Monoclonal antibody-mediated CD200 receptor signaling suppresses macrophage activation and tissue damage in experimental autoimmune uveoretinitis

Macrophage responses are regulated by multiple secreted factors as well as by cell surface receptors, including the inhibitory signals resulting from ligation of myeloid CD200 receptors (CD200R) by the widely distributed CD200. In the absence of CD200, animals display increased susceptibility to autoimmunity and earlier onset aggressive autoimmune disease. In these current experiments, an agonist monoclonal rat anti-mouse CD200R (DX109) antibody delivered a negative signal to bone marrow-derived macrophages, which suppressed interferon (IFN)gamma-mediated nitric oxide (NO) and interleukin-6 production. Experimental autoimmune uveoretinitis (EAU) was used as a model of organ-specific autoimmunity in the eye, a tissue with extensive neuronal and endothelial CD200 expression. In mice lacking CD200 (CD200(-/-)), increased numbers of retina-infiltrating macrophages displaying heightened NO responses were observed during EAU. In addition, we aimed to suppress disease by maintaining tonic suppression of macrophage activation via CD200R. Systemically administered DX109 monoclonal antibody suppressed EAU despite maintained T-cell proliferation and IFNgamma production. Furthermore, locally administered DX109 monoclonal antibody resulted in an earlier resolution of disease. These experiments demonstrate that promoting CD200R-mediated signaling can successfully prevent full expression of IFNgamma-mediated macrophage activation and protect against tissue damage during autoimmune responses.     

Idiotypic expression of antibodies to retinal S-antigen in experimental autoimmune uveoretinitis.

Retinal S-antigen (S-ag), found in the rod photoreceptors of the eye, is a potent autoantigen that is commonly involved in inflammatory eye disease leading to blindness in man. Antibodies, induced in the experimental model by immunizing rats with S-ag purified from porcine retina, were used to prepare heterologous rabbit anti-idiotypic antibodies. The binding of the four rabbit anti-idiotypes to S-ag antibodies was partially inhibitable by porcine S-ag but not by ovalbumin. The idiotypic determinants were localized to the heavy chains by Western blotting with the anti-idiotypes. The presence of the idiotype recognized by the rabbit anti-idiotype was assessed in antisera from various species containing antibodies to S-ag. All rat sera from animals undergoing experimental autoimmune uveoretinitis by immunization with S-ag from porcine or bovine retina contained antibodies that react to varying degrees with the rabbit anti-idiotype. The intraspecies nature of the idiotypic determinants recognized was demonstrated by the fact that none of the anti-idiotypes showed any reactivity with rabbit or murine antisera to S-ag from porcine, bovine or human retina or to human autoantibodies to S-ag from patients with inflammatory eye disease. Thus, all private and recurrent idiotypic determinants induced in rats by immunization with S-ag appear to be restricted to that species.     

Effects of CD8 depletion on retinal soluble antigen induced experimental autoimmune uveoretinitis.

During the later stages of soluble-antigen (sAg)-induced experimental autoimmune uveoretinitis (EAU), an increase in the relative number of CD8+ lymphocytes has been observed at the site of inflammation in the retina. It has been suggested that these late-appearing CD8+ cells might down-regulate this acute disease process. To determine the role of the CD8+ cells in EAU, Lewis rats were depleted of CD8+ cells prior to and during disease and the enucleated eyes examined histologically. The spleen cells from CD8-depleted rats were also examined for their ability to respond to concanavalin A (Con A) and to allogeneic targets as determined by mixed lymphocyte reaction (MLR) and cytotoxicity assays. The results suggest that depleting CD8+ cells had no effect on the course of disease and that CD8+ cells do not play a crucial role in the immunoregulation of EAU.     

Intranasal administration of retinal antigens suppresses retinal antigen-induced experimental autoimmune uveoretinitis.

