The prostaglandin D2 receptor CRTH2 is important for allergic skin inflammation after epicutaneous antigen challenge.

BACKGROUND: Cutaneous prostaglandin (PG) D2 levels increase after scratching. Chemoattractant receptor-homologous molecule expressed on receptor on T(H)2 cells (CRTH2) mediates chemotaxis to PGD2 and is expressed on T(H)2 cells and eosinophils, which infiltrate skin lesions in patients with atopic dermatitis. OBJECTIVE: We sought to examine the role of CRTH2 in a murine model of atopic dermatitis. METHODS: CRTH2(-/-) mice and wild-type control animals were epicutaneously sensitized by means of repeated application of ovalbumin (OVA) to tape-stripped skin for 7 weeks and then challenged by means of OVA application to tape-stripped previously unsensitized skin for 1 week. Skin histology was assessed by means of hematoxylin and eosin staining and immunohistochemistry. Cytokine mRNA expression was examined by means of quantitative RT-PCR. Levels of PGD2, antibody, and cytokines were measured by means of ELISA. RESULTS: PGD2 levels significantly increased in skin 24 hours after tape stripping, although not in skin subjected to repeated sensitization with OVA. Allergic skin inflammation developed normally at sites of chronic epicutaneous sensitization with OVA in CRTH2(-/-) mice but was severely impaired in previously unsensitized skin challenged with OVA, as evidenced by significantly decreased skin infiltration with eosinophils and CD4(+) cells and impaired T(H)2 cytokine mRNA expression. Impaired skin inflammation at sites of acute OVA challenge in CRTH2(-/-) mice was not due to an impaired systemic response to epicutaneous sensitization because OVA-specific IgG1 and IgE antibody levels and OVA-driven splenocyte secretion of cytokines in these mice were comparable with those seen in wild-type control animals. CONCLUSIONS: CRTH2 promotes allergic skin inflammation in response to cutaneous exposure to antigen in previously sensitized mice.

Toll-like receptor 2 is important for the T(H)1 response to cutaneous sensitization.

BACKGROUND: Atopic dermatitis and allergic contact dermatitis are skin disorders triggered by epicutaneous sensitization with protein antigens and contact sensitization with haptens, respectively. Skin is colonized with bacteria, which are a source of Toll-like receptor (TLR) 2 ligands. OBJECTIVE: We sought to examine the role of TLR2 in murine models of atopic dermatitis and allergic contact dermatitis. METHODS: TLR2(-/-) mice and wild-type littermates were epicutaneously sensitized with ovalbumin (OVA) or contact sensitized with oxazolone (OX). Skin histology was assessed by means of hematoxylin and eosin staining and immunohistochemistry. Ear swelling was measured with a micrometer. Cytokine mRNA expression was examined by means of quantitative RT-PCR. Antibody levels and splenocyte secretion of cytokines in response to OVA stimulation were measured by means of ELISA. Dendritic cells were examined for their ability to polarize T-cell receptor/OVA transgenic naive T cells to T(H)1 and T(H)2. RESULTS: In response to OVA sensitization, TLR2(-/-) mice experienced skin infiltration with eosinophils and CD4(+) cells, as well as upregulation of T(H)2 cytokine mRNAs that was comparable with that seen in wild-type littermates. In contrast, epidermal thickening, IFN-gamma expression in the skin, IFN-gamma production by splenocytes, and IgG2a anti-OVA antibody levels were impaired in TLR2(-/-) mice. After OX ear challenge, contact sensitized TLR2(-/-) mice exhibited defective ear swelling with impaired cellular infiltration, decreased epidermal thickening and local IFN-gamma expression, and impaired OX-specific IgG2a responses. Dendritic cells from TLR2(-/-) mice induced significantly lower production of IFN-gamma but normal IL-4 and IL-13 production in naive T cells. CONCLUSIONS: These results indicate that TLR2 promotes the IFN-gamma response to cutaneously introduced antigens.

