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.

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.

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.

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.

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.

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.

Suppression of experimental immune-mediated blepharoconjunctivitis in Brown Norway rats by topical application of FK506.

BACKGROUND: Experimental immune-mediated blepharoconjunctivitis (EC) in Brown Norway (BN) rats, which is inducible by transfer of antigen-specific T cells, is a model for human allergic conjunctivitis. We investigated the possible inhibition of EC in BN rats by topical application of FK506, which is an immunosuppressive agent that mainly targets T cells. METHODS: To induce EC by active immunization, ovalbumin (OVA) adsorbed to alum was injected into the hind footpads of BN rats. Three weeks after the initial immunization, rats were challenged with OVA by eye drops. Twenty-four hours later, lids including conjunctivas, lymph nodes (LNs), and sera were harvested for histology or reverse transcriptase PCR, proliferation assays, and measurement of IgE titer, respectively. For passive immunization, rats were intravenously injected with 10 million of in vitro-stimulated OVA-primed LN cells. Four days after the transfer, rats were challenged with OVA and evaluated as above. The rats were divided into two groups. One group received topical FK506 treatment three times per day from 15 to 21 days after active immunization or from 1 to 4 days after transfer. The other group was treated with vehicle as above. RESULTS: FK506 treatment suppressed infiltration of both lymphocytes and eosinophils in the conjunctiva either by active or passive immunization (P<0.002). No differences were noted in antigen-specific cellular and humoral immune responses. Concerning cytokine expression in the conjunctiva, a prominent difference was noted only with IL-4, which was more abundantly detected in the vehicle-treated group. CONCLUSION: Topical FK506 treatment suppressed EC in BN rats, possibly by inhibition of IL-4 in the conjunctiva.