Local Anterior Chamber Production of IL–10 and Not IFN During Corneal Graft rRjection

Abstract

Abstract: : Purpose: To determine cell–surface and inflammatory cytokine phenotype of cells infiltrating the anterior chamber (AC) during corneal graft rejection. Methods: Corneas from LEW strain rat were transplanted to PVG strain recipients. Animals were killed and perfused with PBS at the time of endothelial rejection (day 15 after transplantation). Cells were removed from the AC of transplanted eyes (pooled from 4–6 animals on each occasion), stained with a range of antibodies to cell surface markers and the cytokines TNFα, IFNγ and IL–10 and examined by flow cytometry. Staining with each combination of antibodies was repeated at least twice. For cytokine detection cells were pre–incubated for 2.5–3h in culture medium containing brefeldin A. Results:Proportions of leukocyte subtypes by single staining were as follows: αßTCR 52% (range 42–62), CD4 29% (27–30), CD8 47% (45–48), CD25 13% (8–18), MHC class II 30%, CD161 (NK) 27% (25–28), CD68 (myeloid cell) 21%, CD163 (tissue macrophage) 11% (10–12) and granulocyte 12% (10–13). 65% of ED2+ macrophages were CD4⁺ and at least 90% were MHC II⁺. The CD161⁺ population comprised a 1:1 ratio of aßTCR⁺CD161^low to αß TCR^–CD161^high cells and 50% of CD161⁺ cells were also CD8⁺. 80–90% of αß TCR⁺ cells, CD161⁺ cells and macrophages expressed TNFα and at least 20% of each cell type expressed IL–10, suggesting co–expression of TNF and IL–10 in some cells. Both TNF and IL–10 expression could be blocked by pre–incubation of cells with an excess of unlabelled antibody or recombinant cytokine. In contrast there was negligible IFNγ expression. However, stimulation of AC cells for 4 hours with PMA and ionomycin induced IFNγ in 23% of cells, 43% of which were CD4⁺. Conclusions:The profile of cells infiltrating the AC is different from that in the stroma (fewer macrophages and granulocytes, more NK cells and lack of IFNγ). Local production of IFNγ is not essential for endothelial rejection. The presence of large numbers of IL–10⁺ cells in combination with a lack of IFNγ is suggestive of immunoregulation occurring in the AC environment, which nevertheless does not prevent endothelial rejection.

Keywords: transplantation • immunomodulation/immunoregulation • anterior chamber