Tamaki Sumi

Notch ligand Delta-like4 inhibits the development of murine experimental allergic conjunctivitis.

BACKGROUNDnTo investigate the involvement of Notch ligands in the development of experimental allergic conjunctivitis (EC) in mice.nnnMETHODSnTo induce EC, wild-type (WT) or IFN-gamma-deficient (GKO) BALB/c mice were immunized with ragweed (RW) in alum followed by RW challenge in eye drops. Twenty-four hours after RW challenge, the conjunctivas, spleens and blood were harvested to evaluate conjunctival eosinophil infiltration, RW-specific cytokine production and serum Ig levels, respectively. Abs against Notch ligands (anti-Jagged1, anti-Jagged2, anti-Delta-like (Dll)1 and anti-Dll4) were injected intraperitoneally into EC-developing mice during the induction or effector phase. As a control, normal hamster IgG (nhIgG) was injected.nnnRESULTSnTreatment with anti-Dll4 Ab but not the other Abs during the induction phase significantly augmented the severity of EC, as measured by the conjunctival eosinophil infiltration. Anti-Dll4 Ab treatment also significantly upregulated RW-recall IL-4 production and suppressed serum IgE and IgG1 levels. However, anti-Dll4 Ab treatment during the induction phase did not significantly affect the severity of EC in GKO mice. None of the Abs significantly affected the severity of EC, splenocyte cytokine production, or serum Ig levels when administered during the effector phase.nnnCONCLUSIONSnThese observations suggest that Dll4, a Notch ligand, plays a role in suppressing the development of EC, possibly by providing a negative signal for Th2 development during the induction phase. In addition, IFN-gamma may participate in the augmentation of EC by anti-Dll4 Ab treatment.

Thymus-Derived CD4+ CD25+ T Cells Suppress the Development of Murine Allergic Conjunctivitis

CD4+ CD25+ T cells regulate various kinds of immune-mediated diseases. Here, we sought to clarify whether CD4+ CD25+ T cells also regulate the development of experimental allergic conjunctivitis (EC). Thymectomized BALB/c mice, treated with anti-CD25 antibody (PC61), normal rat immunoglobulin G (nrIgG) or left untreated were immunized with short ragweed pollen (RW). Ten days later, the mice were challenged with RW in eye drops, and 24 h later, the conjunctivas, blood and spleens were harvested. The severity of EC, as evaluated by conjunctival eosinophil numbers, was significantly higher in the PC61-treated group as compared with the other two groups. The PC61-treated group also had significantly higher RW-specific IgE and IgG1 levels and displayed RW-specific splenocyte proliferation and RW-induced splenocyte T helper cell 2 cytokine production. However, PC61 treatment of unthymectomized mice did not affect the severity of EC. Thus, thymus-derived CD25+ T cells regulate the development of EC. Furthermore, transfer of Foxp3-expressing CD4+ CD25+ T cells from naïve mice into RW-sensitized mice suppressed the development of EC in these mice after RW challenge. Taken together, these results suggest that CD4+ CD25+ T cells regulate the development of EC.

Roles of galectin-9 in the development of experimental allergic conjunctivitis in mice.

Background: Galectin-9 was recently identified as a ligand for Tim-3, which negatively regulates Th1 cells. Here, we investigated whether administration of anti-galectin-9 antibodies (Abs) affects the development of murine experimental allergic conjunctivitis (EC), a Th2-mediated disease. Methods: EC was induced in BALB/c mice by active immunization with ragweed (RW) followed by RW challenge in eye drops. Normal rat IgG (nrIgG) or anti-galectin-9 Ab was injected intraperitoneally into the mice either during the induction phase treatment or effector phase treatment. Alternatively, Abs were injected into the subconjunctival space during the effector phase. To evaluate in vitro effects of anti-galectin-9 Abs, splenocytes from RW-sensitized mice were cultured in vitro with RW in the presence of nrIgG or anti-galectin-9 Abs. To induce EC, these splenocytes were transferred into naïve BALB/c mice followed by RW challenge. Results: Treatment with anti-galectin-9 Abs in vivo during either the induction or effector phase did not significantly affect the severity of EC. However, when the splenocytes from RW-primed mice treated with anti-galectin-9 Abs during the induction phase were stimulated in vitro and adoptively transferred into naïve recipients, they induced significantly severer EC. In contrast, when RW-primed splenocytes were restimulated in vitro with RW in the presence of anti-galectin-9 Abs and then adoptively transferred, they induced less severe EC and produced significantly less IL-5 and IL-13 and more IFN-γ compared to nrIgG-treated control splenocytes. Conclusions: These observations may reflect the fact that galectin-9 not only regulates T-cell function, it is also involved in many other biological functions.

