Inhibition of complement alternative pathway suppresses experimental autoimmune anterior uveitis by modulating T cell responses

Abstract

The objective of the current study was to delineate the pathway of complement activation that is crucial for the induction of experimental autoimmune anterior uveitis (EAAU). We studied the development of EAAU in melanin-associated antigen (MAA)-sensitized Lewis rats treated with antibody against C4 or factor B. Control animals received isotype IgG control. Antibody against C4 had no effect on EAAU, and all of the animals developed EAAU similar to those injected with control IgG. In contrast, EAAU was completely inhibited in all MAA-sensitized Lewis rats injected with factor B antibody. Treatment with anti-factor B antibody resulted in suppression of ocular complement activation. Adoptive transfer of T lymphocytes harvested from draining lymph nodes of donor animals treated with anti-factor B did not transfer EAAU to naïve syngenic rats. Anti-factor B antibody inhibited the ability of MAA-specific CD4(+) T cells to proliferate (in vitro) in response to MAA in a dose-dependent manner. Level of TNF-α and IFN-γ decreased in the presence of anti-factor B. Collectively, our results provide the novel finding that complement activation via the alternative pathway contributes to intraocular inflammation in EAAU, and anti-factor B-mediated inhibition of EAAU is due to diminished antigen-specific CD4(+) T cell responses to MAA. Our findings explain the interactions between the complement system and T cells that are critical for the induction of EAAU and may lead to the development of therapy for idiopathic anterior uveitis based on selective blockade of the alternative pathway.

FIGURE 1.

Expression of complement components C4 and factor B in naïve rat eye. A, C4 and factor B mRNA were analyzed by RT-PCR. Representative ethidium bromide-stained agarose gel (2%) shows C4 (290 bp, lane 1) and factor B (412 bp, lane 2) transcripts in the eye of naïve Lewis rats. β-Actin (318 bp, lane 3) was used as the positive control. B and C, total protein from serum and rat intraocular tissue was separated on 12% SDS-PAGE. B and C, representative Western blots shown were probed with polyclonal antibody against human C4 (B) and human factor B (C). B, Western blot probed with anti-human C4 antibody revealed three bands with molecular masses of 97, 75, and 33 kDa in human serum (lane 1, positive control) and naive rat eye (lane 2) under reducing condition. C, under nonreducing conditions, factor B is visualized at the expected molecular mass of 93 kDa in human serum (lane 1, positive control) and naïve rat eye (lane 2). D and E, immunofluorescence micrographs show staining for C4 (D) and factor B (E) within the eyes of naïve Lewis rats. F, no staining was observed in the control sections. Co, cornea; I, iris; AC, anterior chamber. Objective magnification, ×40. The results shown are representative of three independent experiments.

FIGURE 2.

Assessment of inhibitory activity of anti-human C4 and anti-human factor B on rat serum complement activity in vitro. MAA-sensitized Lewis rats were sacrificed at day 19 post-immunization (peak of EAAU), and blood was collected. Rat serum was treated with different concentrations (0, 0.5, 1, and 2 μg) of anti-C4 (A) or anti-factor B antibody (B and C). Anti-C4-treated (■) serum samples were used in the classical pathway hemolytic assay (A). Anti-factor B-treated (■) serum samples were used in the alternative pathway activity assay (B) and the classical pathway hemolytic assay (C). The rat serum treated with the same concentrations (0, 0.5, 1, and 2 μg) of isotype IgG (□) was used as control in all of the experiments. *, p < 0.05.

FIGURE 3.

Effect of anti-C4 and anti-factor B on EAAU. MAA-sensitized Lewis rats treated intraperitoneally with anti-human C4 (A), anti-human factor B (C), or isotype IgG control (B and D) were sacrificed at day 19 post-immunization (peak of EAAU), and the eyes were subjected to histopathological examination after hematoxylin and eosin staining. Representative histologic sections of rat eyes from different experimental groups stained with hematoxylin and eosin are shown. Severe EAAU developed in animals injected intraperitoneally with anti-C4 antibody (A). The iris (I), the CB, and the anterior chamber (AC) were infiltrated with inflammatory cells. In contrast, no inflammation was observed in the eyes of Lewis rats treated similarly with anti-factor B antibody (C). B and D represent histopathology of eyes from MAA-sensitized Lewis rats treated with isotype IgG control. Objective magnification, ×10.

FIGURE 4.

Effect of alternative pathway inhibition on ocular complement activation. MAA-sensitized Lewis rats treated intraperitoneally with anti-factor B (A and C) or isotype IgG control (B and D) were sacrificed 19 days after EAAU induction. Paraffin sections of rat eyes were subjected to immunohistochemical staining with antibodies to rat C3 (A and B) and rat C9 (C and D). Immunofluorescence analysis revealed very faint staining for C3 (A) and MAC (C) in the eye of animals treated with anti-factor B antibody. At this time point, high levels of C3 (B) and MAC (D) were detected in the eyes of Lewis rats injected with isotype IgG control.

FIGURE 5.

Effect of anti-factor B antibody on the proliferation of MAA-specific T cells. Cells from lymph nodes at day 7 post-immunization were used. Density plots (A–D and F–I) show representative flow cytometric data, and E and J represent cumulative data from three separate experiments. Treatment with 0.5 μg/ml (B and E), 1 μg/ml (C and E), and 2 μg/ml (D and E) anti-factor B antibody inhibited the proliferation of CD3⁺ T cells compared with the treatment with isotype control (2 μg/ml; A and E) in a dose-dependent manner. A similar effect was observed on CD4⁺ T cells when treated with 0.5 μg/ml (G and J), 1 μg/ml (H and J), and 2 μg/ml (I and J) anti-factor B antibody compared with isotype control (2 μg/ml; F and J). *, p < 0.05.

FIGURE 6.

Effect of anti-factor B antibody on cytokine expression. Popliteal lymph node cells from MAA-sensitized Lewis rats produce/secrete less TNF-α (A) and IFN-γ (B) in the presence of anti-factor B antibody compared with when cultured in the presence of isotype IgG control as determined by ELISA. *, p < 0.05.