Biomedical subjects

Waka Ishida

Publications and source records attributed to Waka Ishida.

17 recordsLinked to original sources

PubMed2006

Role of VLA-4 in the development of allergic conjunctivitis in mice.

PURPOSE: The severity of allergic conjunctivitis (AC) correlates with the degree of eosinophil infiltration into the conjunctiva, which is believed to be mediated by chemokines and adhesion molecules. The adhesion molecule very late antigen (VLA)-4 and its ligand, vascular cell adhesion molecule (VCAM)-1, are known to play important roles in eosinophil infiltration. However, the expression and function of VLA-4 in AC have not been investigated in detail. We sought to characterize VLA-4-expressing cells in the conjunctivas of mice that are developing experimental AC (EC) and to determine whether the interaction between VLA-4 and VCAM-1 is needed for the infiltration of eosinophils into the conjunctiva in AC. METHODS: EC was induced in Balb/c mice by active immunization with ragweed (RW) or adoptive transfer of RW-primed splenocytes, followed by RW challenge. Twenty-four hours after RW challenge, the conjunctivas were harvested. The conjunctivas from naive mice or mice developing EC were evaluated for VLA-4 and VCAM-1 expression by immunohistochemistry and immunofluorescent analyses. To investigate whether the interaction between VLA-4 and VCAM-1 is needed for the genesis of AC, mice developing EC were treated with anti-VLA-4 or anti-VCAM-1 antibodies two h before and after RW challenge. As a control, EC-developing mice were treated with normal rat IgG. Twenty-four hours after RW challenge, the conjunctivas were harvested for histological analysis. RESULTS: Upon induction of EC, VLA-4-expressing cells infiltrated the conjunctiva but the constitutive VCAM-1 expression around conjunctival vessels was not augmented. Immunofluorescent analyses demonstrated that most of the T cells infiltrating the conjunctiva expressed VLA-4 but only half of the infiltrating eosinophils expressed it. Treatment with both anti-VLA-4 and anti-VCAM-1 antibodies significantly suppressed the infiltration of eosinophils into the conjunctiva that was induced by either active immunization or splenocyte transfer. CONCLUSIONS: These results confirm that VLA-4-expressing cells infiltrate the conjunctiva and that the interaction between VLA-4 and VCAM-1 is needed for the development of EC.

Adoptive Transfer↗

PubMed2006

Roles of OX40 in the development of murine experimental allergic conjunctivitis: exacerbation and attenuation by stimulation and blocking of OX40.

PURPOSE: To investigate the roles of interaction between OX40 and OX40 ligand (OX40L) in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: BALB/c mice actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0, 2, 4, 6, and 8 with agonistic anti-OX40 Ab, blocking anti-OX40L Ab, or normal rat (nr)IgG. On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas, spleens, and blood were harvested for analyses. For examination of the effects of the Abs during the late induction (or effector) phase, actively immunized mice were treated with the Abs just before or at the same time as the challenge. In addition, splenocytes from RW-primed mice were transferred into syngeneic naïve mice, and the recipients were treated with Abs twice (on days 2 and 4). On day 4, the mice were challenged with RW and evaluated. RESULTS: When the treatments were performed during the induction phase, anti-OX40 Ab treatment significantly increased clinical EC and eosinophil infiltration into the conjunctiva, whereas anti-OX40L Ab treatment significantly reduced eosinophil infiltration. Compared with splenocytes from nrIgG-treated mice, splenocytes from anti-OX40 Ab-treated mice proliferated vigorously against RW and produced significantly higher amounts of IL-2, -4, and -5 by RW stimulation but a significantly lesser amount of IFN-gamma after Con A stimulation. In contrast, splenocytes from anti-OX40L Ab-treated mice produced significantly less IL-5 with RW stimulation and IL-2 and IL-5 with Con A stimulation, whereas significantly more IFN-gamma was induced by Con A stimulation. Treatment with anti-OX40 and anti-OX40L Abs during the late induction or effector phase of EC did not affect eosinophil infiltration. CONCLUSIONS: Blocking of the interaction between OX40 and OX40L in vivo inhibits the development of EC. In contrast, forced stimulation of OX40 in vivo significantly exacerbates EC by activating T cells, especially Th2 cells. These effects were noted only in the induction phase of EC, suggesting that the interaction between OX40 and OX40L is important in the generation of Th2 immune responses in the development of EC.

