Anti-D initially stimulates an Fc-dependent leukocyte oxidative burst and subsequently suppresses erythrophagocytosis via interleukin-1 receptor antagonist

Previous results have demonstrated that anti-D therapy in children with chronic auto-immune thrombocytopenic purpura (AITP) induced a significant increase in several pro- and anti-inflammatory plasma cytokines within 2 hours of administration. To investigate the biologic basis of these early in vivo responses, we developed a flow cytometric assay to measure Fc-dependent responses of human peripheral leukocytes with fluorescently labeled and anti-D–opsonized red blood cells (RBCs). When anti-D–opsonized RBCs were incubated with peripheral blood leukocytes, the earliest detectible event observed was a significant oxidative burst in both monocytes (P < .05) and granulocytes (P < .0001), characterized by the production of hydrogen peroxide (H2O2), peroxynitrite (ONOO–), superoxide (O –2), and hydroxyl (OH) by 10 minutes which declined by 1 hour. By 2 hours, the opsonized RBCs were phagocytosed, particularly by granulocytes (P < .001), but the phagocytosis subsequently declined by 6 hours of incubation. The decline in phagocytosis was correlated with a significant production of interleukin-1 receptor antagonist (IL1ra) by both monocytes (P = .036) and granulocytes (P = .0002) within 4 hours. None of these events occurred if the RBCs were coated with anti-D F(ab)′2 fragments. When recombinant IL1ra was titrated into the assay, phagocytosis of the opsonized RBCs was significantly inhibited (P = .002). Taken together, these results suggest that at least one mechanism of action of anti-D is via the production of the anti-inflammatory cytokine IL1ra which can negatively regulate the ability of leukocytes to phagocytose opsonized cells.