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J-H Sohn, H-J Suk, PS Bora, Y Wang, HJ Kaplan, NS Bora; Anterior Chamber Associated Immune Deviation (ACAID) is Complement Dependent . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2284.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To explore whether suppression of DTH associated with ACAID is complement dependent – i.e. requires iC3b, the activation product of C3. Methods: . iC3b coated EA (SRBC-IgM) were generated by incubating EA with 10% C5-deficient human serum while control EA (without opsonized iC3b) were generated by incubation with C3-deficient human serum. EA-iC3b or control EA were added to PEC (20 EA-iC3b/PEC or 20 EA/PEC) at the «Preparation of APC» step of in vitro replication of ACAID. PEC were cultured overnight with OVA in RPMI and were then co-cultured with naïve syngenic splenocytes for 5-7 days. Non-adherent cells were collected and mixed with equivalent number of primed syngenic splenocytes. This cell mixture (2x106 cells) was injected into the footpad and footpad swelling was measured at 24 h. In some experiments the purified IgG fraction of neutralizing Ab to IL-10, TGF-ß2 or rat CR3 (iC3b receptor, clone OX-42) was added to the PEC culture media and the reaction was allowed for 1 h before incubation with EA-iC3b. Goat IgG or UPC-10 was used as the control for IL-10, IL-12 or OX-42. All cultures were performed under serum free condition. Culture supernatants were collected for ELISA for IL-10 and IL-12. RNA obtained from PEC cultures was used in RT-PCR for IL-10 and TGF-ß2. Experiments were repeated five times. Results: iC3b was required for the suppression of DTH responses in the in vitro model of ACAID and this effect was abrogated by the presence of neutralizing antibody to rat IL-10, TGF-ß2 or CR3. Additionally, iC3b caused the upregulation of TGF-ß2(mRNA) and IL-10(mRNA and protein) sequentially, and the downregulation of IL-12. The administration of anti-rat CR3 reversed this cytokine profile. Conclusion: Our results suggest that iC3b must bind to CR3 on PECs to initiate the cytokine cascade responsible for the induction of ACAID.
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