Transcript CFA

Th1/Th2 balance in animal models of polyarthritis
(adjuvant-induced arthritis, oil-induced arthritis and
pristane-induced arthritis)
J. Basek, C.L. Zheng, M.A. Hossain, A. Kukita, K. Ohki, O. Kohashi
Saga Medical School
Saga, Japan (15')
Molecular Medicine
# 453
Oil-induced arthritis and Adjuvant-induced arthritis
Are Th1-mediated autoimmune diseases
CFA (complete Freund adjuvant)
Oil Vehicles
LEW No arthritis
DA rat
Th1
Th2
Th1
Th1
OIA
IL-2 ↑,
IFNγ↑
TNF
Lewis rat
Th2
CFA Challenge
Th2
IL- 4↑
Th1
AA
IL2↓ TNFα↓
Prevention of AA
IL-2 ↑,
IFNγ↑
TNF
Chemical structures of various vehicles
CH3
CH3
n-Hexadecane
CH3
( C16 H34 )
CH3
CH3
Pristane
CH3
CH3
CH2CH2OH
Squalane
N
CH3
( C30 H62 )
CH3OOC
C18H37
CH2
N
CH2CH2OH
Avridine
( C41H86N2O2
)
Composition of IFA
CH 3
Methyl oleate
CH3
( C19H40 )
C18H37
CH3
CH3
CH3
CH3
CH3
CH3
CH3
( C19 H36 O2 )
1) Mineral oil (85%) :
Mixture of hydrocarbons (CnH2n+2)
n=mostly 14-18
2) Mannide monooleate (15%)
Part 1
The pretreatment of IFA can
completely prevent AA in Lew rats
Hexadecane,
metheyl oleate,
pristane,
squalane
Clinical score
9
7
mRNA expression or
Adoptive transfer
6
5
4
3
CFA alone
IFA pretreatment
then CFA challenge
LNCs
Day11
2
1
IFA
-28
8
0
-14
-7
10
CFA
12
14
16
18
20
22
24
26
28 (day)
Day after intradermal inoculation of CFA
( ) : 50 l IFA was intradermally injected at the left foot pad 28 days before CFA challenge.
( ) : CFA (0.5 mg) was intradermally injected at the base of tail without IFA pretreatment.
Each group consists of 15 rats.
mRNA expression of cytokines in the LNCs of AA rats
CFA
IL-2
IFA/
CFA
IFA
Naive
LEW Rats
CFA
IFA/CFA
IFA
(day 11)
(day 11)
(day 28)
Naive
IFN-
TNF-
Inguinal lymph node
1.0x107 LNCs
IL- 4
Total RNA extraction
IL- 5
RT-PCR
GADPH
AA
+
mRNA expression of cytokines in the LNCs of OIA rats
Rat strain
DA
LEW
DA
LEW
IFA
IFA
Naive
Naive
LEW
CFA
IL-2
IFN-
TNF-
IL- 4
IL- 5
GADPH
OIA
+
AA
+
IFA/
CFA
Summary of cytokine profiles of LNCs
The rats with either AA or OIA) showed
1) increased mRNA expression of IFN-α, IL-2 and TNF-α
2) no mRNAexpression of IL-4
indicating the intimate relationship between the increased expression
of Th1 cytokines and disease development
The pretreatment of IFA prevented AA in Lew rats whose cytokine
profiles of LNCs showed
1) increased expression of IL-4 and IFN-α
2) no expression of IL-2 and TNF-α, suggesting the higher
expression of Th2 than Th1 cytokines
It is thus concluded that the balance of Th1/Th2 cytokines is intimatly involved in
progression or suppression of Adjuvant-induced Arthritis (AA) and Oil-induced
Arthritis (OIA) of Lewis and DA rats
Part 2
PIA can be completely suppressed by CFA
CFA
7
Arthritis score
6
Pristane
***
PBS
5
***
4
*
3
2
1
0
-12
0
CFA
15
30
45
60
70
Days after CFA treatment
Rats were injected with 0.2 ml of pristane and on day 12 after pristane injection (before onset of
PIA), they were randomly separated into two groups. One group of rats was injected with 0.2 ml
(0.2mg) of CFA at the back and other group of rats was injected with 0.2 ml of PBS
PIA can be cured by CFA
CFA
PBS
6
Arthritis score
**
Pristane
-18
5
**
4
*
3
2
1
CFA
0
16
37
64
94
125
Days after CFA treatment
Rats were injected with 0.2 ml of pristane and on day 18 after pristane injection (initial peak
severity of PIA), they were separated into treatment and control groups.
Treatment group; 0.1 ml (0.1mg) of CFA was injected at the base of tail
Control group ; 0.1 ml of PBS was injected at the base of tail
mRNA expression of cytokines in the LNCs of PIA rats
with or without CFA treatment
Exp.1
Exp.2
0.1
PIA/
CFA
PIA CFA
0.5
CFA
IFA/
CFA
IL-2
IFN
TNF
IL- 4
IL- 5
GADPH
PIA
-
+
AA
(very mild)
AA
/severe
IFA
Part 3
PIA can be prevented completely by CFA
A
Arthritis score
6
pristane
B
7
CFA
PBS
Reactive Nitrogen Intermediate
25
A
** p < 0.005
20
5
15
4
3
10
B
2
C
5
1
CFA
-d18
0
3
5
Days after CFA treatment
7
0
PIA/CFA
PIA/PBS
Naive
Serum RNI of Lewis rats 7 days after CFA treatment
Rats were injected with 0.2 ml of pristane at the base of the tail. On day 18 after pristane injection, the rats rats
were grouped with equal average arthritis score, then the rats in CFA-treated group were injected with 0.2 ml
CFA (Mt, 0.2 mg). The rats in the control group were injected with the same volume of PBS. Each group
consisted of 7 rats. P < 0.01.
iNOS inhibitors exacerbate EAE or AA possibly through inhibition of glucocorticoide production, while higher levels of iNOS or
RNI are related to suppression of EAE or AA. NO is a 'double edged sword'
Summary of Part 2 &3
Pristane-induced arthritis (PIA) is believed to be Th1-mediated autoimmune
disease but nobody succeed to transfer PIA by LNCs.
