Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT‐PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.
The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen‐specific immune tolerance in animals pre‐sensitized with melanin‐associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post‐immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4 + T cells and CD8 + T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non‐tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase‐8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4 + T cells and CD8 + T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non‐tolerized animals. The level of interferon (IFN)‐γ and IL‐2 decreased, whereas TGF‐β2 was elevated in the state of tolerance. Furthermore, the number of CD4 + CD25 + FoxP3 + regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non‐tolerized animals. In conclusion, our results suggest that deletion of antigen‐specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.
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. 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.
Membrane cofactor protein (MCP; formerly termed glycoprotein 45-70 to indicate its Mr) of complement is a widely distributed iC3/C3b binding protein with co-factor activity. On human mononuclear cells and cell lines and platelets, MCP is a doublet. The two forms differ in Mr by approximately 5 k and the upper species is predominant in most individuals. To further characterize these two forms, limited proteolytic digestions were performed. Of the four peptides produced, three have identical Mr indicating that the molecules are similar proteins. Both forms also have acidic isoelectric points and shift to a less acidic isoelectric point after treatment with neuraminidase. Glycosidase digestions indicate that both species contain N- and O-linked oligosaccharides but that the quantity of sialic acid is greater on the larger one. Pulse-chase experiments demonstrate approximately equal quantities of two precursor forms with Mr of 41 and 43 k. These two precursors possess N-linked high-mannose type of oligosaccharides and chase into the mature molecules which have complex sugars. The smaller precursor chases at a slower rate, possibly accounting for the reduced quantity of the smaller form of the mature form of MCP. These experiments indicate that the two forms of MCP are structurally similar and are derived from two distinct precursors. They also suggest that variations in the rate of processing of two intracellular precursors may account for the different quantities of the mature forms of this membrane protein.
PURPOSE. Experimental autoimmune anterior uveitis (EAAU) can be induced in Lewis rats with bovine melanin associated antigen (MAA) extracted from the iris/ciliary body (CB) and does not require adjuvant. The present investigation was undertaken to study the expression of various cytokines in EAAU. METHODS. Lewis rats were immunized with bovine MAA and sacrificed at various time points. The iris/CB and popliteal lymph nodes were harvested, and total RNA isolated. The reverse transcription polymerase chain reaction (RT-PCR) was utilized to determine the mRNA expression of IFN-?, TNF-a, IL-2, IL-4, IL-6 and IL-10. RESULTS. TNF-a mRNA levels in iris/CB paralleled the course of EAAU and increased dramatically at the peak of disease. However, mRNA levels of TNF-a demonstrated little change in the popliteal lymph node. IFN-? mRNA was barely detectable in the iris/CB and increased only slightly at the peak of disease. In contrast, IFN-? mRNA levels in the popliteal lymph node paralleled the course of disease and increased during the peak of disease. IL-10 mRNA did not change in the iris/CB but increased modestly in the popliteal lymph node. IL-2, IL-4, and IL-6 mRNA levels did not change during the course of EAAU in either tissue. CONCLUSIONS. Our study reveals an interesting correlation between the expression of TNF-a, IFN-? and disease progression in EAAU. Furthermore, they suggest that TNF-a is an important cytokine in the target tissue, while IFN-? is in the draining lymph node.