CD8+ T cells play disparate roles in the induction and the effector phases of murine experimental allergic conjunctivitis.

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8α‐deficient (CD8KO) mice and wild‐type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW‐containing eye drops. Alternatively, EC was induced by transferring RW‐primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL‐5 and IL‐6 expression and greater upregulation of IL‐4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW‐primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.

Roles of CD4+CD25+ T cells in the development of experimental murine allergic conjunctivitis

BackgroundCD25+ regulatory T (T reg) cells play a suppressive role in experimental autoimmune uveoretinitis as well as experimental airway inflammation but their involvement in the development of allergic conjunctivitis (AC) remains unclear. We therefore investigated whether T reg cells play a role in the development of experimental AC (EC).MethodsBALB/c and C57BL/6 mice were actively immunized with ragweed (RW). The mice were treated with an anti-CD25 Ab (PC61) or control normal rat IgG (nrIgG) either 2xa0days prior to active immunization or during the induction phase (daysxa00, 2, 4, 6 and 8). Ten days after active immunization, the mice were challenged with RW-containing drops. Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis of eosinophil infiltration, and the spleens were harvested for cell culture for splenocyte transfer. Cultured splenocytes were transferred into syngeneic mice, and 4xa0days after the transfer, the recipient mice were challenged with RW. Twenty-four hours after the challenge, conjunctivas were collected for histological analysis.ResultsPretreatment with PC61 did not affect EC in either strain of mice; however, treatment with PC61 during the induction phase significantly suppressed EC in C57BL/6 mice. In contrast, transfer of RW-primed splenocytes from mice treated with PC61 induced EC that was significantly more severe regardless of strain and treatment protocol.ConclusionsThe finding that T reg cells play a suppressive role in the development of EC in splenocyte transfer experiments suggests that modulation of T reg cells may be a possible therapy for AC.

Endogenous IL-17 does not play a significant role in the development of experimental murine allergic conjunctivitis.

Background: Endogenous IL-17 is needed for the Ag sensitization that results in murine allergic asthma, a Th2-mediated disease. Here, we aimed to investigate the role IL-17 plays in the development of murine experimental allergic conjunctivitis (EC) which is also a Th2-mediated disease.Methods:To induce EC, wild-type (WT) and IL-17-deficient (IL-17 KO) mice on the BALB/c and C57BL/6 backgrounds were immunized with ragweed (RW) in alum and challenged with RW in eye drops. Alternatively, EC was induced by adoptively transferring RW-primed splenocytes followed by challenge with RW-containing eye drops. Twenty-four hours after the RW challenge, the conjunctivas and spleens were harvested for histological analyses and cytokine assays, respectively. Results: The WT and IL-17 KO mice on both backgrounds did not differ in terms of the severity of actively induced EC, as evaluated by the conjunctival eosinophil infiltration. They also did not differ with regard to the phenotypes of the inflammatory cells infiltrating the conjunctivas, although primed IL-17 KO splenocytes stimulated in vitro with RW extract did produce significantly higher amounts of IL-4, IL-13 and IFN-γ than WT splenocytes. Reciprocal adoptive transfer experiments also demonstrated that the IL-17 from both the donor splenocytes and the recipient mice is not involved in the development of EC. Conclusions: Endogenous IL-17 does not appear to play a significant role in the development of EC.

CD27 and CD70 do not play a critical role in the development of experimental allergic conjunctivitis in mice

CD27, which belongs to the TNF receptor family, is a costimulatory molecule that participates in T-cell activation. Unlike costimulatory molecules such as OX40 and 4-1BB, little is known about the role CD27 plays a role in the development of experimental diseases. We asked whether CD27 and its ligand CD70 participate in the development of experimental allergic conjunctivitis (EC) in BALB/c mice, which is generated by immunization with ragweed (RW) in alum and challenged 10 days later with RW in eye drops. The roles of CD27 and CD70 were tested by intraperitoneally injecting the mice with anti-CD27, anti-CD70 or a control Ab during the induction or effector phase. Twenty-four hours after challenge, the conjunctivas, blood and spleens were harvested for histological analysis, measuring Ig levels and cytokine analysis, respectively. Regardless of when the mice were treated, anti-CD27 or anti-CD70 Ab treatment did not significantly affect the severity of EC as evaluated by conjunctival eosinophil numbers. However, anti-CD27 or anti-CD70 Ab treatment during the induction phase did significantly modulate systemic humoral and cellular immune responses. In vitro treatment of RW-primed splenocytes with anti-CD27 or anti-CD70 Ab did not affect the EC-inducing capability of the splenocytes. Taken together, CD27 and CD70 do not play a critical role in the development of EC.