Bovine retinal extract (RE) is a heterologous mixture of highly uveitogenic proteins including S-Antigen (S-Ag), interphotoreceptor retinol binding protein (IRBP) and rhodopsin, and is a potent inducer of experimental autoimmune uveoretinitis (EAU). Intranasal inoculation of Lewis rats with RE performed daily for 10 days prior to immunization with RE suppresses both the severity and the incidence of the clinical response and histopathological changes in EAU. Significant suppression of the disease in treated animals could be achieved with a total (cumulative) intranasal inoculum of 42 micrograms of antigen. Animals which were treated with extract exhibited a normal total antibody response to S-Ag, IRBP and retinal extract when compared with controls [phosphate-buffered saline (PBS) treated] animals. The antibody response in tolerized animals was predominantly anti-S-Ag IgG2a with suppression of anti-S-Ag IgM response. Treated animals had a significantly suppressed delayed-type hypersensitivity (DTH) response to retinal extract but normal response to purified protein derivative (PPD) compared to control animals. Adoptive transfer of splenocytes from treated animals also demonstrated some protection against RE-induced EAU. These results demonstrate that tolerance induction impairs the onset and severity of EAU by inhibiting the DTH response to heterologous mixture of retinal antigens.     

鼻腔内给予视网膜抗原抑制实验性自身免疫性色素膜网膜炎

６一８周龄雌性Ｌｅｗｉｓ大鼠，在用视网膜抽提物（ＲＥ）或Ｓ－Ａｇ加弗氏完全佐剂（ＣＦＡ）免疫制成实验性自身免疫性色素膜网膜炎（ＥＡＵ）动物模型之前鼻腔内给予相同抗原每天１次，共１０天。结果表明：①该耐受疗法可有效地抑制临床疾病的发生率和严重程度；②耐受组疾病的抑制与不同淋巴器官中γ干扰素（ＩＦＮ－γ）分泌细胞数的减少和ＲＥ特异性皮肤迟发性过敏反应（ＤＴＨ）的抑制相一致；③病程中血清抗体的变化与对照组差异无显著性。提示鼻腔给予ＲＥ或Ｓ－Ａｇ抑制ＥＡＵ与其诱导的致病性Ｔ淋巴细胞的抑制有关。     

CD45 isoform expression in microglia and inflammatory cells in HIV-1 encephalitis

CD45 is a membrane tyrosine phosphatase that modulates the function of the hematopoietic cells. In vitro, agonist antibodies to CD45RO or CD45RB isoforms have been shown to suppress microglial activation, but whether microglia in vivo express these isoforms in HIV encephalitis (HIVE) is unknown. Brain sections from control and HIVE were immunostained for CD45 isoforms using exon-specific antibodies (RA, RB, RC and RO). RA and RC were limited to rare lymphocytes, while RB expression was robust in microglia and inflammatory cells. RO was low in control microglia, but increased in HIVE. RO was also localized to macrophages and CD8+ T cells. Targeting CD45 in vivo with isoform-specific antibodies remains a therapeutic option for neuroinflammatory diseases.     

Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations

Experimental autoimmune uveoretinitis (EAU) is an animal model for human intraocular inflammatory disease. EAU is induced in B10.RIII mice by immunization with RBP-3 161–180 peptide and intraperitoneal pertussis toxin and is mediated by CD4⁺ T cells that generate a clinically monophasic disease peaking approximately 2 weeks post-immunization. Collagenase digestion of retinal tissue allowed the quantification and characterization of leukocytes in the inflamed retina during disease progression. Using this method we identified three stages of disease. Initially there is a prodromal phase where we found significant changes in the number of leukocytes in the eye as early as 5 days post-immunization. This effect was, in part, non-antigen specific as a small increase in retinal leukocytes was also observed following immunization with OVA peptide. Following the prodrome there is a primary peak of infiltration including both CD4⁺ T cells and CD11b⁺ cells. This coincides with an early influx of neutrophils and is associated with a peak in IL-17-producing T cells. The neutrophils in the eye are CD11b⁺ and Gr1⁺ but can be distinguished from other myeloid cells by their high expression of Ly6G. The remaining CD11b⁺Gr1⁺ cells can suppress proliferation and are analogous to myeloid derived suppressor cells which are found in tumors. The inflamed eye also contains a considerable proportion of FoxP3⁺ regulatory cells. Following peak disease, the retina does not return to its pre-disease phenotype. Instead, fluctuations in infiltrating leukocyte numbers and changes to their relative composition continue, indicating that clinical recovery does not equate to the restoration of a normal retinal leukocyte population.     

Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations

Experimental autoimmune uveoretinitis (EAU) is an animal model for human intraocular inflammatory disease. EAU is induced in B10.RIII mice by immunization with RBP-3 161-180 peptide and intraperitoneal pertussis toxin and is mediated by CD4(+) T cells that generate a clinically monophasic disease peaking approximately 2 weeks post-immunization. Collagenase digestion of retinal tissue allowed the quantification and characterization of leukocytes in the inflamed retina during disease progression. Using this method we identified three stages of disease. Initially there is a prodromal phase where we found significant changes in the number of leukocytes in the eye as early as 5 days post-immunization. This effect was, in part, non-antigen specific as a small increase in retinal leukocytes was also observed following immunization with OVA peptide. Following the prodrome there is a primary peak of infiltration including both CD4(+) T cells and CD11b(+) cells. This coincides with an early influx of neutrophils and is associated with a peak in IL-17-producing T cells. The neutrophils in the eye are CD11b(+) and Gr1(+) but can be distinguished from other myeloid cells by their high expression of Ly6G. The remaining CD11b(+)Gr1(+) cells can suppress proliferation and are analogous to myeloid derived suppressor cells which are found in tumors. The inflamed eye also contains a considerable proportion of FoxP3(+) regulatory cells. Following peak disease, the retina does not return to its pre-disease phenotype. Instead, fluctuations in infiltrating leukocyte numbers and changes to their relative composition continue, indicating that clinical recovery does not equate to the restoration of a normal retinal leukocyte population.     

嗅鞘细胞移植实验性自身免疫性脑脊髓炎后髓鞘碱性蛋白的表达和小胶质细胞的捕获

目的:研究嗅鞘细胞(OECs)移植实验性自身免疫性脑脊髓炎(EAE)后髓鞘碱性蛋白(Myelin basic protein,MBP)的表达及小胶质细胞捕获OECs情况。方法:用新生近交系Wistar大鼠嗅球培养出OECs,经荧光染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记后注入同系EAE Wistar大鼠侧脑室,免疫组化检测OECs髓鞘碱性蛋白(MBP)、单核巨噬细胞诱导分子1(ED1)和CFSE共表达情况。结果:培养OECs不表达MBP,经侧脑室移植后在EAE大鼠脑内出现多量MBP+CFSE+细胞,同时在大脑广泛区域及血管周围间隙出现多量ED1+CFSE+细胞,与对照组相比差异显著。结论:OECs在EAE环境下表达MBP分子,其抗原能被脑内小胶质细胞或巨噬细胞捕获。     

The effect of retinal S-antigen-specific monoclonal antibody therapy on experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP).

We induced an autoimmune uveitis and pinealitis in Lewis rats by inoculating them with bovine S-antigen. This type of uveitis forms a useful experimental model of human chronic intra-ocular inflammation. Induction of experimental autoimmune uveitis (EAU) but not of experimental autoimmune pinealitis (EAP) could be prevented by the administration of S-antigen-specific monoclonal antibody simultaneously with the S-antigen. Inhibition of EAU was associated with significantly raised levels of anti-S antibodies during the first 2 weeks post-immunisation. Immunocytochemical staining for lymphocyte subsets, monocytes and macrophages showed that eyes of monoclonal antibody treated animals contained no immunocompetent inflammatory cells unless they also had clinical signs of inflammation. In contrast, the inflammatory exudate in the pineal glands of both treated and untreated animals contained equal numbers of infiltrating lymphocytes and monocytes in the same relative proportions. These results indicate that the inhibitory effect of the monoclonal antibody S2.4.C5 may be directed towards the effector arm of the immune-mediated cytotoxic response.     

Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis

We have shown previously that complement factor H (CFH) and complement factor B (CFB) are constitutively expressed by retinal pigment epithelial cells and their production is regulated by inflammatory cytokines, suggesting that the alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study, we further investigated the role of the AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Mice with EAU show increased levels of C3d deposition and CFB expression in the retina. Retinal inflammation was suppressed clinically and histologically by blocking AP‐mediated complement activation with a complement receptor of the Ig superfamily fusion protein (CRIg‐Fc). In line with reduced inflammation, C3d deposition and CFB expression were markedly decreased by CRIg‐Fc treatment. Treatment with CRIg‐Fc also led to reduced T‐cell proliferation and IFN‐γ, TNF‐α, IL‐17, and IL‐6 cytokine production by T cells, and reduced nitric oxide production in BM‐derived macrophages. Our results suggest that AP‐mediated complement activation contributes significantly to retinal inflammation in EAU. CRIg‐Fc suppressed retinal inflammation in EAU by blocking AP‐mediated complement activation with probable direct effects on C3/C5 activation of macrophages, thus leading to reduced nitric oxide production by infiltrating CRIg^? macrophages.     

Injury of Müller cells increases the incidence of experimental autoimmune uveoretinitis

Müller cells have been shown to have a dual effect in vitro on autoimmune T helper lymphocytes. In a coculture system, Müller cells have a primary inhibitory effect on the proliferation of T lymphocytes. In conditions where their inhibitory action is suppressed, Müller cells can, however, stimulate T cells. In the present study we evaluated the in vivo effect of Müller cells on actively induced experimental autoimmune uveoretinitis (EAU). Ten millimoles of L-alpha-aminoadipic acid (L-AAA), a specific gliotoxic agent, was injected into the vitreous of one eye of Wistar-Furth (WF) rats (a low EAU responder) on the day of immunization. Control rats were injected similarly with phosphate-buffered saline alone. The rats were immunized with S-antigen in CFA or in CFA alone. The results demonstrate that the incidence of EAU increases twofold in the eyes receiving an intravitreal injection of L-AAA in comparison to the contralateral eyes not receiving an injection. No such difference in EAU incidence was observed in control animals. Some rats that had been immunized with CFA alone after an intravitreal injection of L-AAA demonstrated a small amount of retinal perivascular inflammatory cell infiltrate but did not develop typical EAU lesions. The retinal vasculature was normal on examination by fluorescein angiography after injection of L-AAA. These data suggest that Müller cells can influence the course of uveoretinitis through their interaction with T cells.     

Effect of anti-macrophage inflammatory protein-1alpha on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), on leukocyte behavior at the blood-retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP-1alpha has not previously been thought to be effective under conditions of physiological shear flow. However, short-term anti-MIP-1alpha treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood-retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post-capillary venules which have been shown to be the main site of passage of leukocytes across the blood-retina barrier. Long-term anti-MIP-1alpha treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood-retina barrier breakdown.     

Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells

To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161c-treated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1(+) NK cells.     

Induction of experimental autoimmune uveoretinitis in Lewis rats with purified recombinant human retinal S-antigen fusion protein.

Full-length human retinal cDNA for S antigen (S-ag) and for the alpha subunit of transducin (alpha-Td) were subcloned into a bacterial expression plasmid vector to generate recombinant fusion proteins with glutathione-S-transferase (GST). The recombinant GST-S-ag and rGST-alpha-Td fusion proteins were purified from bacterial extracts by continuous flow preparative gel electrophoresis under denaturing conditions, and were assessed for their ability to induce experimental autoimmune uveoretinitis (EAU). Immunization of Lewis rats with single doses of 10 micrograms-100 micrograms rGST-S-ag in Freund's complete adjuvant supplemented with Bordetella pertussis readily induced clinical signs of EAU. Immunization with GST alone did not induce EAU indicating that disease activity was ascribable to the S-ag residues in the fusion protein. Although the alpha-Td shares limited sequence homology with S-ag, the rGST-alpha-Td fusion protein was also not uveitogenic in Lewis rats. The clinical severity of EAU in Lewis rats sensitized with rGST-S-ag was found to be milder than that induced with native S-ag preparations purified from human retina. However, humoral antibody responses to sensitization with the recombinant S-ag fusion protein were of a higher magnitude than with native S-ag. The availability of recombinant preparations of human S-ag protein will be of value in studying its processing and presentation to T cells derived from patients with autoimmune retinal vasculitis.     

