Abstract

Autoantibody-opsonized platelets in immune thrombocytopenia (ITP) are thought to be destroyed primarily by macrophage Fc gamma receptor (FcγR)-mediated phagocytosis in the spleen. Blockade of splenic macrophage FcγRs has been proposed as a therapeutic mechanism for ITP intervention. Unfortunately, the contribution of specific FcγRs to disease in ITP remains unknown. Our objective was to determine which FcγRs are responsible for the phagocytosis of ITP autoantibody-opsonized platelets by splenic macrophages. Splenic macrophages were purified by CD14 positive selection from spleens of splenectomized ITP patients, and were treated with blocking antibodies to FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII. Blocking antibodies were deglycosylated to prevent non-specific blocking effects by their Fc region. Two separate ITP sera confirmed positive for anti-GPIIb/IIIa autoantibodies by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to opsonize healthy donor human platelets. Phagocytosis was determined by confocal microscopy and non-phagocytosed (external) platelets were differentiated by an anti-platelet antibody stain following macrophage fixation. Human ITP splenic macrophages were found to express FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII, and expression was not significantly different compared to healthy (trauma) controls (n=5). The two anti-GPIIb/IIIa-positive ITP sera induced a mean 3.7- and 4.2-fold increase of platelet uptake by ITP splenic macrophages relative to normal human serum controls (n=3 each, p<0.001). Blockade of all FcγRs significantly reduced phagocytosis of serum-opsonized platelets for both ITP sera as compared to an IgG control (p<0.001) down to background (non-opsonized) levels. Using single blocking antibodies, inhibition of FcγRI reduced splenic macrophage phagocytosis of platelets by 45% and 37% for the two respective ITP sera as compared to IgG controls (n=3 for each patient sera, p<0.01), while inhibition of FcγRIII reduced phagocytosis of platelets by 43% and 44% (n=3 each, p<0.05). Blockade of FcγRIIa or FcγRIIa/b/c only marginally inhibited splenic macrophage phagocytosis and was not significant. An Fab-like FcγRIII-blocking antibody with a mutant Fc region completely deficient for FcγR binding demonstrated equal FcγRIII blockade compared to the deglycosylated anti-FcγRIII antibody. In comparison to ITP sera-opsonized platelets, FcγRI had a greater involvement in ITP splenic macrophage phagocytosis of anti-D-opsonized human erythrocytes as FcγRI blockade inhibited phagocytosis by 70% (n=5, p<0.001), while FcγRIII blockade inhibited phagocytosis by only 30% (p<0.001) and FcγRIIa or FcγRIIa/b/c blockade had no significant effect. This work demonstrates that FcγRI and FcγRIII are the primary phagocytic receptors on splenic macrophages for anti-GPIIb/IIIa autoantibody-opsonized platelets in human ITP and suggests that FcγRI and FcγRIII are the best targets for FcγR blockade as a potential therapeutic maneuver in the treatment of ITP.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.