Animal models of atopic dermatitis.

Atopic dermatitis (AD) is characterized by allergic skin inflammation. A hallmark of AD is dry itchy skin due, at least in part, to defects in skin genes that are important for maintaining barrier function. The pathogenesis of AD remains incompletely understood. Since the description of the Nc/Nga mouse as a spontaneously occurring model of AD, a number of other mouse models of AD have been developed. They can be categorized into three groups: (1) models induced by epicutaneous application of sensitizers; (2) transgenic mice that either overexpress or lack selective molecules; (3) mice that spontaneously develop AD-like skin lesions. These models have resulted in a better understanding of the pathogenesis of AD. This review discusses these models and emphasizes the role of mechanical skin injury and skin barrier dysfunction in eliciting allergic skin inflammation.

IL-21R is essential for epicutaneous sensitization and allergic skin inflammation in humans and mice.

Atopic dermatitis (AD) is a common allergic inflammatory skin disease caused by a combination of intense pruritus, scratching, and epicutaneous (e.c.) sensitization with allergens. To explore the roles of IL-21 and IL-21 receptor (IL-21R) in AD, we examined skin lesions from patients with AD and used a mouse model of allergic skin inflammation. IL-21 and IL-21R expression was upregulated in acute skin lesions of AD patients and in mouse skin subjected to tape stripping, a surrogate for scratching. The importance of this finding was highlighted by the fact that both Il21r-/- mice and WT mice treated with soluble IL-21R-IgG2aFc fusion protein failed to develop skin inflammation after e.c. sensitization of tape-stripped skin. Adoptively transferred OVA-specific WT CD4+ T cells accumulated poorly in draining LNs (DLNs) of e.c. sensitized Il21r-/- mice. This was likely caused by both DC-intrinsic and nonintrinsic effects, because trafficking of skin DCs to DLNs was defective in Il21r-/- mice and, to a lesser extent, in WT mice reconstituted with Il21r-/- BM. More insight into this defect was provided by the observation that skin DCs from tape-stripped WT mice, but not Il21r-/- mice, upregulated CCR7 and migrated toward CCR7 ligands. Treatment of epidermal and dermal cells with IL-21 activated MMP2, which has been implicated in trafficking of skin DCs. These results suggest an important role for IL-21R in the mobilization of skin DCs to DLNs and the subsequent allergic response to e.c. introduced antigen.

Epicutaneous antigen exposure induces a Th17 response that drives airway inflammation after inhalation challenge.

IL-17 has been implicated in a number of inflammatory diseases, but the conditions of antigen exposure that drive the generation of Th17 responses have not been well defined. Epicutaneous (EC) immunization of mice with ovalbumin (OVA), which causes allergic skin inflammation with many characteristics of the skin lesions of atopic dermatitis, was found to also drive IL-17 expression in the skin. EC, but not i.p., immunization of mice with OVA drove the generation of IL-17-producing T cells in draining lymph nodes and spleen and increased serum IL-17 levels. OVA inhalation by EC-sensitized mice induced IL-17 and CXCL2 expression and neutrophil influx in the lung along with bronchial hyperreactivity, which were reversed by IL-17 blockade. Dendritic cells trafficking from skin to lymph nodes expressed more IL-23 and induced more IL-17 secretion by naïve T cells than splenic dendritic cells. This was inhibited by neutralizing IL-23 in vitro and by intradermal injection of anti-TGFbeta neutralizing antibody in vivo. Our findings suggest that initial cutaneous exposure to antigens in patients with atopic dermatitis may selectively induce the production of IL-17, which, in turn, drives inflammation of their airways.

Phenotypes, distribution, and morphological features of antigen-presenting cells in the murine cornea following intravitreal injection.