CD200/CD200R paired potent inhibitory molecules regulating immune and inflammatory responses; Part I: CD200/CD200R structure, activation, and function

CD200/CD200R are highly conserved type I paired membrane glycoproteins that belong to the Ig superfamily containing a two immunoglobulin-like domain (V, C). CD200 is broadly distributed in a variety of cell types, whereas CD200R is primarily expressed in myeloid and lymphoid cells. They fulfill multiple functions in regulating inflammation. The interaction between CD200/CD200R results in activation of the intracellular inhibitory pathway with RasGAP recruitment and thus contributes to effector cell inhibition. It was confirmed that the CD200R activation stimulates the differentiation ofT cells to the Treg subset, upregulates indoleamine 2,3-dioxygenase activity, modulates cytokine environment from a Thl to a Th2 pattern, and facilitates an antiinflammatory IL-10 and TGF-beta synthesis. CD200/CD200R are required for maintaining self-tolerance. Many studies have demonstrated the importance of CD200 in controlling autoimmunity, inflammation, the development and spread of cancer, hypersensitivity, and spontaneous fetal loss.     

CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid beta-peptide

Although deposition of amyloid beta-peptide (Abeta) as Abeta plaques involves activation of microglia-mediated inflammatory responses, activated microglia ultimately fail to clear Abeta plaques in the brains of either Alzheimer's disease (AD) patients or AD mouse models. Mounting evidence suggests that chronic microglia-mediated immune response during Abeta deposition etiologically contributes to AD pathogenesis by promoting Abeta plaque formation. However, the mechanisms that govern microglia response in the context of cerebral Abeta/beta-amyloid pathology are not well understood. We show that ligation of CD40 by CD40L modulates Abeta-induced innate immune responses in microglia, including decreased microglia phagocytosis of exogenous Abeta(1-42) and increased production of pro-inflammatory cytokines. CD40 ligation in the presence of Abeta(1-42) leads to adaptive activation of microglia, as evidenced by increased co-localization of MHC class II with Abeta. To assess their antigen-presenting cell (APC) function, cultured microglia were pulsed with Abeta(1-42) in the presence of CD40L and co-cultured with CD4(+) T cells. Under these conditions, microglia stimulate T cell-derived IFN-gamma and IL-2 production, suggesting that CD40 signaling promotes the APC phenotype. These data provide a mechanistic explanation for our previous work showing decreased microgliosis associated with diminished cerebral Abeta/beta-amyloid pathology when blocking CD40 signaling in transgenic Alzheimer's mice.     

Time-course expression of CNS inflammatory, neurodegenerative tissue repair markers and metallothioneins during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS). EAE and MS are characterized by CNS inflammation, demyelination and neurodegeneration. The inflammatory response occurring within the CNS leads to glial activation, dysfunction and death, as well as axonal damage and neurological deficit. Although the pathogenic mechanisms involved in EAE/MS are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development, and contributes to axonal dysfunction and degeneration. Metallothionein-I and -II are anti-inflammatory, neuroprotective, antioxidant proteins expressed during EAE and MS, in which they might play a protective role. The present study aimed to describe the expression profile of a group of inflammatory, neurodegenerative and tissue repair markers as well as metallothioneins during proteolipid protein-induced EAE, and to establish the time-relationships these molecules had during EAE. Interestingly, we found two marker expression profiles. In the first, marker expression increased as clinical signs worsened and reverted to baseline expression during recovery; in the second, marker expression increased at a later point during relapse, peaked at highest clinical score, and remained elevated throughout recovery. Of note, metallothionein expression was found to be related to the second profile, which would suggest that metallothionein proteins are implicated in the clinical recovery of EAE and perhaps these antioxidant proteins may provide therapeutic benefits in MS.     

Complement activation and expression during chronic relapsing experimental autoimmune encephalomyelitis in the Biozzi ABH mouse

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti‐myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1‐related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti‐complement agents might be of benefit in MS.