Background

Clearance of anti-HPA-1a antibody-opsonized fetal platelets by phagocytes represents the major mechanism of fetal and neonatal alloimmune thrombocytopenia. According to the general idea, opsonized fetal platelets are cleared in the spleen by tissue-resident macrophages via Fc-dependent mechanism. However, previous studies indicated the role of blood phagocytes on platelet clearance. In this study, anti-HPA-1a mediated platelet phagocytosis by blood phagocyte subsets was investigated.

Methods

Platelet phagocytosis caused by anti-HPA-1a standard sera was analyzed by Whole Blood Platelet Phagocytosis Assay (WHOPPA) using pH sensitive dye (pHrodo-SE) labeled platelets. PHrodo opsonized platelets were incubated with whole blood and the rates of platelets engulfed by neutrophils and monocyte subsets were measured by flow cytometry using gating strategy as previously described (Fujimoto et al., 2000). The expression of FcγRs and HLA-DR on neutrophils and monocytes subsets was analyzed using anti-FcγRI, -II, III and anti-HLA-DR specific monoclonal antibodies.

Results

Anti-HPA-1a mediated platelet phagocytosis was only observed in monocytes, but not in neutrophils. In the control experiments, monocytes and neutrophils engulfed pHrodo labelled E. coli bacteria. Analysis of monocyte subsets showed that not only the most abundant conventional monocytes (CD16-; around 85%), but also other monocytes subsets (CD16+; around 12%) including non-classical, intermediate monocyte subsets were able to engulf anti-HPA-1a opsonized platelets. Interestingly, shifted monocyte subset (around 5%) with high phagocytosis rate was found. Furthermore, analysis of FcγRs and HLA-DR on monocytes showed significant decreased of FcγRI and increased of HLA-DR surface expression.

Conclusion

In this study, we showed by WHOPPA that monocyte subsets are the most dominant phagocytes in blood cells responsible for the clearance of anti-HPA-1a opsonized platelets in vitro. The significant expression changes of FcγRI and HLA-DR, formation of previously unknown shifted monocyte subset underline the importance of FcγRI and FcγRIII in platelet phagocytic process and subsequent immune response. This assay may allow us to study on antibody mediated platelet phagocytosis in ex vivo conditions.

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