Mechanistic Insights into Redox-Dependent Macropinocytosis in Primary Human Neutrophils

Macropinocytosis is an actin-driven endocytic process mediating nonspecific uptake of extracellular fluid through membrane ruffling. Neutrophil macropinocytosis has been reported only in limited descriptive studies, and its signaling mechanisms have not been defined. Here, we provide a characterization of macropinocytosis in primary human neutrophils and investigate signaling pathways that contribute to the regulation of this process. Quantitative flow cytometry using a high-molecular-weight fluid-phase tracer showed that the diacylglycerol (DAG) mimetic 4β-phorbol 12-myristate 13-acetate (4β-PMA) induces macropinocytic activity in primary human neutrophils. Granulocyte macrophage-colony stimulating factor (GM-CSF) and hepatocyte growth factor (HGF) also promoted fluid-phase uptake. Inhibition of actin polymerization or macropinocytosis reduced tracer uptake, confirming dependence on actin-driven machinery. Scanning electron microscopy revealed dorsal membrane ruffling and cup-like structures after stimulation. Mechanistically, DAG-dependent activation of protein kinase C beta (PKCβ) acted upstream of NADPH oxidase 2 (NOX2)-derived superoxide anion production, driving membrane remodeling. Collectively, these findings define a DAG-PKCβ-NOX2-superoxide signaling axis, as a key regulator of macropinocytosis in primary human neutrophils and provide the first mechanistic framework for its redox-dependent regulation.