The complement system plays a critical role in the development of experimental autoimmune anterior uveitis

Abstract

Purpose: The role of complement in ocular autoimmunity was explored in a experimental autoimmune anterior uveitis (EAAU) animal model.

Methods: EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen. Complement activation in the eye was monitored by Western blot for iC3b. The importance of complement to the development of EAAU was studied by comparing the course of intraocular inflammation in normal Lewis rats (complement-sufficient) with cobra venom factor-treated rats (complement-depleted). Eyes were harvested from both complement-sufficient and complement-depleted rats for mRNA and protein analysis for IFN-gamma, IL-10, and interferon-inducible protein (IP)-10. Intracellular adhesion molecule (ICAM)-1 and leukocyte-endothelial cell adhesion molecule (LECAM)-1 were detected by immunofluorescent staining. OX-42 was used to investigate the importance of iC3b and CR3 interaction in EAAU.

Results: There was a correlation between ocular complement activation and disease progression in EAAU. The incidence, duration, and severity of disease were dramatically reduced after active immunization in complement-depleted rats. Complement depletion also completely suppressed adoptive transfer EAAU. The presence of complement was critical for local production of cytokines (IFN-gamma and IL-10), chemokines (IP-10), and adhesion molecules (ICAM-1 and LECAM-1) during EAAU. Furthermore, intraocular complement activation, specifically iC3b production and engagement of complement receptor 3 (CR3), had a significant impact on disease activity in EAAU.

Conclusions: The study provided the novel finding that complement activation plays a central role in the pathogenesis of ocular autoimmunity and may serve as a potential target for therapeutic intervention.

Figure 1

Relationship between ocular complement activation and disease activity in EAAU. Semiquantitative Western blot and densitometric analyses of polypeptide chains corresponding to iC3b was used to measure intraocular complement activation after immunization of Lewis rats with MAA and BSA. The intensity of the iC3b protein bands was quantitated, and the relative intensity was expressed as ratio of the intensity of the bands from the experimental (MAA- or BSA-injected) animals to those of the naïve rats (experimental/naïve). In animals immunized with MAA, the levels of iC3b within the eye paralleled the course of disease in EAAU (A). In these rats there was a significant increase in the levels of iC3b at days 12 to 16 compared with days 8, 10, 23, and 30. Low levels (similar to naïve rats) of iC3b were detected in the eyes of BSA-injected animals at all time points (B). The bar charts represent the relative intensity of iC3b bands and the line chart shows the disease activity in MAA- (A) and BSA-injected (B) rats. Data are presented as the mean ± SD and are representative of six independent experiments. *P < 0.05.

Figure 2

Effect of CVF on EAAU. Lewis rats immunized with MAA received a single intraperitoneal injection of PBS (A) or CVF (B) at day 9 and were killed at the peak of EAAU (day 19 after immunization) to study the effect of complement depletion on histopathological changes within the eye. Severe EAAU was observed in the MAA-injected complement-sufficient (PBS-treated) animals (A). The anterior chamber, the iris (arrow), and the ciliary body (arrowhead) were infiltrated by inflammatory cells. EAAU did not develop in complement-depleted (CVF-treated) Lewis rats immunized with MAA (B). Mag-nification, ×20. A single intraperitoneal injection of CVF in MAA-injected (C) rats resulted in almost complete complement depletion for 5 days. In these animals, serum complement activity returned to the basal levels on day 16 and remained at this level on days 19 to 30. Disease activity in these animals was zero. The bar charts represent serum complement activity and the line chart shows the disease activity (C). C3 split products in the eye of naïve (lane 1) and MAA-injected, CVF-treated (lane 2) rats at day 19 after immunization (D). The data shown are representative results in three different experiments.

Figure 3

Effect of systemic complement depletion on intraocular IFN-γ, IL-10, and IP-10 mRNA expression during EAAU. Eyes (n = 6/time point) were harvested from MAA-injected, complement-sufficient (PBS-treated) and -depleted (CVF-treated) Lewis rats killed at different time points for semiquantitative RT-PCR analysis. PCR products were analyzed on a 2% agarose gel. (A) mRNA expression of IFN-γ and IL-10 in complement-sufficient, MAA-injected rats, and (B) represents mRNA expression of these cytokines in complement-depleted, MAA-injected rats. (C) shows IP-10 mRNA expression in both CVF- and PBS-treated, MAA-injected rats. There was decreased production of IFN-γ and IL-10 mRNA in complement-depleted animals (B) compared with complement-sufficient rats (A). IP-10 mRNA expression was abolished by CVF treatment (C). A strong band at 335 bp for β-actin indicated an equal amount of RNA in each lane (A, B). Shown are ethidium-bromide–stained bands for PCR products after UV exposure. Images are representative of results in three independent experiments.

Figure 4

Effect of complement on intraocular IFN-γ, IL-10, and IP-10 protein production during EAAU. Eyes (n = 6/time point) were harvested from MAA-injected, complement-sufficient (■) and complement-depleted (▧) rats killed at different time points, and cytokine production was assessed by protein-specific ELISA. Compared with complement-sufficient rats, IFN-γ (A) and IL-10 (B) protein levels in complement-depleted rats were significantly reduced on days 12 to 19 and days 14 to 19, respectively. Results shown are the mean ± SD of results in triplicate independent analyses. *P < 0.05. (C). On Western Blot analysis, IP-10 was barely detected in complement-depleted Lewis rats compared with complement-sufficient animals. Control blot incubated with equivalent concentration of irrelevant antibody (rabbit IgG isotype control) did not show any reactivity (D). (C, D) Representative of results in three separate experiments.

Figure 5

Effect of complement depletion on the ocular expression of ICAM-1 and LECAM-1 at the peak of EAAU. Paraffin-embedded sections were prepared from eyes harvested from MAA-injected, complement-sufficient (A, D) and complement-depleted (B, E) Lewis rats at day 19 after immunization (the peak of EAAU). (C, F) Images from naïve animals. Sections were stained with FITC-labeled monoclonal antibodies against ICAM-1 (A–C) and LECAM-1 (D–F). At the peak of EAAU, strong expression of ICAM-1 (A) and LECAM-1 (D) was observed on the iris (arrow) and the ciliary body (arrowhead). Expression of these adhesion molecules was suppressed in complement-depleted rats (B, E). Weak staining for ICAM-1 (C) and LECAM-1 (F) was detected in naïve rat eye. Each micrograph is representative of imaging in three separate experiments. Magnification, × 40.

Figure 6

CR3 staining during EAAU (A). Eyes harvested from complement-sufficient Lewis rats killed at the peak of EAAU (day 19 after immunization) were stained with OX-42 (anti-CR3). Strong expression of CR3 by infiltrating cells at the peak of disease was observed (A). No fluorescence was detected in the sections stained with isotype control antibody (B). CR3-positive cells in the spleen of naïve Lewis rat injected with PBS (C) and OX-42 (D). Magnification: (A, B) × 40; (C, D) × 100.