Amelioration Of Immune Thrombocytopenia Research Articles

GM-CSF and IL-4 are not involved in IVIG-mediated amelioration of ITP in mice: a role for IL-11 cannot be ruled out.

Previously, we have reported that interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11, but not IL-33, are up-regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL-4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL-4 and, in addition, GM-CSF and IL-11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL-4 receptor knock-out (IL-4R-/- ), IL-11 receptor knock-out (IL-11Rα-/- ) and GM-CSF knock-out (Csf2-/- ) mice. We also used a neutralizing antibody to IL-11 and recombinant human IL-11 (rhIL-11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL-4 and GM-CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL-11 in these processes was less clear. Even though IL-11Rα-/- mice with ITP responded to IVIg similarly to wild-type (WT) mice, treatment of ITP WT mice with rhIL-11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti-IL-11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL-4, basophils or GM-CSF have roles in IVIg amelioration of ITP; however, a role for IL-11 requires further study.

Amelioration of Immune Thrombocytopenia in Human CD44 Transgenic Mice By Anti-Human CD44 Antibodies

Selected monoclonal antibodies to murine CD44 are able to successfully ameliorate murine immune thrombocytopenia (ITP) and other inflammatory diseases at a 3 log fold lower dose than IVIg. Since not all murine CD44 antibodies were successful and, like IVIg, the anti-inflammatory mechanism involved is speculative, progress toward human antibodies has been stymied. To potentially develop a therapeutic antibody for patients, we generated transgenic mice expressing the extracellular and transmembrane sequences of human CD44 linked to the murine intracellular CD44 sequence (huCD44) with knockout of murine CD44. Flow cytometric analysis revealed that these mice express huCD44 on leukocytes, but not on platelets or red blood cells.HuCD44 mice injected with the anti-platelet antibody MWReg30 develop significant thrombocytopenia comparable to normal mice. Utilizing the huCD44 mouse model, we tested 5 monoclonal antibodies reactive with human CD44 of different isotypes; mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG2a, and rat IgG2b. We report here that pretreatment of mice with all of these antibodies, with the exception of the mouse IgG2b clone all ameliorated thrombocytopenia to the same extent as IVIg, but given at a 3 log-fold lower dosage than IVIg. Interestingly, the mouse IgG2b antibody that was not successful bound to white blood cells as well as the successful antibodies. When mice were re-injected with anti-platelet antibody 24 hr post anti-huCD44 antibody injection, they were still protected from thrombocytopenia at 48 hr, suggesting that these antibodies exhibit durable protection.The clinical effectiveness of these antibodies does not appear to be related to their extent of huCD44 binding or IgG subtype. In addition, the effectiveness of these antibodies may not be restricted or related to specific activating IgG Fc receptors (FcγR) as the successful mouse IgG1 subtype is only known to interact with FcγRIII, while the IgG2b subtype can bind activating FcγRI, III and IV with only one-of-two of this subtype having therapeutic activity.In conclusion we have developed, to our knowledge, the first huCD44 transgenic mouse and demonstrate herein that selected antibodies to huCD44 can ameliorate immune thrombocytopenia. These data suggest that some anti-CD44 antibodies may offer a new therapeutic modality for patients with ITP. DisclosuresNo relevant conflicts of interest to declare.

Unraveling the IVIG mystique

Unraveling the <scp>IVIG</scp> mystique

Amelioration of Murine Immune Thrombocytopenia by CD44 Antibodies: a Potential Therapy for ITP?

AbstractAbstract 2528Intravenous immunoglobulin (IVIg) is used to treat autoimmune diseases such as ITP. IVIg is a limited resource and its dosage and cost are both high. Although considered safe, it will always carry a theoretical risk of transferring infectious disease. Thus it would thus be highly desirable to improve the efficacy of IVIg or develop monoclonal antibodies, capable of mimicking the clinical effects of IVIg. Work by others has successfully improved the efficaciousness of IVIg in murine models of autoimmunity: It has been shown that enriched sialylated IVIg has a therapeutic effect at a 1 log fold lower dosage than IVIg; and a sialylated recombinant Fc fragment of IgG functions successfully at a 2 log fold lower dosage than IVIg.CD44 is a widely expressed cell surface polymorphic glycoprotein. It is involved in many processes including tumour metastasis and inflammation. CD44 antibodies have been successfully used to treat several murine autoimmune disease models, including inflammatory arthritis and experimental autoimmune encephalomyelitis, although their mechanism of action remains unclear. To explore the potential for monoclonal antibodies as a treatment for ITP as well as further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP, and found 4 antibodies which could successfully ameliorate ITP; 2 of these antibodies function at a full 3 log fold lower dosage as compared to IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg, i) the expression of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function and ii) complement-depleted mice also responded to anti-CD44 treatment. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required: an F(ab')2 fragment of antibody KM114 also significantly ameliorated thrombocytopenia at an equivalent molar concentration as intact KM114. Thus while KM114 functions by an FcγRIIB sensitive mechanism, FcγRIIB is unlikely a direct target of the Fc region of this antibody and likely plays a downstream role in the amelioration of immune thrombocytopenia, similar to IVIg. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and we speculate that they could potentially provide a very low dose recombinant therapy for the treatment of ITP. Disclosures:No relevant conflicts of interest to declare.

Monoclonal IgG Specific for the CD44 Antigen Mimics the Action of IVIg in the Amelioration of ITP.

Previous work in our laboratory has shown that both IVIg and “anti-D like” erythrocyte-reactive antibodies ameliorate immune thrombocytopenia (ITP) in a murine model. However, they appear to function through different mechanisms: IVIg is dependent upon the inhibitory Fc receptor, FcγRIIB in the amelioration of ITP, but the anti-erythrocyte antibodies function independently of FcγRIIB expression. We have also demonstrated that anti-erythrocyte antibodies down-modulate the expression of the activating Fc receptor, FcγRIIIA in splenic macrophages during the amelioration of ITP. A monoclonal antibody to CD44, cell surface protein involved in cell homing, cancer progression and inflammation, can also ameliorate murine ITP. Elucidation of the mechanisms of action of anti-CD44 antibodies in the amelioration of ITP would be critical to the successful application of this potential therapy. In this report, two monoclonal antibodies against CD44, KM114 and IM7 were studied in C57BL/6 and FcγRIIB−/− mice to assess their therapeutic activity in treating ITP. Both KM114 and IM7 bound well to splenic cells from all mice tested. Neither KM114 nor IM7 demonstrated any in vitro anti-idiotypic activity which could neutralize the binding of the thrombocytopenia-inducing anti-platelet antibody. At the dose that protected against thrombocytopenia, KM114 did not mediate RES blockade. In contrast, IM7, which did not at all protect against thrombocytopenia, blocked RES. Since FcγRIIB has been documented to play a key role in the function of IVIg in the amelioration of ITP, we questioned the significance of it in the function of KM114. Surprisingly, KM114 was not able to ameliorate ITP in mice genetically deficient in FcγRIIB. These results suggest that, like IVIg, monoclonal antibody against CD44 mediates amelioration of murine thrombocytopenia in a manner dependent upon the inhibitory FcγRIIB rather than via RES blockade.