Mouse Model Of Immune Thrombocytopenia Research Articles

Lack of a Requirement for the Inhibitory Fcγ Receptor In IVIG-Mediated Treatment of ITP

AbstractAbstract 3338One of the most quoted articles in publications and at International meetings regarding the mechanism of intravenous immunoglobulin (IVIg) therapy in autoimmune and inflammatory diseases is that by Samuelsson et al., Science 291, 484 (2001). This article deals with the mechanism of effect of IVIg being due to an increase in the expression and function of the inhibitory Fcγ receptor (FcγRIIB) in splenic macrophages. The conclusions in this Science paper are based primarily on the use of an experimental mouse model of immune thrombocytopenia (ITP) and mice made deficient in FcγRIIB (FcγRIIB-/- knockouts). We have not been able to support these previous findings but instead find the presence of FcγRIIB receptor not necessary for successful treatment of experimental ITP through IVIg treatment. Administration of IVIg to wild-type Balb/c mice previously made thrombocytopenic using an escalating dose of antiplatelet antibody, MWReg30, leads to amelioration of experimental ITP while in untreated mice, platelet counts stay close to nadir. Similar dynamics of the amelioration of ITP were found using splenectomized or FcγRIIB-/- knockout (Taconic Labs) Balb/c mice. However, as previously published (Blood 15, 558 (2003)), IVIg does not work with FcγRIIB-/- knockout mice that are on a mixed B6:129S4 background, obtained from the Jackson Labs. Indeed, B6 (129S4-Fcgr2btm1Rav/J) FcγRIIB-/- knockout mice made thrombocytopenic are completely unresponsive to IVIg treatment. However, surprisingly, when using the recommended control wild-type mice for this knockout, the B6.129SF2 mouse, we found that this wild-type FcγRIIB+/+ mouse also does not respond to IVIg treatment. Confirmation of genotype was done; thus, we suggest that something about the 129S background prevents a response to IVIg therapy and this phenomenon is independent of the FcγRIIB. We have confirmed that wild-type 129S4 mice made thrombocytopenic do not respond to IVIg. We have also examined the B6 FcγRIIB-/- knockout mice from Taconic Labs which are purported to be fully congenic and these mice also do not respond to IVIg. Because of the lack of response to IVIg of wild-type 129S4 mice, we believe that the Taconic B6 FcγRIIB-/- knockout mice may, in fact, be mixed background as are the Jackson mice. Thus, we plan to use SNP protocols for determination of congenicity, comparing C57BL/6 and 129S4 strains to Taconic and Jackson Labs B6 FcγRIIB-/- knockout mice. We expect to show that both B6 FcγRIIB-/- knockout animals are not fully congenic, being a mixture of both C57BL/6 and 129S4 strains. Previous publications that indicated a lack of response to IVIg using FcγRIIB-/- knockout mice either did not test the fully congenic Taconic Balb/c knockouts and/or failed to use the most suitable control animals with the nonfully congenic B6 knockouts, using instead, C57BL/6 wild-type mice which respond well to IVIg treatment. In order to avoid misinterpretation of results, all experiments testing the role for FcγRIIB in the mechanism of IVIg should be done using Taconic Balb/c FcγRIIB-/- knockout mice until congenicity can be established with the B6 knockout animals. These data taken together lead us to the conclusion that the presence of the FcγRIIB receptor is not important for the therapeutic action of IVIg. Disclosures:No relevant conflicts of interest to declare.

Improved mouse models for the study of treatment modalities for immune‐mediated platelet destruction

We found when using a mouse model of immune thrombocytopenia (ITP) that platelet (PLT) nadir could not be maintained in the face of daily PLT antibody, making interpretation of treatment modalities difficult. This finding was documented to be at least in part due to increased thrombopoiesis as a result of a compensated thrombocytolytic state. Thus, it was important to develop an improved mouse model of human ITP so as to maintain PLT nadir over time. To maintain PLT nadir, we have developed two mouse models. One model uses single-dose sublethal total body gamma irradiation (TBI) in combination with daily low-dose PLT antibody administration while the second model uses escalation of the dose of PLT antibody over time. Both models maintain PLT nadir and allow for the study of treatment modalities without interference by marrow compensation. Surprisingly, intravenous immune globulin (IVIG) shows no efficacy when using the TBI combination model but works well using the dose-escalation mouse model. In contrast, anti-TER-119 shows efficacy using either mouse model. Our results indicate that the mechanism of action of IVIG requires a functional marrow and/or involves a radiosensitive regulatory cell. However, IVIG works using the dose-escalation model without TBI and the increase in PLT counts correlates directly with reticulated PLTs suggesting that the IVIG mechanism involves effects on megakaryopoiesis/thrombopoiesis. These mouse models should be useful for investigators wishing to maintain PLT nadir over prolonged periods of time for the study of mechanism and efficacy of various treatments for ITP.

Mannose Labeled Liposome Containing DXR Administration Is Effective in Reversing Thrombocytopenia in the Model Mouse of Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is classified as an autoimmune disease in, which antibody-coated platelets are phagocytosed by macrophages in the reticuloendothelial system (RES) through Fc gamma receptor-mediated or complement-mediated pathways. Based on the the pathophysiology of ITP, described above, and evidences that macrophages expresses mannose receptor on their surface and that mannose-terminated human glucocerebrosidase is highly effective in ameliorating many of the clinical manifestations of Gaucher's disease, we devised mannose labeled liposome containing DXR to eliminate specifically macrophages. In this study, we evaluated the effects of mannose labeled liposome containing DXR (liposome-DXR) in a mouse model of ITP.1.In vitro experiments: Murine peritoneal macrophages were obtained by the intra-peritoneal injection of thioglycolate (TGC) to ddY mice.a.The uptake of fluorescence by peritoneal macrophages was measured using rhodamine-liposome by flow cytometry. The macrophages were incubated with mannose labeled rhodamine-liposome {mannose(+)-rhodamine-liposome} or mannose unlabeled rhodamine-liposome {mannose(−)-rhodamine-liposome} for 30 min at 37°C. After washing with PBS, they were applied for flow cytometry. Rhodamine fluorescence intensity in macrophages was higher in incubation with mannose(+)-rhodamine-liposome than in incubation with mannose(−)-rhodamine-liposome treatment.b.The viability of peritoneal macrophages was measured by MTT assay after 30 min incubation with liposome-DXR. The viability in the incubation with mannose labeled liposome-DXR {mannose(+)-liposome-DXR} was much less than in the incubation with mannose unlabeled liposome-DXR {mannose(−)-liposome-DXR} (Figure 1).In vivo experiment: Anti-mouse platelet serum (APS) was injected intra-peritoneally to ddY mice at 24 hr after intravenous injection of mannose(+)-liposome-DXR or mannose(−)-liposome-DXR.Platelet counts were measured at 12 hr after APS injection. They were 89.3±22.1 × 104 /μl (n=6, p<0.01) in mannose(+)-liposome-DXR treatment, while 4.5±2.0 × 104 /μl (n=8) in mannose(−)-liposome-DXR treatment (Figure 2). The platelet counts were 3.2±0.5 × 104 /μl (n=4), 96.1±15.0 × 104 /μl (n=4) or 99.3±26.2 × 104/μl (n=4) in mice with only APS administration, mannose(+)-liposome-DXR+saline or mannose(−)-liposome-DXR+saline administrations instead of APS, respectively. These in vivo data indicated that APS induced thrombocytopenia was not observed in mice treated with mannose(+)-DXR-liposome, because of the destruction of macrophages by mannose(+)-DXR-liposome administration. These in vitro and in vivo data strongly suggest that mannose(+)-liposome-DXR may specifically delete the macrophages and and can be effective in the management of experimental ITP model mice. [Display omitted] [Display omitted]

Pharmacokinetic/Pharmacodynamic Modeling of IVIG Effects in a Murine Model of Immune Thrombocytopenia

