Flow cytometric analysis of defensins in blood and marrow neutrophils

Polymorphonuclear neutrophils (PMN) are vital in host defense against micro bial infections. This study provides a flow cytometric method for the quant itative analysis of microbicidal peptides (defensins) in cells of PMN linea ge. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing I-selectin. PMN lineage counts were ma de on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alka line phosphatase technique. The results show that marrow PMN express higher levels of NP-2 and NP-5 than blood PMN, p < 0.001 and that these levels ar e associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence in tensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most I-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surfa ce. Furthermore, defensins are not detected in monocytes, eosinophils, lymp hocytes and erythrocytes. The how cytometric method described here provides a novel means of quantitating host natural defenses, allows the characteri zation of PMN subpopulations and has clinical applications.