Adoptive Transfer↗

PubMed2005

TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance.

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-gamma knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.

Animals↗

PubMed2005

The interaction between ICOS and B7RP-1 is not required for the development of experimental murine allergic conjunctivitis.

It is still unclear whether the interaction between inducible costimulator (ICOS) and its ligand, B7 related protein (B7RP)-1, is important for the development of allergic diseases. We investigated whether blocking the ICOS/B7RP-1 interaction affects the development of murine experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice either by active immunization of ragweed (RW) or by transferring RW-primed splenocytes, followed by challenge with RW-containing eye drops. The mice were treated with anti-B7RP-1 antibody (Ab) or normal rat immunoglobulin G (IgG) during either the induction or effector phase. Regardless of the induction method or when the animals were treated, eosinophil infiltration into the conjunctiva was not affected by the anti-B7RP-1 Ab treatment. Splenocyte responses were not largely affected by this treatment. However, serum Ig levels were significantly reduced. These data suggest that blocking the ICOS/B7RP-1 in allergic diseases may not always be therapeutic.

Allergens↗

PubMed2005

Engagement of 4-1BB inhibits the development of experimental allergic conjunctivitis in mice.

The 4-1BB receptor acts as a costimulator in CD8(+) T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-gamma production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-gamma knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-gamma.

Animals↗

PubMed2005

The immunization protocol determines whether endogenous interferon-gamma suppresses the infiltration of eosinophils into the conjunctiva.

BACKGROUND: Studies with interferon-gamma knockout (GKO) mice showed that endogenous IFN-gamma suppresses the infiltration of inflammatory cells into the conjunctiva. To examine whether this phenomenon is universally true, we induced conjunctival inflammation by four different immunization protocols. METHODS: Both wild type (WT) and GKO mice (C57BL/6 background) were immunized with ragweed (RW) in aluminum hydroxide (alum). Two different immunization protocols were used: either the emulsion was injected into only the left hind footpad or it was also injected into the tail base (50 microg RW in 2mg alum per injection site). In addition, to compare the effects of the immunization dose of RW and the immunization site, 100 microg RW in 2mg alum was injected into only the left hind footpad and 25 microg RW in 2mg alum per injection site was injected into both the left hind footpad and the tail base. Ten days later, the mice were challenged with 2mg RW in 10 microl PBS. Twenty-four hours later, the conjunctivas were analyzed histologically, and the cellular and humoral immune responses in the spleens and sera were determined, respectively. RESULTS: Similar to a previous report, GKO mice showed significant eosinophilic infiltration into the conjunctiva after the footpad only injection of 50 microg RW. However, injection of 50 microg RW per injection site into the footpad plus the tail base resulted in comparable levels of eosinophilic infiltration in WT and GKO mice. On the contrary, either immunization of 100 microg RW in 2mg alum into only the left hind footpad or that of 25 microg RW in 2mg alum into both the left hind footpad and the tail base induced significant infiltration of eosinophils into the conjunctiva of GKO mice, compared to WT mice. CONCLUSIONS: These results show that the immunization protocol employed has a marked effect on the severity of eosinophilic infiltration. These observations indicate that in interpreting experimental results in the study of EC, the immunization protocol employed must be considered.

Allergens↗

PubMed2005

Genetic background determines susceptibility to experimental immune-mediated blepharoconjunctivitis: comparison of Balb/c and C57BL/6 mice.

Balb/c and C57BL/6 mice have been reported to be biased towards Th2 and Th1 immune responses, respectively. We investigated which strain is more susceptible to the development of experimental immune-mediated blepharoconjunctivitis (EC), which is predominantly mediated by Th2 immune responses. EC was induced by three different methods in Balb/c and C57BL/6 mice using ragweed (RW) as the antigen. The mice were thus either actively immunized with RW, passively immunized by transfer of RW-primed T cells, or passively immunized by transfer of RW-specific IgE, followed by RW challenge in eye drops. Twenty-four hours after the challenge, conjunctivas, sera and spleens were harvested for histological analysis, measurement of serum IgE and assessment of cellular immune responses, respectively. The responses of the Balb/c and C57BL/6 mice were compared. In addition, to assess the involvement of IFN-gamma in the development of EC in the two strains, IFN-gamma knockout (GKO) mice of the two strains were actively immunized and evaluated as above. Regardless of the method of induction, EC, as determined by the degree of eosinophil infiltration into the conjunctiva, was more severe in Balb/c mice than in C57BL/6 mice. Moreover, more IgE was produced by actively immunized Balb/c mice than C57BL/6 mice and RW-primed splenocytes from Balb/c mice produced more IL-4 but less IFN-gamma than those from C57BL/6 mice. EC could be induced in the GKO mice of both strains. However, when their EC was compared to that in WT mice, significantly less infiltration of eosinophils was noted in the Balb/c GKO mice. Taken together, Balb/c mice are more susceptible to EC than C57BL/6 mice and this higher susceptibility might be related to the Th2 immune response bias of Balb/c mice. Furthermore, the involvement of endogenous IFN-gamma in the development of EC in these two strains differs.

Adoptive Transfer↗

PubMed2005

Mice lacking the IFN-gamma receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses.

Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-gammaR and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-gammaR and fyn may differ.

Animals↗

PubMed2005

Suppression of macrophage infiltration into the conjunctiva by clodronate liposomes in experimental immune-mediated blepharoconjunctivitis.

Macrophages infiltrate the conjunctiva in severe cases of allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC). We established experimental immune-mediated blepharoconjunctivitis (EC) in Brown Norway (BN) rats as a model for severe types of AC. We investigated whether macrophage infiltration in the conjunctiva in this EC model is inhibited by clodronate liposomes (CL2MDP-lip). The numbers of ED1-positive but not ED2-positive macrophages in the conjunctivas were increased by the induction of EC. Subconjunctival injection of CL2MDP-lip decreased the number of ED2-positive but not ED1-positive macrophages in the conjunctivas of naive rats. CL2MDP-lip did not affect macrophages in the spleen. Subconjunctival injection of CL2MDP-lip into EC-developing BN rats decreased the number of ED2-positive macrophages at all the time points. ED1-positive cell infiltration was inhibited when treatment was administered just prior to OVA challenge. Intravenous injection of CL2MDP-lip decreased the number of ED2-positive cells in the conjunctiva. Thus, we conclude that CL2MDP-lip inhibits infiltration of macrophages into the conjunctiva within 24 h of antigen challenge.

Animals↗

PubMed2005

Systemic interferon-gamma suppresses the development of endotoxin-induced uveitis in mice.

PURPOSE: It has been shown that interferon (IFN)-gamma is involved in the development of endotoxin-induced uveitis (EIU), but its exact role is unclear. We aimed to elucidate the role that endogenous systemic IFN-gamma plays in EIU pathogenesis. METHODS: EIU was induced in wild-type (WT) or IFN-gamma knockout (GKO) mice on the C57BL/6 background by injecting Salmonella typhimurium endotoxin into a hind footpad. Twenty-four hours later, the eyes were harvested for histological analysis, and the serum was collected for cytokine ELISAs. WT and GKO mice were also intraperitoneally injected with 1 microg of recombinant murine IFN-gamma (rmIFN-gamma) just after and 6 h after EIU induction, and their eyes and sera were evaluated 24 h after EIU induction, as above. RESULTS: The GKO mice had significantly more severe EIU as determined by the number of ocular infiltrating cells and lower serum IL-6 levels after EIU induction compared to WT mice. The injection of rmIFN-gamma suppressed the severity of EIU and increased the serum IL-6 levels in both the WT and GKO mice. CONCLUSIONS: Endogenous IFN-gamma suppresses EIU pathogenesis. In addition, the systemic administration of IFN-gamma suppresses EIU. The suppressive mechanism involved is unclear but may relate to the production of IL-6.

Animals↗

PubMed2005

Interleukin-4-mediated infiltration of eosinophils into the conjunctiva and its suppression by interferon-gamma.

PURPOSE: Interleukin (IL)-4 is a T helper (Th)2 cytokine that plays an important role in the development of allergic reactions. It has been suggested that IL-4 is responsible for the infiltration of eosinophils into the conjunctiva during the development of allergic conjunctivitis. However, it is still unclear whether IL-4 is able to induce this eosinophilic infiltration on its own. We investigated whether subconjunctival injection of IL-4 can induce eosinophils to infiltrate into the conjunctiva. METHODS: Brown Norway rats were subconjunctivally injected with IL-4, eotaxin, or phosphate buffered saline (PBS), and the conjunctivas were harvested for histologic analysis (including immunohistochemistry) 6, 12, 18, and 24 hr later. The harvested conjunctivas were also subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to examine chemokine expression. In a separate experiment, the effect of coadministering interferon-gamma (IFN-gamma) along with IL-4 was examined. RESULTS: The subconjunctival injection of IL-4 induced eosinophil infiltration into the conjunctiva in a dose-dependent manner. IL-4 was as potent as eotaxin. The eosinophilic infiltration started 6 hr after the injection and persisted for up to 24 hr after the injection. Other infiltrating cell phenotypes were noted but were also observed in conjunctivas injected with PBS alone. RT-PCR analysis demonstrated that IL-4 injection did not upregulate chemokine RNA expression in the conjunctiva. Coinjection of IFN-gamma suppressed the infiltration of eosinophils into the conjunctiva induced by IL-4 injection. CONCLUSIONS: The subconjunctival injection of IL-4 specifically induces eosinophils to infiltrate into the conjunctiva. In addition, IFN-gamma in the conjunctiva can counteract this effect of IL-4.

Animals↗

PubMed2005

Dissection of antigen-specific humoral and cellular immune responses for the development of experimental immune-mediated blepharoconjunctivitis in C57BL/6 mice.

PURPOSE: Allergic conjunctivitis is characterized by allergen-specific IgE in the serum and infiltration of eosinophils into the conjunctiva. However, it remains unclear whether early-phase reaction (EPR) mediated by Ag-specific IgE links to late-phase reaction (LPR) in the conjunctiva. We aimed to investigate whether LPR is mediated by either cellular or humoral immune responses. METHODS: Experimental immune-mediated blepharoconjunctivitis (EC) was induced in C57BL/6 mice by either active immunization or passive immunization by transfer of ragweed (RW)-primed lymphocytes and RW-specific IgE, followed by RW challenge onto the conjunctiva. Transferring RW-primed lymphocytes were prepared from RW-primed splenocytes which were stimulated in vitro with RW for 3 days. Fifteen minutes after RW challenge, clinical findings were evaluated and 24 hr after challenge, the conjunctivas and sera were harvested for histologic analysis and measurement of IgE, respectively. RESULTS: EPR was most prominent when EC was induced by transfer of RW-specific IgE. EPR was hardly detectable if EC was induced by transfer of RW-primed lymphocytes. Mild EPR was noted when EC was induced by active immunization. LPR, evaluated by infiltration of eosinophils into the conjunctiva, was most severe when EC was induced by transfer of RW-primed lymphocytes. Minimal, but definite LPR was induced when EC was induced by transfer of RW-specific IgE. Intermediate severity of LPR was induced when EC was induced by active immunization. CONCLUSIONS: LPR in the conjunctiva is dominantly mediated by cellular immune responses, whereas EPR in the conjunctiva is putatively mediated by humoral immune responses. Importantly, LPR in the conjunctiva is inducible by Ag-specific IgE alone, although minute.

Allergens↗

PubMed2004

Detection of antigen-specific T cells in experimental immune-mediated blepharoconjunctivitis in DO11.10 T cell receptor transgenic mice.

Antigen (Ag)-specific T cells are thought to play a key role in pathogenesis of chronic allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). In order to investigate the trafficking of Ag-specific T cells in experimental immune-mediated blepharoconjunctivitis (EC), we established a novel AC model in DO11.10 T cell receptor (TcR) transgenic (Tg) mice. DO11.10 TcR-Tg mice were challenged with eye drops of whole OVA protein, OVA peptide 1-15, 321-335, or 323-339. Their eyes were histologically examined. Conventional proliferation assay was performed against each Ag. Phenotypes of infiltrating cells and kinetics of Ag-specific T cells were investigated by immunohistochemistry. Adoptive transfer of CD4(+) Ag-specific T cells from DO11.10 TcR-Tg to WT mice was performed. The distribution of KJ1-26(+) cells was investigated in recipient mice. The challenge of OVA peptide 323-339 induced infiltration of inflammatory cells in conjunctivae in a dose dependent manner, accompanied by the proliferative responses of splenocytes. Immunohistochemical analysis revealed Agspecific/ non-Ag-specific T cells, macrophages, and eosinophils in conjunctivae. Infiltration of Ag-specific T cells increased 24 hr later. Transfer of CD4(+) cells from DO11.10 TcR-Tg to WT mice induced EC depending on the number of transferred cells. Ag-specific T cells were detected in the conjunctivae and spleens of recipient mice, though its numbers were significantly smaller compared to DO11.10 TcR-Tg mice. The challenge of OVA peptide 323-339 induced EC in DO11.10 TcR-Tg mice without prior sensitization. The response was mediated by CD4(+) Ag-specific T cells. The trafficking of Ag-specific T cells in EC was clearly visualized.

Adoptive Transfer↗

PubMed2003

Ag-specific recognition, activation, and effector function of T cells in the conjunctiva with experimental immune-mediated blepharoconjunctivitis.

PURPOSE: To investigate antigen (Ag) specificity, activation, and effector function of the Ag-specific T cells involved in the development of experimental immune-mediated blepharoconjunctivitis (EC), an experimental conjunctivitis. METHODS: EC was induced in Brown Norway rats by injection of ovalbumin (OVA)-specific T cells followed by OVA challenge with eye drops. Eyes, including the conjunctivas, were harvested at different time points after challenge. The dependence of EC onset on the challenging Ag was assessed by challenge with an irrelevant Ag or stimulatory OVA peptides. To show the infiltration of transferred T cells into the conjunctiva, T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before transfer. The activation of T cells in the conjunctiva was assessed by measuring phosphorylation of Lck-associated molecules by Western blot analysis. Conjunctivas were also examined by immunohistochemistry and used for reverse transcription-polymerase chain reaction to determine the phenotype of the infiltrating cells and cytokine, chemokine, and chemokine receptor expression. To investigate infiltration of non Ag-specific T cells into the conjunctiva, ragweed (RW)-primed lymphocytes were transferred into OVA-specific T-cell receptor transgenic (DO11.10) mice. The mice were then challenged with RW and the conjunctivas were harvested for immunohistochemistry to detect T cells derived from DO11.10 mice. RESULTS: EC was induced only when challenged with OVA protein or stimulatory OVA peptides, and CFSE-labeled transferred cells were found in the conjunctiva. Phosphorylation of Lck and an 85-kDa Lck-associated molecule were observed in the conjunctiva 6 hours after challenge. Many cytokines and chemokines began to be expressed at 6 hours, and individual expression patterns over time correlated well with the infiltration patterns of different inflammatory cells. In DO11.10 mice that received RW-primed lymphocytes, T cells derived from the recipient mice infiltrated the conjunctiva after RW challenge. CONCLUSIONS: Ag-specific T cells initiate EC by first infiltrating the conjunctiva, where they become activated by the specific Ag in the conjunctiva.

Animals↗

PubMedDate not supplied

Phenotypic changes and inflammatory cell distribution in the cornea during development of experimental immune-mediated blepharoconjunctivitis.

PURPOSE: To investigate the morphological changes in the cornea during the development of experimental immune-mediated blepharoconjunctivitis (EC). METHODS: EC was induced in Brown Norway (BN) rats by active immunization with ovalbumin (OVA) emulsified in complete Freund's adjuvant and a subsequent challenge by OVA eyedrops. The corneas were analyzed immunohistochemically. RESULTS: Before the induction of EC, cells stained with OX6 (rat MHC class 2, RT1B), ED1 (tissue macrophages), ED2 (resident macrophages), CD4, or major basic protein were present in the peripheral corneal stroma. ED1- and OX6-stained cells were also observed in the central corneal stroma, and their number increased after the antigen challenge. Infiltration of cells stained with ED1, ED2, OX62 (dendritic cells), CD4, or CD3 (T cells) from the limbus to the peripheral corneal stroma started 6 h after the antigen challenge. Expression of MHC class 2 molecules was induced on the corneal epithelium by the antigen challenge. CONCLUSIONS: The present study demonstrates for the first time the phenotypic changes and distribution of inflammatory cells in the cornea during the development of EC.

Animals↗

PubMedDate not supplied

Analysis of Ag-presenting cells in the conjunctiva during the development of experimental immune-mediated blepharoconjunctivitis.

PURPOSE: The aim of this study was to investigate the phenotypes of antigen (Ag) presenting cells (APCs) in the conjunctiva during the development of experimental immune-mediated blepharoconjunctivitis (EC), which serves as a model for investigation of severe types of human allergic conjunctivitis. METHODS: Brown Norway rats treated by ovalbumin (OVA) were used in this study. To confirm the restriction of MHC class II by OVA-specific T cells, monoclonal Abs against MHC class II were added to the conventional proliferation assay. To evaluate the MHC class II expression in the conjunctiva during the development of EC, an immunohistochemical analysis, either as the single or double staining, was performed. Conjunctival fibroblast cell lines were established from naive rats and the MHC class II expression was evaluated by flow cytometric analysis. To examine the roles of costimulatory molecules, OVA-specific T cells were stimulated with anti-TcR Ab and anti-CD28 Ab and then subjected for Western blotting to evaluate the ERK phosphorylation. Finally, in vivo expression of B7 molecules was examined immunohistochemically. RESULTS: OVA-specific T cells recognized OVA in the context of MHC class II. MHC class II was expressed in conjunctival macrophages but not in fibroblasts. EC induction was accompanied by abundant infiltration of macrophages positive for MHC class II. MHC class II was also expressed in conjunctival epithelial cells by EC induction. Stimulation from CD28 was necessary for ERK phosphorylation. B7-2, but not B7-1, was expressed in the conjunctiva. CONCLUSION: Conjunctival macrophages may represent a major source of APCs for the induction of EC in the conjunctiva.

Animals↗

PubMedDate not supplied

Cyclosporin A inhibits eosinophilic infiltration into the conjunctiva mediated by type IV allergic reactions.

BACKGROUND: Eosinophils are important effector cells in severe allergic conjunctivitis such as vernal keratoconjunctivitis. Infiltration of eosinophils into the conjunctiva is mediated by type I and type IV allergic reactions. Cyclosporin A (CsA) eye drops are administered therapeutically for severe allergic conjunctivitis, but the mechanism by which CsA acts, that is, by inhibiting type I, type IV or both types of allergic reactions, is not known. We investigated whether CsA eye drops inhibit type I, type IV or both types of allergic reactions in the conjunctiva. METHODS: Experimental immune-mediated blepharoconjunctivitis (EC) was induced in BALB/c mice by either active immunization or passive immunization by transfer of ragweed (RW)-primed splenocytes and RW-specific IgE, followed by RW challenge to the conjunctiva. These mice were treated in eye drops with vehicle, 0.1% CsA, 0.5% CsA or 0.1% betamethasone five times (1 and 2 h before RW challenge and 1, 2 and 3 h after RW challenge). Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis to evaluate eosinophilic infiltration. To evaluate effects of CsA eye drops on systemic immune responses, sera and spleens were collected from actively immunized mice at the time of sacrifice to examine serum IgE levels and cellular immune responses, respectively. RESULTS: CsA eye drops significantly inhibited eosinophilic infiltration into the conjunctiva in actively immunized EC-developing mice compared with vehicle-treated mice. The CsA-induced inhibition was similar to inhibition induced by 0.1% betamethasone. Serum IgE levels and splenocyte responses in CsA-treated mice were equivalent to those in vehicle-treated mice. Betamethasone treatment inhibited eosinophilic infiltration into the conjunctiva induced by both splenocyte transfer and IgE transfer, while CsA treatment inhibited infiltration induced by splenocyte transfer. CONCLUSIONS: CsA eye drops inhibited eosinophilic infiltration into the conjunctiva without affecting systemic immune responses. CsA predominantly inhibits eosinophilic infiltration by interfering with the type IV allergic reaction in the conjunctiva.

Animals↗