Costimulatory Molecules Cd30/cd30l Mediated Murine Experimental Allergic Conjunctivitis Immune Mechanisms And Their Biological Significance

Objective:1. To study and evaluate the mechanic and roles of the costimulatory molecules CD30 and CD30L in the development of experimental allergic conjunctivitis (EC) in mice.2. To research and analyse the influence made by expression of the costimulatory molecules CD30 and CD30L on EC in mice.Methods:Seventy-two (72) Wild-type (WT) mice were randomly divided into three groups (N=24), They are induction phase group A, effector phase group B, immunization group C respectively; The mice in every group were randomly divided into three groups (n=8), nrIgG group(A1,B1, C1),anti-CD30 Ab group(A2, B2, C2) and anti-CD30L Ab group (A3, B3,C3). Forty-eight(48) naive mice were randomly divided into two groups (N=24), They are the vivo of adoptive transfer group D, and the vitro of adoptive transfer group E, The mice in every group were randomly divided into three groups (n=8), nrIgG group (D1, E1), anti-CD30 Ab group (D2,E2) and anti-CD30L Ab group(D3,E3). Sixteen(16) Wild-type (WT) mice(Group F) were randomly divided into two groups (N=24), They are experiment group(F1) and control group(F2).1. Induction phase group A1.1 The WT mice in induction phase actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0,2,4,6, and 8 with normal rat(nr) IgG, anti-CD30 Ab, anti-CD30L Ab, (200μg, n=8). 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.1.2 The splenocytes from the induction group were stimulated in vitro with RW and then transferred into naive mice (Group D). On day 4, the mice were challenged with RW in eye drops. Twenty-four hours later, conjunctivas were harvested for histologic analysis and the measurement of eosinophils through the Giemsa stain in electron microscopy.2.Effector phase group BThe WT mice in induction phase actively immunized with short ragweed pollen (RW) were intraperitoneally injected on day 10 with agonisticnormal rat (nr)IgG, anti-CD30 Ab, anti-CD30L Ab (200μg, n=8).At the same time,the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas were harvested for related analyses.3.Immunization group CThe mice were immunized with RW in alum and not treated with any Abs. Ten days later, their spleens were harvested, and the splenocytes were stimulated in vitro with RW and then transferred into naive mice (Group E). The mice were intraperitoneally injected with 200μg nrIgG, anti-CD30 Ab or anti-CD30L Ab twice (on days 2 and 4, n=8 per group). Two hours after the injection of Abs on day 4, the mice were challenged with RW in eye drops. Twenty-four hours later, conjunctivas were harvested for histologic analysis and the measurement of eosinophils.4.IHC(immunohistichemistry) FThe WT mice in induction phase actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0,2,4,6, and 8 with anti-CD30 Ab, (200μg,n=8). On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas were harvested for testing CD4+T cell by IHC.5. The same detections were used for conjunctivitis tissue among the group A, B, D. And test the level of IgE in the blood from the induction group A, in addition, detect the change of CD4+T cells visa IHC in the group F.Results:1.Clinical signs:Severe lid swelling (+++) and discharge (+++) were observed only in the mice treated with anti-CD30 Ab (A2)in the induction group,whereas nrIgG(A1) and anti-CD30L-treated mice (A3) showed mild conjunctival hyperemia(+).2. Induction phase group A2.1 The results of eosinophils (ES) through the Giemsa stain in electron microscopy Show:Compared with the nrIgG-treated mice(A 1), Counting of infiltrating eosinophils confirmed that there was significantly more eosinophil infiltration in anti-CD30-treated mice(A2)(p<0.01); there was significantly less eosinophil infiltrationin anti-CD30L-treated mice (A3)than in nrIgG-treated mice (P<0.05).2.2With ELTSA, compared with nrIgG-treated mice(A1), the IgE levels in the serum demonstrated that the light difference was observed in anti-CD30 Ab mice(A2), and the lower significant change was observed in anti-CD30L Ab mice(A3)(p<0.01).3. Effector phase group BThe numbers of infiltrating eosinophils into the conjunctiva were not significant among B1,B2,B3, although anti-CD30-treated mice(B2) had lightly more eosinophilic infiltration.4. the vivo of adoptive transfer group DCompared with nrIgG-treated mice(D1), Obviously increased eosinophilic infiltration is counted on adoptive transfer of splenocytes from the mice treated with anti-CD30 Ab in vivo(D2) (p<0.01). There is no significant differences between D1 and D3.5. the vitro of adoptive transfer group EThere were no significant differences in the numbers of infiltrating eosinophils into the conjunctiva among the three groups(E1,E2,E3) on adoptive transfer of splenocytes in vitro.6.IHC(immunohistichemistry) FCompared with the control group(F2), more CD4+T cells infiltrated into the conjunctivitis of The experimental group(F1) obviously by IHC.(Picture2,3)Conclusions:1. The costimulatory molecules CD30 and CD30L in the development of EC in mice induce immune responses by activating CD4+T cells.2.The expression of CD30L inhibits the development of EC in mice.