IVIG may achieve its beneficial effects in immune thrombocytopenia (ITP) patients via several mechanisms; however, little is known of the relative importance of various mechanisms associated with IVIG action in ITP. The purposes of this study were to develop a pharmacokinetic/pharmacodynamic (PK/PD) model relating an anti‐platelet antibody, MWReg30, to platelet counts in a mouse model of sustained ITP, to use modeling to characterize effects of IVIG on MWReg30 elimination, and to use PK/PD modeling to assess the contribution of IVIG effects on MWReg30 disposition to the effects of IVIG on MWReg30‐induced thrombocytopenia in mice. A pharmacokinetic model, based on the competitive occupancy of protective FcRn receptors, was used to characterize the effects of IVIG on MWReg30 pharmacokinetics. The relationships between MWReg30 plasma concentrations to MWReg30‐induced thrombocytopenia, in the presence and absence of IVIG treatment, were characterized using an indirect response model. The pharmacokinetic model well‐captured MWReg30 plasma concentration‐time profiles, with and without administration of IVIG. The indirect response model accurately characterized the effects of IVIG on MWReg30‐induced thrombocytopenia in mice. Using these models, it was estimated that 43 ± 5% of overall effects of IVIG on MWReg30‐induced thrombocytopenia in mice could be accounted for by the IVIG‐mediated increases in MWReg30 clearance.

Macrophage depletion following liposomal-encapsulated clodronate (LIP-CLOD) injection enhances megakaryocytopoietic and thrombopoietic activities in mice.

Megakaryocytopoiesis is the cellular process by which stem cells progress through commitment, proliferation and differentiation, leading to the production of platelets. In the mouse, this process is accomplished within the bone marrow (BM) and spleen microenvironment and is carried out by regulatory molecules and accessory cells, including macrophages, fibroblasts and endothelial-like cells. Previously, we demonstrated that specific macrophage depletion, using liposomal-encapsulated clodronate (LIP-CLOD), induced a rapid recovery of the platelet count in a mouse model of immune thrombocytopenia. We now show that LIP-CLOD treatment also provoked enhancement of both megakaryocytopoiesis and thrombocytopoiesis. In fact, a dose-dependent increase in the number of BM and spleen megakaryocytes was detected after treatment and this pattern correlated inversely to the macrophage count detected in these organs. Furthermore, the mice treated with the higher dose of LIP-CLOD showed signs of enhanced thrombopoiesis as they had an increased frequency of reticulated platelets and an improvement in the total platelet count 2 d later. In addition, the in vitro cytokine-induced megakaryocytopoiesis in BM and spleen cell cultures was significantly augmented in the presence of LIP-CLOD. Taken together, these results suggest that BM and spleen microenvironmental macrophages could be involved in the regulation of megakaryocyte and platelet production.

Rapid recovery of platelet count following administration of liposome-encapsulated clodronate in a mouse model of immune thrombocytopenia

Immune thrombocytopenic purpura (ITP) is a haematological disorder characterized by increased platelet consumption. The destruction of platelets is mediated by the reticulo-endothelial system (RES), particularly by splenic and hepatic macrophages. Previously, we demonstrated in a mouse model of thrombocytopenia that the depletion of these cells by liposome-encapsulated clodronate (LIP-CLOD) induces the recovery of the platelet count. We now report that LIP-CLOD is capable of reversing the thrombocytopenia with minimal effects on both, functional RES integrity and platelet functionality. Our data indicate that thrombocytopenic mice treated with low doses of LIP-CLOD/body weight increase the platelet count to haemostatically safe values within 18 h of treatment. The predictable bleeding time was significantly decreased in these mice, suggesting that the circulating platelets have enhanced haemostatic capacity. Platelet functionality measured through the ADP-induced fibrinogen-binding assay showed normal platelet activation after treatment. Regarding immunological competence, mice treated with LIP-CLOD showed similar antibody titres against sheep red blood cells. However, antibody-dependent cell-mediated cytotoxicity carried out by splenocytes was reduced. All these data demonstrate that LIP-CLOD deserves consideration as a potential therapeutic approach in thrombocytopenic states in which the rapid increase of platelet count is the primary goal.

Rapid recovery of platelet count following administration of liposome-encapsulated clodronate in a mouse model of immune thrombocytopenia

Rapid recovery of platelet count following administration of liposome-encapsulated clodronate in a mouse model of immune thrombocytopenia