Treatment with FTY720 during the induction or effector phase suppresses the development of experimental allergic conjunctivitis in mice

Purpose: To investigate whether experimental allergic conjunctivitis (EC) can be suppressed by treatment with the immunomodulatory drug FTY720, which reduces the recruitment of effector T cells into inflammatory sites.

Regulatory T cells participate in 4-1BB-mediated suppression of experimental allergic conjunctivitis.

Background: We showed previously that treatment with an agonistic anti-4-1BB Ab during the induction phase of murine experimental allergic conjunctivitis (EC) suppresses the development of this model disease. It was reported that 4-1BB promotes the expansion of regulatory T cells. Here we asked whether the suppression of EC by agonistic anti-4-1BB Ab treatment is mediated by regulatory T cells. Methods: Neonatal BALB/c mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in alum. As a control, immunocompetent BALB/c mice were immunized. Ten days later, the mice were challenged with RW in eye drops and 24 h later, the conjunctivas and spleens were harvested for histological and flow-cytometric analyses, respectively. The agonistic anti-4-1BB Ab or control normal rat IgG was injected intraperitoneally during the induction phase of EC. Results: With regard to immunocompetent mice, anti-4-1BB Ab treatment significantly suppressed the severity of EC as evaluated by conjunctival eosinophil numbers. In contrast, in thymectomized and anti-CD25 Ab-treated mice in which CD4+CD25+ regulatory T cells were efficiently depleted, anti-4-1BB Ab treatment did not affect the severity of EC. Conclusions: These results indicate that CD4+CD25+ regulatory T cells play a critical role in the suppression of EC by anti-4-1BB Ab treatment.

Depletion of Thymus-Derived CD4^+CD25^+ T Cells Abrogates the Suppressive Effects of α-galactosylceramide Treatment on Experimental Allergic Conjunctivitis

BACKGROUNDnWe showed previously that α-galactosylceramide (α-GalCer) treatment elevated splenic CD4+ CD25+Foxp3+ T-cell numbers and suppressed the development of experimental allergic conjunctivitis (EC). Here, we investigated whether CD4+CD25+Foxp3+ T cells mediate the suppressive effects of α-GalCer treatment on EC.nnnMETHODSnTo deplete CD4+CD25+Foxp3+ T cells, neonatal mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in aluminum hydroxide. Ten days later, the mice were challenged with RW in eye drops and 24 hours later, the conjunctivas and spleens were harvested for histological and flow cytometric analyses, respectively. α-GalCer or vehicle was injected 2 hours prior to RW challenge. In addition, α-GalCer was injected into thymus-intact EC-developing mice that had not been treated with anti-CD25 Ab.nnnRESULTSnα-GalCer treatment significantly suppressed EC in the thymus-intact mice that had not been treated with anti-CD25 Ab. In contrast, α-GalCer treatment of thymectomized and anti-CD25 Ab-treated mice did not affect the severity of EC or splenic CD4+CD25+Foxp3+ T-cell numbers. However, α-GalCer treatment did significantly increase splenic CD4+CD25+Foxp3+ T-cell numbers in thymectomized mice that had not received anti-CD25 Ab.nnnCONCLUSIONSnα-GalCer treatment during the effector phase of EC increased CD4+CD25+Foxp3+ T-cell numbers, which in turn suppressed the development of EC.BACKGROUNDnWe showed previously that alpha-galactosylceramide (alpha-GalCer) treatment elevated splenic CD4+CD25+Foxp3+ T-cell numbers and suppressed the development of experimental allergic conjunctivitis (EC). Here, we investigated whether CD4+CD25+Foxp3+ T cells mediate the suppressive effects of alpha-GalCer treatment on EC.nnnMETHODSnTo deplete CD4+CD25+Foxp3+ T cells, neonatal mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in aluminum hydroxide. Ten days later, the mice were challenged with RW in eye drops and 24 hours later, the conjunctivas and spleens were harvested for histological and flow cytometric analyses, respectively. alpha-GalCer or vehicle was injected 2 hours prior to RW challenge. In addition, alpha-GalCer was injected into thymus-intact EC-developing mice that had not been treated with anti-CD25 Ab.nnnRESULTSnalpha-GalCer treatment significantly suppressed EC in the thymus-intact mice that had not been treated with anti-CD25 Ab. In contrast, alpha-GalCer treatment of thymectomized and anti-CD25 Ab-treated mice did not affect the severity of EC or splenic CD4+CD25+Foxp3+ T-cell numbers. However, alpha-GalCer treatment did significantly increase splenic CD4+CD25+Foxp3+ T-cell numbers in thymectomized mice that had not received anti-CD25 Ab.nnnCONCLUSIONSnalpha-GalCer treatment during the effector phase of EC increased CD4+CD25+Foxp3+ T-cell numbers, which in turn suppressed the development of EC.