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Bharati Matta, Purushottam Jha, Puran S. Bora, Nalini S. Bora; Role of iC3b-CR3 interaction in Experimental Autoimmune Anterior Uveitis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6237.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the role of interaction between complement activation product - iC3b and its receptor CR3 in experimental autoimmune anterior uveitis (EAAU).
Male Lewis rats (5-6 weeks old) were immunized with bovine melanin associated antigen (MAA) emulsified (1:1) in complete Freund’s adjuvant (CFA) in the hind footpad. Animals were sacrificed on different days post-immunization and lymphocytes were purified from the popliteal lymph nodes (LNs) using histopaque gradient. Lymphocytes were stained with anti-CD4 APC and anti-iC3b followed by staining with FITC labeled secondary antibody (Ab). In a separate set of experiment, cells were stained with anti-CD11b/c PE, anti-CD3-APC, anti-CD45RA-FITC and anti-iC3b followed by staining with PerCP labeled secondary Ab. CFSE labeled lymphocytes purified from the popliteal LNs on day 5 post-immunization were plated in serum free CellGroR Medium containing rat iC3b in the presence of different concentration of anti-CD11b and anti-iC3b Abs separately for 5 days in the presence of bovine MAA (20 µg/ml). On day 6 non-adherent cells were collected and stained with anti-CD4-APC. The proliferation data was collected using FACS Calibur and was analyzed using Win MDI.
During the induction of EAAU, iC3b is deposited on antigen presenting cells (APCs) and CD4+ T cells present in the popliteal LNs. Out of total CD4+ T cells, the percentage of CD4+ T cells that are positive for iC3b started to increase as early as day 2, peaked on day 7 post-immunization and started to decrease after that. Out of total CD11b/c+ CD3- CD45RA- cells, the percentage of cells that are iC3b positive sharply increased at day 2 and peaked on day 7 post-immunization. Treatment with anti-CD11b and anti-iC3b Abs separately inhibited the proliferation of CD4+ T cells in response to MAA in a dose dependent manner.
Our results demonstrate that during the induction phase of EAAU, iC3b is deposited on CD4+ T cells and CD11b/c+ CD3- CD45RA- APCs that are present in the popliteal LNs of MAA sensitized Lewis rats. Our data further suggest that the interaction between iC3b and its receptor CR3 modulates CD4+ T cells responses in EAAU.
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