CFA can completely prevent the induction of PIA
and also suppress the ongoing PIA (cure PIA).
Nitric oxide induced by CFA may play some critical role in the recovery of
PIA. One can ask whether iNOS inhibitors may reverse or flare up the PIA
suppressed by CFA, but at present we faile.
The mRNA expression of LNCs can not show clear Th1 or Th2 profiles.
Since the increased mRNA expression of IFNγalways be apparent in PIA or
PIA, IFNγ may play an important regulatory role in preogression or
suppression of PIA.
Hypothetical network of Th1/Th2 differentiation and balance in AA
and PIA in Lew rats.
IL-2
CD8+
IFN-
DC
IFN-
CFA , Oils or Pristane
IL-12
TNF-b
Th 1
(Microbe/allergen)
OIA, AA
IL-12 R
IL-4 R
inhibit
IL-12Rb2↑
and PIA
IL-2
Thp
IL-4
TCR
Naive
IL-4 R
CD4+T cell
IFN-γ
inhibit
IL-4
IL-5
IL-4
IL-4
IL-10
Th 2
DC
CD8-
IL-4 expressed in Th2, mast cells, NK T cells, Basophils,
And d T cells. Suppressed by CsA and FK506.
Complex regulation of IL-4 production, impaired or overproduction
IL-12Rb2↓
IL-13
Antibodies
Mast cell degranulation
Eosinophil activation
PCR primers used for RT-PCR analysis
Cytokines
Primer sequence
Base pairs
IFN-
5'-CCCTCTCTGGCTGTTACTGC-3’
5'-CTCCTTTTCCGCTTCCTTAG -3'
415
IL-2
5' -TACAGCATGCAGCTCGCATCCTGTG-3’
5' -CAGAAATTCCACCACAGTTGCTGGC-3'
414
TNF-
5'-GGCAGGTCTACTTTGGAGTCATTGC-3’
5'-ACATTCGGGGATCCAGTGAGTTCCG-3'
319
IL-4
5'-ACCTTGCTGTCACCCTGTTCTGC-3’
5'-GTTGTGAGCGTGGACTACTTCACG-3'
352
IL-5
5'-TGACGAGCAATGAGACGATG-3’
5'-TCATCACGCCAAGGAACTCT-3'
248
GADPH
5'-CATGGAGAAGGCTGGGGCTC-3’
5'-AACGGATACATTGGGGTAG-3'
414
Group 1 (Adjuvant-Induced Arthritis)
Group 2 (MDP-Induced Arthritis)
Muramyl Dipeptide (MDP)-Induced Arthritis (Kohashi et. al. 1980).
MDP is considered to be the minimum essential structure for adjuvanticity of CFA.
Acetylated MDPs in PBS could induce acute and chronic arthritis indistinguishable to AA
Group 3 (Chemical Compounds-Induced Arthritis)
1. Avridine-Induced Arthritis (Chang et. al. 1980).
Avridine: C43H90N2
2. Pristane-Induced Arthritis (Wooley et. al 1989; Vingsbo et. al. 1996).
Pristane: C19H40
3. Adjuvant Oil (IFA)-Induced Arthritis (OIA) (Kleinau et. al. 1991).
IFA: Mixture of hydrocarbons (C=14-18)+Surfactant (Mannide Monooleate).
4. CpG Oligodeoxynucleotides -induced Arthritis
5. Dimethyl Dioctadecyl Ammonium Bromide (DDA)-Induced Arthritis
(Mia et al. 2000)
Balance of GATA3 and T-bet-model for Th1/Th2 polerization
IL-12
IL-4
Epitope
IL-12R
IL-4R
TCR
T-box expressed in T
cells (T-bet)
iscorrelates with the
expression of IFN-
Day 0
1
2
3
4
5
1 2 1 2 1 2 1 2 1 2
Stat 4
Stat 6
m-T-bet
T-bet
GATA3
m-GATA3
NFAT C-Maf
NAFT NF-k B
IFN-, IL-4, IL-5
Th 1
IL-4, IL-5, IFN-
Transduction of T-bet into
Th2 cells converts into IFN secreting Th1 cells and
repress the Th2 cytokines
IL-4 and IL-5.
Th 2
Role of heat shock protein (HSP) in AA
Heat shock proteins (HSP) are highly immunogenic proteins,
up-regulated under various kinds of stress.
CFA
No disease
hsp 65/IFA
Pretreatment
LEW
Tregulatory cell responds to HSP
Bacterial hsp 65 cross-reacts with rat self HSP or rat proteoglycan
Tpath
Induction
Arthritogenic
Resistant
180-188
256-270
4 amino acid homology
Rat proteoglycan
9 amino acid homology
Rat hsp 60
Treg
Prevention