PURPOSE: To study the phenotypes, distribution, and morphologies of different antigen-presenting cells (APCs) in the murine cornea. METHODS: Intravitreal injection of fluorescently tagged ovalbumin (OVA) or antibodies to MHC-II (I-A(d)), F4/80, CD11c, B7-1, and B7-2 was performed to label cells in the murine cornea. Light and transmission electron microscopy were used to examine corneal histology. Intravital microscopy, epifluorescence microscopy, and confocal microscopy were used to evaluate the labeled cells. In vitro staining was performed to validate the in vivo staining and localize the labeled cells. Three-dimensional rotatable images were taken to evaluate relationships between two differently labeled cells. RESULTS: Histological examination revealed no observable change in the cornea following intravitreal injection. In vivo staining showed that OVA+ cells and cells positive for MHC-II, F4/80, CD11c, B7-1, or B7-2 were noted throughout the cornea with a decreasing density from limbus toward the central cornea. Two populations with distinct morphological features were identified among these APCs. Labeled cells were found beneath the epithelium or in the shallow stroma in the central and paracentral cornea, but in all layers in the peripheral cornea. A number of F4/80+ and CD11c+ cells were also positive for OVA, MHC-II, B7-1, or B7-2. Rotatable images showed a close contact between two differently labeled cells. CONCLUSIONS: Intravitreal injection of labeled antibodies can be adapted to visualize labeled cells in the cornea. APCs with distinct morphologies, phenotypes, and distribution may contribute to the immunologically privileged feature of the cornea.

IL-23 promotes CD4+ T cells to produce IL-17 in Vogt-Koyanagi-Harada disease.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease is a systemic refractory autoimmune disease. IL-23 has been thought to play a critical role in autoimmune disease through inducing the development of IL-17-producing CD4(+) T cells. OBJECTIVE: To investigate the expression of IL-23 and IL-17 and the influence of IL-23 on IL-17 production in patients with VKH disease. METHODS: Blood samples were taken from 25 patients with VKH disease and 16 healthy controls. Peripheral blood mononuclear cells (PBMCs) were subjected to analysis of IL-23p19 mRNA and IL-23 protein expression using RT-PCR and ELISA, respectively. The IL-17 levels in the supernatants of PBMCs and CD4(+) T cells cultured in the absence or presence of recombinant (r)IL-23, rIL-12, or anti-IFN-gamma were determined by ELISA. RESULTS: The patients with VKH disease with active uveitis showed an elevated level of IL-23p19 mRNA in PBMCs, higher IL-23 in the serum and supernatants of PBMCs, and increased production of IL-17 by polyclonally stimulated PBMCs and CD4(+) T cells. Recombinant IL-23 significantly enhanced IL-17 production, whereas rIL-12 and IFN-gamma inhibited IL-17 production. More importantly, IL-17 production was significantly increased in patients with active uveitis in the presence of rIL-23. Both rIL-23 and rIL-12 enhanced IFN-gamma production. CONCLUSION: The results suggest that IL-23-stimulated production of IL-17 by CD4(+) T cells may be responsible for the development of uveitis seen in patients with VKH disease. CLINICAL IMPLICATIONS: This study provides a new insight into the mechanism involved in the development of VKH disease.

Indoleamine 2,3-dioxygenase (IDO) is involved in promoting the development of anterior chamber-associated immune deviation.

Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the tryptophan catabolism, has been shown to play an important role in various forms of immune tolerance. Since anterior chamber associated immune deviation (ACAID) is a systemic immune tolerance elicited by introducing exogenous antigens into the anterior chamber of the eye, we investigated the expression and function of IDO in the development of this ocular tolerance. ACAID was induced in BALB/c mice by an intracameral injection of 50mug ovalbumin (OVA). The IDO expression in the splenocytes during ACAID was determined by fluorescent quantitative real-time PCR, Western blot analysis and immunohistochemistry. The development of ACAID was evaluated by the delayed-type hypersensitivity (DTH) response after intraperitoneal injection of an IDO inhibitor 1-methyl-dl-tryptophan (1-MT). Secretion of IFN-gamma and IL-4 by splenocytes and lymph node cells from the mice treated with or without 1-MT were also evaluated using intracellular cytokine staining. Our results showed that the IDO expression was significantly increased at both mRNA and protein levels following OVA intracameral injection. Inhibition of IDO with 1-MT prevented the development of ACAID, which was indicated by the re-appearance of the OVA-specific DTH response. IL-4 was significantly reduced and IFN-gamma was partially recovered after the treatment of 1-MT. Our study reveal that IDO is up-regulated during ACAID and IDO inhibitor prevents ACAID generation, suggesting that IDO is involved in the development of this immune tolerance.

