Transcript Document

Contribution of Programmed Death
Ligand-1 Expression to Corneal
Avascularity
Yiping Jin,1 Sunil K. Chauhan, 1 Arlene Sharp, 2 Reza Dana1
1.Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary,
2. Department of Pathology, Harvard Medical School,
The authors have no financial interest in the subject matter of this poster.
Support
NIH R01-EY12963
Introduction
The cornea possesses an extensive array of mechanisms by which immune
effectors and neovascularization (NV) are regulated, or even silenced,
including an intact corneal epithelial layer, epithelial expression of membrane
and soluble VEGF receptors and the high expression of FasL and
thrombospondin-1.
Recently, an immunoregulatory molecule, programmed death ligand-1
(PDL-1, CD274, B7-H1), has been shown to be expressed at high levels by
corneal tissue by our lab and others. PDL-1 is a member of the B7-CD28
family, which plays important roles in regulating T cell activation and
tolerance.
Several immunoregulatory factors, such as FasL and thrombospondin-1,
also possess a regulatory function on corneal NV. However, the antiangiogenic effect of PDL-1 has not been demonstrated to date. Here, we
provide novel evidence for the regulatory function of PDL-1 in corneal
angiogenesis in vivo.
Methods
• Animals: PDL-1-/- C57BL/6 mice, C57BL/6 mice
• Three intrastromal sutures were placed to induce corneal
neovascularization.
• The numbers of infiltrating CD11b+ and Gr-1+ cells were
measured by flow cytometry.
• The expression levels of VEGR-2, IL-1α, IL-1β and TNF-α
in corneas were analyzed by real-time PCR.
• Angiogenesis was quantified by biomicroscopically and by
measuring the blood vessel area spanned by CD31+ cells on
whole-mount corneas.
Results
PDL-1 suppresses corneal neovascularization
A.
9
8
7
score
NVscore
NV
B.
WT
6
5
4
3
KO
WT
2
1
0
KO
D3
CD31
D5
D7
D9
D11
Days after Days
suture placement
*
C.
% of cornea area
D.
WT
KO
WT
KO
Figure 1. (A) Corneal neovascularization (NV) was induced in WT and PDL-1 KO mice after sutureplacement. (B) In a masked fashion, corneal NV was scored biomicroscopically with a slit-lamp using
a grid system. Values are expressed as the mean (+ SEM) of 6 corneas. (C) On Day 14, whole corneas
were dissected and stained with anti-CD31-FITC (Green) (D) The density of blood vessels (CD31+)
covering the cornea was analyzed. Values are expressed as the mean (+ SEM) of 6 corneas of per
treatment group (* P< 0.001 vs. vehicle-treated group).
Results
PDL-1 promotes the expression level of VEGFR-2, but
not inflammatory cytokines, in suture-placed corneas
TNF-α
mRNA Relative expression level
mRNA Relative expression level
VEGFR2
**
9
*
6
3
0
WT
KO
WT
0.75
0.50
0.25
KO
mRNA Relative expression level
1.00
0.75
0.50
0.25
WT
KO
24 hr
WT
KO
WT
KO
24 hr
WT
KO
72 hr
IL-1β
1.25
normal
normal
72 hr
IL-1α
mRNA Relative expression level
1.00
0.00
normal
24 hr
0.00
1.25
WT
KO
72 hr
400
300
200
100
0
normal
WT
KO
24 hr
WT
KO
72 hr
Figure 2. Three days after suture placement, PDL-1 KO mice showed a
significantly higher mRNA expression level of VEGFR-2, comparing to the
WT mice. However, there were no significant differences in the mRNA
expression levels of IL-1α, IL-1β or TNF-αbetween both groups.
Results
PDL-1 does not affect the infiltration of innate
immune cells in suture-placed corneas
Isotype control
PDL-1KO
1044
10
0.21
1044
10
1.03
CD11b-PE
PE
1033
10
1022
10
1011
10
1000
10
10
1000
1011
10
1022
Fitc
1033
1044
8.84
WT
104
10
22.01
1033
10
103
10
1022
10
102
10
1011
10
101
10
1000
10
0 0
1010
10
1011
10
1022
10
1033
10
1044
100
10
9.43
00
1010
10
1011
20.30
10
102
10
103
10
104
Gr-1-Fitc
Figure 3. Corneas were harvested from WT and PDL-1 KO mice at 72 hr after
suture placement and digested by collagenase D. Cells were double-stained
with anti-CD11b-PE and anti-Gr-1-FITC Abs, and analyzed by flowcytometry.
Gr-1+CD11b+ neutrophil and Gr-1-CD11b+ macrophage populations were
compared between WT and PDL-1 KO groups.
Conclusion
• PDL-1 expression in the cornea contributes
to the inhibition of corneal angiogenesis.
• The anti-angiogenic effect of PDL-1 is not
related to its immune regulatory function.
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