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J. Sohn, H. Suk, P.S. Bora, Y. Wang, H.J. Kaplan, N.S. Bora; ACAID is Complement-Dependent . Invest. Ophthalmol. Vis. Sci. 2003;44(13):753.
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Purpose: We previously observed that the systemic depletion of complement with cobra venom factor abrogated ACAID. To explore the mechanism by which ACAID is complement-dependent we investigated the binding of iC3b to CR3 on the antigen-presenting cell (i.e. peritoneal exudate cell – PEC) in both the "in vitro" and "in vivo" model of suppressed DTH. Methods: The monoclonal antibody OX-42, which prevents the binding of iC3b to CR3 as well as both "in vivo" and "in vitro" models of ACAID were used to investigate this possibility. The "in vivo" model of ACAID was induced by the AC injection of OVA on day 0 and the measurement of DTH (foot pad swelling assay) on day 7. The purified IgG (500 ug) fraction of OX-42, which prevents the binding of iC3b to CR3, was administered to Lewis rats (n=5) 24 h prior to the AC injection of OVA. Normal mouse IgG2a was used as the control for OX-42. The "in vitro" model of ACAID involved the following procedures. 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. The role of CR3 was studied by the addition of anti-rat CR3 (4.0 ug/ml) to OVA and PEC for 1 h before the addition of iC3b (using EA-iC3b). Cultures were performed under serum free conditions. Results: Experimental and control groups consisted of six animals each. Experiments were repeated five times with similar results. Blockade of CR3 (the iC3b receptor) by OX-42 reversed (p<0.01) iC3b induced suppression of DTH in both the "in vivo" and "in vitro" models of ACAID. In contrast, DTH was inhibited when normal mouse IgG2a (isotype control) was used. Conclusions: Our data demonstrate the novel finding that iC3b bound to CR3 on the APC is central to the induction of tolerance associated with ACAID.
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