Flow cytometric analysis of defensins in blood and marrow neutrophils

Abstract

Polymorphonuclear neutrophils (PMN) are vital in host defense against microbial infections. This study provides a flow cytometric method for the quantitative analysis of microbicidal peptides (defensins) in cells of PMN lineage. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing 1-selectin. PMN lineage counts were made on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alkaline 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 are associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence intensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most 1-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surface. Furthermore, defensins are not detected in monocytes, eosinophils, lymphocytes and erythrocytes. The flow cytometric method described here provides a novel means of quantitating host natural defenses, allows the characterization of PMN subpopulations and has clinical applications.

C‐reactive protein and leucocyte activation in psoriasis vulgaris according to severity and therapy

“…Therefore, we calculated the ratios of elastase/neutrophil and lactoferrin/neutrophil and found that both were significantly increased compared with controls, suggesting that neutrophil degranulation was hyperstimulated and/or the granule content was enriched, turning neutrophils more prepared to inflammatory response. A stimulated granulocytopoiesis is usually associated with more immature circulating neutrophils, containing a higher number of elastase containing granules 29,30 . The release of these neutrophil activation products has to be counterbalanced by well‐known endogenous systems to reduce or to avoid the inflammatory process.…”

Systemic upregulation of neutrophil -defensins and serine proteases in neutrophilic asthma

“…30 Circulating mature neutrophils contain substantial amounts of the formed mature peptides, but are no longer transcribing their mRNA. 31 High levels of defensins in marrow neutrophils are associated with a high ratio of immature to mature neutrophilic granulocytes, 32 suggesting that the upregulation of defensin and protease mRNA expression in NA may represent the presence of immature neutrophils in the circulation. Under normal conditions granulocyte precursors do not leave the bone marrow.…”

Dynamic Changes in Neutrophil Defensins during Endotoxemia

“…CHANGES IN PMN DEFENSINS DURING ENDOTOXEMIA 7797 (16). In the present study, a rise in defensins is likely due to maturational and/or conformational changes in the peptide precursors of less mature PMN.…”

“…Cells were then labeled (1 h) with a 1/50 dilution from stock of fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Sigma). After fixing with 0.8% PFA, and to exclude cell aggregates, cells were stained (15 min) with 4 g of propidium iodide per ml (16). The mean fluorescence intensity of 5,000 to 30,000 cells was measured using analysis gates for PMN in a flow cytometer (Epics XL; Beckman Coulter, Inc.).…”

“…Defensins are known to mediate inflammation (40) and cause damage to endothelial (29), epithelial (23,24), and tumor (20) cells. We have previously shown that immature marrow PMN contain elevated levels of NP-2 and NP-5 (16), and it has been shown that PMN transit time through the marrow decreases with bacterial infection (34,37). The present study was designed to extend these observations by determining the level of defensins and the functional activity of circulating PMN during endotoxemia.…”