Redundant and unique regulation of activated mouse B lymphocytes by IL-4 and IL-21.

IL-21 distinctively regulates B cell growth and death, and it redundantly functions with IL-4 for IgG production. B cells likely encounter IL-4 and IL-21 in vivo, as both are secreted by activated T cells. Therefore, the action of both these cytokines was investigated during activation of B cells. IL-21 or the combination of IL-4 and IL-21 inhibited proliferation by purified mouse B cells to LPS or CpG DNA, whereas these cytokines enhanced proliferation after engaging the BCR or CD40. Although B cell subsets expressed somewhat varied levels of the IL-21 receptor, LPS-stimulated follicular and marginal B cell subsets were also dominantly susceptible to IL-21-induced growth arrest and cell death. After activation of B cells with CD40 and LPS, IL-4 and IL-21 distinctively regulated the expression of CD23, CD44, and CD138, and they cooperatively promoted IgG1 class-switching and synthesis. These findings support a model in which the presence of IL-4 and IL-21 inhibits B cells activated by polyclonal innate signals, and they promote B cell expansion and differentiation during T cell-dependent antibody responses, although the individual responses to IL-4 and IL-21 do not always overlap.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis.

BACKGROUND: T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. METHODS: EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). RESULTS: After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. CONCLUSION: Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.

Inducible co-stimulator (ICOS) is upregulated in experimental autoimmune uveoretinitis.

PURPOSE: To study the expression of inducible co-stimulator (ICOS) and its association with T cell effector function in experimental autoimmune uveoretinitis (EAU). METHODS: Eighteen Lewis rats were immunized by retinal S-antigen (50 microg) emulsified in complete Freund's adjuvant (CFA). Twelve normal rats served as normal controls and 18 receiving injection of CFA and PBS as CFA controls for studying the influence of CFA on the expression of ICOS in CD4+CD25+ T cells. ICOS expression on cells from the spleens, inguinal nodes and retinae on day 0 (normal rats), 7, 13 and 21 was investigated using fluorescent quantitative real-time-PCR and Western blot. Expression of B7RP-1, an ICOS ligand, was also studied by Western blot. The phenotype of the cells from the aforementioned three tissues was identified with flow cytometry using antibodies to ICOS, CD4 and CD25. ICOS+ cells from the lymph nodes, and spleens on day 13 were magnetically sorted and cultured with S-antigen to study the cytokines production with enzyme-linked immunosorbent assay. RESULT: An obvious uveitis was induced in all the immunized rats on day 13 after S-antigen immunization. The mRNA and protein of ICOS were scarcely detectable in normal rat spleens. In EAU rats, an up-regulation of ICOS could be observed on day 7 and was very pronounced on day 13, followed by a decrease on day 21 in the spleens, draining nodes and retinae. Similarly, B7RP-1 expression seemed to be up-regulated during EAU. Flow cytometry showed that ICOS+ cells were mostly CD4 positive. Kinetics of ICOS+CD4+CD25+ T cells was similar to that of ICOS+ cells. CFA alone was also able to induce increased expression of ICOS in CD4+CD25+ T cells. IFN-gamma was secreted predominantly by ICOS+ T cells. CONCLUSION: ICOS expression is up-regulated in association with T cell effector capacity in EAU. It is presumed that the ICOS/B7RP-1 costimulatory pathway may play a role in the development of EAU.

