Figure 1
Figure 1. Antibodies to CD44 can ameliorate murine ITP and do so in a manner dependent on the presence of the inhibitory FcγRIIB but independent of the Fc region of IgG. (A) Mice were injected with 50 μg CD44 antibody (5035-41.1D, KM114, KM81, KM201, 9A4, IOD1, IRAWB14, or IM7) or 50 mg IVIg (∼ 2 g/kg of body weight). Twenty-four hours later, mice were injected with 2 μg antiplatelet antibody to induce thrombocytopenia. Mice were bled after a further 24 hours, and the platelet count was determined. Horizontal bars represent the range of the mean platelet count (± 1 SEM) of naive, antiplatelet antibody injected mice, and IVIg treated mice. n = 9 mice for each group from 3 independent experiments. *P < .01 versus ITP. **P < .001 versus ITP. (B) CD44 binding to splenic leukocytes was assessed as follows: RBC-depleted mouse splenic leukocytes were incubated with the indicated CD44 antibody followed by the appropriate fluorescein isothiocyanate-labeled secondary anti-IgG and assessed for mean log fluorescence intensity (MLFI) by flow cytometry. The values are the mean of 2 independent experiments. (C) To determine the extent of reactivity of murine platelets with the CD44 antibodies used in this study, murine platelets were incubated with the indicated CD44 antibody followed by appropriate fluorescein isothiocyanate-labeled secondary anti-IgG and assessed for MLFI by flow cytometry. The values are the mean of 4 independent experiments. (D) Mice were injected with 50 μg of the indicated CD44 antibodies to assess the ability of each antibody to induce thrombocytopenia in the absence of antiplatelet antibody. Platelet counting was evaluated before injection and after 24, 48, 72, and 96 hours. n = 6 mice for each group from 2 independent experiments. *P < .05 versus prebleed values. **P < .001 versus prebleed values. (E) C57BL/6 mice (columns 1-4) were treated and bled and platelets counted as in panel A, except that mice were treated with either 50 μg intact KM114 or 37 μg F(ab′)2 KM114 (the equivalent molar concentration). n = 6 mice for each group from 3 independent experiments. Mice expressing human FcγRIIA (columns 5 and 6) were injected with antiplatelet antibody alone or antiplatelet antibody plus KM114 as in panel A and platelets were counted as in panel A. n = 6 mice for each group from 2 independent experiments. (F) Wild-type mice (FcγRIIB+/+) or (G) mice genetically deficient in the inhibitory Fcγ receptor RIIB (FcγRIIB−/−) were injected with 50 mg IVIg, 50 μg anti-RBC monoclonal antibody (TER119), or 50 μg of the indicated CD44 antibodies (KM114 or 5035–41.1D). Twenty-four hours later, all mice were given 2 μg antiplatelet antibody to induce thrombocytopenia. After a further 24 hours, mice were bled for platelet counting. Normal and ITP are described as in panel A. n = 9 mice for each group from 3 independent experiments.

Antibodies to CD44 can ameliorate murine ITP and do so in a manner dependent on the presence of the inhibitory FcγRIIB but independent of the Fc region of IgG. (A) Mice were injected with 50 μg CD44 antibody (5035-41.1D, KM114, KM81, KM201, 9A4, IOD1, IRAWB14, or IM7) or 50 mg IVIg (∼ 2 g/kg of body weight). Twenty-four hours later, mice were injected with 2 μg antiplatelet antibody to induce thrombocytopenia. Mice were bled after a further 24 hours, and the platelet count was determined. Horizontal bars represent the range of the mean platelet count (± 1 SEM) of naive, antiplatelet antibody injected mice, and IVIg treated mice. n = 9 mice for each group from 3 independent experiments. *P < .01 versus ITP. **P < .001 versus ITP. (B) CD44 binding to splenic leukocytes was assessed as follows: RBC-depleted mouse splenic leukocytes were incubated with the indicated CD44 antibody followed by the appropriate fluorescein isothiocyanate-labeled secondary anti-IgG and assessed for mean log fluorescence intensity (MLFI) by flow cytometry. The values are the mean of 2 independent experiments. (C) To determine the extent of reactivity of murine platelets with the CD44 antibodies used in this study, murine platelets were incubated with the indicated CD44 antibody followed by appropriate fluorescein isothiocyanate-labeled secondary anti-IgG and assessed for MLFI by flow cytometry. The values are the mean of 4 independent experiments. (D) Mice were injected with 50 μg of the indicated CD44 antibodies to assess the ability of each antibody to induce thrombocytopenia in the absence of antiplatelet antibody. Platelet counting was evaluated before injection and after 24, 48, 72, and 96 hours. n = 6 mice for each group from 2 independent experiments. *P < .05 versus prebleed values. **P < .001 versus prebleed values. (E) C57BL/6 mice (columns 1-4) were treated and bled and platelets counted as in panel A, except that mice were treated with either 50 μg intact KM114 or 37 μg F(ab′)2 KM114 (the equivalent molar concentration). n = 6 mice for each group from 3 independent experiments. Mice expressing human FcγRIIA (columns 5 and 6) were injected with antiplatelet antibody alone or antiplatelet antibody plus KM114 as in panel A and platelets were counted as in panel A. n = 6 mice for each group from 2 independent experiments. (F) Wild-type mice (FcγRIIB+/+) or (G) mice genetically deficient in the inhibitory Fcγ receptor RIIB (FcγRIIB−/−) were injected with 50 mg IVIg, 50 μg anti-RBC monoclonal antibody (TER119), or 50 μg of the indicated CD44 antibodies (KM114 or 5035–41.1D). Twenty-four hours later, all mice were given 2 μg antiplatelet antibody to induce thrombocytopenia. After a further 24 hours, mice were bled for platelet counting. Normal and ITP are described as in panel A. n = 9 mice for each group from 3 independent experiments.

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