORDS: immune response, cAMP, IL-10, Fresh Human cells, transcription,

post-transcription, IL-10 mRNA, IL-10 promoter, transcription factos, MEF2, CREB.
153
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162
163

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
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
164
EMSA: Electrophoretic Mobility Shift Assay

Epacs:cAMP-stimulated GDP exchange factors
FITC:Fluorescein isothiocyanate
Foxp3: Forkhead box P3
GDP:Guanosine diphosphate
GEFs:Guanine Exchange Factors
G i -protein: G inhibitory -protein
G s -protein: G stimulatory -protein
GTP: Guanosine-5'-triphosphate
HDACs: Histone deacetylases
HIV:Human Immunodeficiency Virus
HRP: Horse Radish Peroxidase
IFN: Interferon
ITAM : Immunoreceptor Tyrosine Based Activation Motifs
IgG: Immunoglobulin G
IgE : Immunoglobulin E
IFN-: Interferon -gamma
IL-1- IL-24 : Interleukin-1 - 24
IL-10 : Interleukin-10
IL-10R : IL-10 Receptor
IL-10R1 : IL-10 Receptor
IL-10R2 : IL-10 Receptor
IL-2 : Interleukin-2
IL-2R : IL-10 Receptor
Ion : Ionomycin
I: Inhibitor
JAK: Janus Kinases
JNK: Jun N-Terminal Kinases
Lck: leukocyte-specific protein tyrosine kinase
LPS :Lipopolysaccharide
Luc: Luciferase
MADS : MCM1, Agamous, Deficiens, Serum response factor
MAPK : Mitogen-activated protein kinases
MEF2 : Myocyte Enhancer Factor-2
165
MEF2D : Myocyte Enhancer Factor-2D

MHC: Major Histocompatibility Complex
MHC : MHC class I
MHC : MHC class II
mRNA: messenger RNA
nTreg : naturally occurring T regulatory cells
NF-B : Nuclear Factor-B
NK: Natural Killer
PBL: Peripheral Blood Leukocyte
PBMC: Peripheral Blood Mononuclear Cells
PBS: Phosphate Buffer Saline
PCR: Polymerase Chain Reaction
PDEs: Phosphodiesterases
PE:R-Phycoerythrin
PGE 2: Prostaglandin E 2
PKA: Protein Kinase A
PKA I: Protein Kinase A Type I
PKA II: Protein Kinase A Type II
PKC: Protein Kinase C
PKI: Protein Kinase Inhibitor
PMA: Phorbol Myristate Acetate
PMSF: Phenyl-Methyl-Sulphonyl-Fluoride
PTK : Protein Tyrosine Kinase
PP-1:Protein Phosphatase 1
PP-2A: Protein Phosphatase 2A
PP-2B: Protein Phosphatase 2B
PP-2C: Protein Phosphatase 2C
rhIL-2: recombinant human IL-2
RI: Regulatory subunit I
RII: Regulatory subunit II
RLU: Relative Light Units
RNA: Ribonucleic Acid
Rp: Rolipram
Rp-8Br-cAMP:Rp- isomer 8-bromoadenosine-3', 5'cyclic monophosphorothioate
166
RT-PCR: Real Time Polymerase Chain Reaction

SDS: Sodium-Dodecyl-Sulphate
SDS-PAGE: SDS Polyacrylamide Gel Electrophoresis
SOCS-1:Suppressor of cytokine signaling 1
SOCS-3: Suppressor of cytokine signaling 3
SSIIRT:SuperScript II reverse transcriptase
STATs : Signal Transuding Activators of Transcription
TCR: T Cell Receptor
T H 1: T Helper cells 1
Tr1: T Regulatory cells 1
TBS: Tris-Buffered Saline
TGF: Transforming Growth Factor
TNF-: Tumor Necrosis Factor-
U.V.: Ultra Violet (Radiation)
167

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ABSTRACT ___________________________________________________________ 152

(Abbas & Lichtman, 2001) (Goldsby

1-7. APCs, MHC I MHC II ,

(Tr1 cells) CD4+

( 1-11) (Abbas & Lichtman, 2001).

1.1.2.1. -10 (IL-10)

1-21. ATP cAMP .

1-23. cAMP- , PKA.

/ .. CREB, CREM, ATF1, NFB (Fimia et al.,

1-25. cAMP 5-AMP cAMP.

(lipid rafts). PKA,

4 mef2 , mef2a, -b, -c, -d,

1-26. MEF2. MEF2 ,

(Histone deacetylases class II-HDACs) HDAC4,

(basicdomain-leucine-zipper, bZip). , CREB,

1-28. CREB. (Q1

CREB/ATF, PKA (Sands et al., 2008; Torgersen et al., 2002).

1-29. cAMP- CREB.

-32P ATP Amersham Pharmacia Biotech

DNA GIBCO BRL

(Trypan blue) BIOCHROM

Trypan blue ( 0.5 % (/) PBS)

200 ml, AET

2.1.5. L-- (Leucine-methyl-ester).

(Ionomycin) (SIGMA) PMA

PKA IL-10 cAMP,

(RT-PCR Reverse Transcription Polymerase chain reaction)

First Strand Buffer

(RTPCR, Real Time PCR)

Maxim Biotech Inc.

E.coli DH5 (competent cells),

IL-10/Luc, firefly luciferase

BIORAD Protein Assay (5x) ,

, (Turner Designs DT20/20)

DNA (5 end labeling)

4.1.2. cAMP IL-2

4.1.4. cAMP IL-4

DNA (transfection by electroporation)

11: PKA IL-10

Rolipram, mRNA IL-10

12: cAMP mRNA IL10 .

4.2.3. mRNA IL-10

13: cAMP mRNA IL10 .

Rolipram mRNA IL-10 220

4.2.4. cAMP 500

14: cAMP IL-10

Conservation profile of Human_IL10

Conservation profile of Mouse_IL10

5.1.3. IL-2 IL-4

5.2.1. cAMP- IL10

(Skalhegg et al., 1998),

5.2.2. cAMP- IL-2 IL-4

5.3.1. cAMP- IL-10

5.3.2. cAMP- mRNA IL-10

et al., 1998), MEF2

CRE (Fimia et al., 2001; Mayr et

which controls the immune response, while IL-10 is considered to be an important

KEYWORDS: immune response, cAMP, IL-10, Fresh Human cells, transcription,

Bradford MM. A rapid and sensitive method for the quantitation of

Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by

He X, Stuart JM. Prostaglandin E2 selectively inhibits human CD4+ T cells

O'Garra A, Vieira P. Regulatory T cells and mechanisms of immune system