RTCPCR was carried out using the following primers: GAPDH (F-5-TGAAGGTCGGTGT-CAACGGATTTGGC-3, R-5-CATGTAGGCCATGAGGTCCACCAC-3); IL-10 (F-5-AAGGACCAGCTGGACAACAT-3, R-5-AGACACCTTTGTCTTGGAGC-TTA-3); TGF-2 (F-5-CTCCTGCATCTGGTCCCGGT-3, R-5-GCACG-GCGTCTGTCACGTCG-3). Statistics Each group consisted of 6 animals and experiments were repeated 5 times with comparable results. tolerance. Complement is usually a major component of natural immunity. VP3.15 dihydrobromide Well-known effector functions of the complement system include anaphylatoxic activity, VP3.15 dihydrobromide opsonization for phagocytosis and lysis of cells or foreign particles1,2. However, in recent years it has become increasingly evident that complement is also involved in antigen-specific immune responses. These include antigen processing and presentation, T-cell proliferation and differentiation, and B-cell activation3C7. Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site8. The present study was undertaken to determine the role of complement in systemic tolerance induced by the injection of antigen into the vision. The anterior chamber (AC) of the eye is an immune-privileged site8. After the introduction of antigen into the AC, there is a deviant systemic immune response resulting in the generation of VP3.15 dihydrobromide antigen-specific regulatory T cells and the suppression of delayed type-hypersensitivity (DTH)a phenomenon initially described as F1 lymphocyte-immune deviation9,10, and subsequently described as anterior chamber associated immune deviation (ACAID)11. The antigenic signal delivered to the AC is usually carried into the circulation and delivered to the spleen11,12, where regulatory T cells are generated13. CD-1 reactive natural killer T cells are required for the inhibition of DTH after the AC injection of antigen14. DTH is usually a form of T cell-mediated immunity that plays an important role in protection against pathogens9C14. Inhibition of DTH (after intraocular injection) has been reproduced with soluble antigen in the Rabbit Polyclonal to RHOB presence of immunosuppressive factors present in ocular microenvironment18. Previously, we showed that a functionally active complement system is present within the AC of the normal vision19. In the present study we investigated whether the presence and activation of complement are necessary for the induction of antigen-specific systemic tolerance after AC injection. Role of complement in the inhibition of DTH after AC injection The role of complement in the inhibition of DTH was investigated using Lewis rats depleted of systemic complement by cobra venom factor (CVF). Our results VP3.15 dihydrobromide (Fig. 1depletion of systemic complement with CVF, before the injection of OVA, blocked (< 0.001) the inhibition of DTH (group 4) but not OVA-specific DTH (group 2), which was comparable to the normal complement control (group 1). Both groups 2 and 4 received CVF, but the animals in group 4 only were given the AC injection of OVA. A CH50 hemolytic assay20 confirmed the absence of functionally active complement from CVF-injected rats (data not shown) for 5 days19. Thus, complement seemed to be essential for the inhibition of DTH after the AC injection of antigen. Open in a separate windows Fig. 1 Role of complement in the suppression of DTH. < VP3.15 dihydrobromide 0.001) blocked the suppression of DTH to OVA (group 3). CVF injection alone (group 2) did not affect OVA-specific DTH (group 1) in the absence of anterior chamber inoculation of OVA. < 0.001. Role of C3 and iC3b in the inhibition of DTH after AC injection Using C3-deficient mice, we next explored whether the inhibition of DTH required C3. AC inoculation of OVA induced inhibition of DTH in the wild-type control mouse, but not the C3-deficient mouse (Fig. 1and models of suppressed DTH (refs. 15,16), we next studied whether iC3b was required for the inhibition of DTH. In our initial experiments, both polymeric iC3b and iC3b-opsonized erythrocytes (EA-iC3b) were used. Using the model of suppressed DTH, our results exhibited that antigen-pulsed peritoneal exudate cells (PEC) exposed to piC3b inhibited (< 0.01) the development of DTH, whereas in the absence of piC3b this effect was not observed (Fig. 2model employs antigen, PECs and syngeneic splenocytes to induce regulatory T cells that inhibit the DTH response of antigen-specific sensitized T cells. Open in a separate windows Fig. 2 Role of iC3b in suppression of DTH. model. Antigen-pulsed PEC exposed to.