High-Dose IgG Alters the Relative Expression of Fcgamma RIIA and Fcgamma RIIB On Human Macrophages: A Mechanism for IVIG Therapy in Human Immune Thrombocytopenia.

Antibody-mediated phagocytosis of platelets by THP-1 (human macrophage) cells in culture was assayed by co-incubation of THP-1 cells with anti-platelet antibody as a model for human immune mediated platelet destruction. Platelets were labeled with 5-(and 6-) carboxyfluorescein diacetate mixed isomers (CFDA) in the presence of human serum containing anti-HPA 1a IgG antibody. Macrophages that had ingested human platelets were identified by flow cytometry as previously reported. To study the mechanism of action of IVIG therapy for immune thrombocytopenia, in vitro phagocytosis was assayed after the addition of purified IgG (100mg/ml) to the THP-1 cells 30 minutes prior to exposure of the labeled platelets to anti-HPA 1a IgG antibody. The effect of LPS on platelet phagocytosis was studied as a model for infection which can exacerbate immune platelet destruction. Phagocytosis was assayed before and after incubation with LPS (1ug/ml) for 2 hours at 37 degrees Celsius. For each experiment, total FcgammaRIIA (CD32A) and FcgammaRIIB (CD32B) were determined by immunoprecipitation and immunoblotting.

Pretreatment with IVIG had no effect on FcgammaRIIA (n=3) expression; however, FcgammaRIIB expression increased an average of 5 fold (n=6). Addition of LPS alone resulted in marked decrease in expression of FcgammaRIIB for up to 60 minutes; however, FcgammaRIIA did not change. Experiments performed with addition of both IVIG and LPS showed that FcgammaRIIB expression remained elevated 3 fold (n=3). Following increased expression of FcgammaRIIB, IgG exposure resulted in decreased phagocytosis as assayed by flow cytometric analysis of macrophage ingestion of CFDA-labeled platelets. Antibody mediated platelet phagocytosis by THP-1 cells was enhanced 26% by LPS after a 2 hour incubation (median increase in 5 experiments; range = 14-125%.) Conclusions: Exposure to high dose IgG increased the expression of FcgammaRIIB but had no effect on FcgammaRIIA. Therefore, the net functional effect was to tip the balance toward inhibition of antibody-mediated phagocytosis, as was observed in our in vitro assay of human platelet phagocytosis. Furthermore, treatment with IgG was able to partially overcome the negative effects of LPS on expression of the inhibiting receptor, FcgammaRIIB. These data clearly support the hypothesis that the balance of the activating (FcgammaRIIA) and inhibiting (FcgammaRIIB) receptors is important in mediating the therapeutic effects of IVIG as a treatment for human immune thrombocytopenia.

Pels:National Hemophilia Foundation-Baxter: NHF-Baxter Clinical Fellowship Awardee, Research Funding. Beardsley:ITP Foundation: Membership on an entity's Board of Directors or advisory committees; Genzyme: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees.