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The effect of TLR on transplantation and tolerance of the cornea
Mee Kum Kim M.D
1 , Jinho Jeong M.D
1 , Won Ryang Wee M.D
1 , Jin Hak Lee M.D
2
1 Department of ophthalmology, Seoul National University Hospital, Seoul, Korea 2 Department of ophthalmology, Seoul National University Bundang Hospital, Bundang, Korea
The authors have no financial interest in the subject matter of this poster.
Purpose
To investigate
the role of toll-like receptor (TLR)
On the
MHC mismatched allograft rejection in corneal transplantation
On the
immune tolerance of the cornea
Materials and Methods
Mouse cell culture
Group Group K : keratocytes only Group D : dendritic cells only (DC2.4*) Group M : macrophages only (Raw264.7*) Group K+D : keratocytes + dendritic cells Group K+M : keratocytes + macrophages : 1x10 4 cells : 1x10 4 cells : 1x10 4 cells : 0.5x10
4 + 0.5x10
4 cells : 0.5x10
4 + 0.5x10
4 cells
Mouse primary corneal keratocyte DMEM/F12 (1:1) with 10% FBS and 1%PS Mouse DC cell line: DC2.4
*
RPMI1640 with 10%FBS and 1%PS
Mouse macrophage cell line: Raw264.7*
DMEM with 10%FBS and 1%PS
* Kindly provided by Kim TJ
TLR stimulation with agonist LPS (TLR4 agonist) Pam3csk4 (TLR2 agonist) Change in TLR expression and its products of TLR signaling pathway
TLR2 and TLR4
expression RT-PCR Stimulation for 6 and 24 hours with LPS or Pam3csk4
TLR
Phospho-IkappaB-alpha (p-I κB-α)
Western blot Stimulation for 2, 6, and 24 hours with LPS or Pam3csk4 Cytokine assay ELISA Candidate cytokines :
IL-6, TNF α, TGF-ß, IL-10
Stimulation for 24 hours with various concentration LPS : 1, 10, 100, 1000, 10000 ng/ml Pam3csk4 : 1, 5, 10, 50, 100 ng/ml
Corneal transplantation
Group (Donor C3H Recipeint) C57BL/6 (B6) B6 C3H/HeN (C3H) B6 TLR4 knockout (TLR4KO) : Autograft (n=10) : MHC mismatched allograft (n=11) : (n=13)
TLR4 knock-out mice on a C57BL6 (B6 background) were bred at a SPF animal facility and were kindly provided by Dr. S-Y Seong at the Department of Microbiology and Immunology, Seoul National University College of Medicine
Transplantation procedure Donor /recipient : 3.0 / 2.0 mm trephine 8 to 10 interrupted 10-0 Nylon sutures Clinical evaluation Analysis of graft survival using Slit-lamp biomicroscopy Definition of rejection (end point) : 100% graft edema Kaplan-Meier survival analysis (Log rank (Mantel-Cox) test) Immune cell assay Cervical lymph node and spleen 12 days & 4 weeks after transplantation Flow cytometry (FACSCalibur) and FlowJo software CD11c, CD80/CD86: CD4/8, CD44/69: CD4/8, IFN
Results
TLR expression after agonist stimulation
TLR4 in M, D, and K
TLR2 in D and K
P-I κB-α expression after agonist stimulation
P-IκB-α in M, D, and K
P-IκB-α in M,and D
Cytokine secretion after agonist stimulation
After LPS stimulation
Coculture : K + D Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition
Coculture : K + M
K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw 264.7
)
After Pam3csk4 stimulation
Coculture : K + D Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition
Coculture : K + M
K : B6 Keratocyte D : Mouse dendritic cell line (DC 2.4) M : Mouse macrophage cell line (Raw 264.7
)
Corneal allograft survival and immunologic profiling
Median survival time :
C3H
B6 > C3H
TLR4KO P<0.05, Log Rank test
IFN γ secreting CD8 T cell :
C3H
B6 < C3H
TLR4KO
0,7 0,6 0,5 0,4 0,3
CD8 IFN-
in L/N at 12 days
0,2 0,1 0 B6-B6
P<0.05, Mann Whitney U test
CH3-B6 CH3-TLR4KO B6
Lymph nodes CD86 1.90
At 12 days 1.81
3.53
2.84
CD 11c 1.15
At 4 weeks 5.49
2.70
10.64
CD 11c CD80 Spleens CD80 CD86 2.27
3.48
1.57
CD 11c 4.13
2.77
3.17
1.75
CD 11c 1.95
C3H
B6 C3H
TLR4KO C3H
B6 C3H
TLR4KO
0.27
0.31
0.74
0.29
0.29
0.60
0.34
0.52
0.93
C3H
B6 C3H
B6 C3H
TLR4KO
Conclusions
TLR4
is likely to be involved in immune modulation of MHC mismatched corneal allograft rejection