Quantitative assessment concerning the contribution of IL-2Rbeta for superantigen-mediated T cell responses in vivo.

IL-2- and IL-2R-deficient mice readily develop T cell-dependent immune responses in vivo, but the relevance of this finding is complicated by severe concurrent autoimmunity. Furthermore, the detection of such responses does not address whether under normal circumstances IL-2 dominates T cell immunity. In the present report, we investigated the extent IL-2-independent T cell growth is mediated by other cytokines in the IL-2 family and compared such responses to those generated by IL-2/IL-2R-sufficient T cells. T cell expansion and contraction to the superantigen staphylococcal enterotoxin A (SEA) by autoimmune-free IL-2Rbeta-/- CD4 and CD8 T cells were comparable to normal control mice, although their CD8+ T cells did not optimally develop into IFNgamma-producing effector cells. The proliferation by these IL-2Rbeta-deficient T cells in vivo was independent of IL-2, IL-4 and IL-15 and not blocked by mAbs to the common gamma chain. However, in co-adoptive transfer experiments, wild-type T cells exhibited somewhat more extensive proliferation than IL-2Rbeta-deficient T cells to SEA and this difference was almost entirely accounted for by CD8+ T cells. Collectively, these data indicate that substantial T cell proliferation occurs in the absence of responsiveness to cytokines in the IL-2 family, although maximal T cell proliferation and development of IFNgamma-producing effector CD8+ T cells depend upon IL-2Rbeta.

Clinical features of Chinese patients with Fuchs' syndrome.

PURPOSE: To characterize the clinical features of Chinese patients with Fuchs' syndrome. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: One hundred eighteen eyes of 104 consecutive patients with Fuchs' syndrome initially examined between January 1999 and March 2005. METHODS: The history and clinical findings of all consecutive Fuchs' patients attending the Zhongshan Ophthalmic Center were reviewed. Auxiliary examinations, including laser flare-cell photometry, ultrasound biomicroscopy (UBM), fundus fluorescein angiography (FFA), and serologic tests for Toxoplasma gondii, were performed in certain cases. MAIN OUTCOME MEASURES: Patients' demographics, clinical presentation, and auxiliary examination findings. RESULTS: One hundred four patients (49 male, 55 female) were included in this study. Unilateral involvement was noted in 90 patients (86.5%). The most common symptom was blurred or decreased vision (86%). Stellate and medium-sized keratic precipitates (KPs) were noted in 108 eyes (91.5%). A mild anterior chamber (AC) reaction was observed in all the affected eyes. Heterochromia was observed in only 15 affected eyes, although there were varying degrees of iris depigmentation in all patients. Iris nodules, mostly Koeppe, were present in 28.0% of the affected eyes. Complicated cataract, vitreous opacity, and secondary glaucoma were observed in 84 of 118 eyes (70.7%), 31 eyes of 42 eyes (73.8%), and 24 of 118 eyes (23.1%), respectively. The mean laser flare photometry value (6.4+/-2.3 photon counts per millisecond) and the cell number in the AC (1.5+/-1.2 cells per 0.5 mm3) in 25 patients were both significantly higher than those in 25 healthy controls (5.3+/-2.3 photon counts per millisecond and 0.8+/-0.6 cells per 0.5 mm3) (P<0.05). Ultrasound biomicroscopy revealed exudates adjacent to the ciliary body in 18 of 24 patients (75%). Serological tests failed to confirm an association of Fuchs' syndrome with toxoplasmosis. Retinal capillary leakage in the midperipheral fundus and disc staining at the late stage were observed in most of the eyes examined by FFA. CONCLUSION: Fuchs' syndrome in Chinese patients is characterized by a mild uveitis with characteristic KPs, varying degrees of iris depigmentation, and, occasionally, heterochromia. Exudates adjacent to the ciliary body and subclinical retinal and optic nerve involvement were common in the patients who were studied by UBM and FFA.

Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis.

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.