Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation.

Pember SO, Barnes KC, Brandt SJ, Kinkade JM
Blood. 1983 61 (6): 1105-15

PMID: 6839018

When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10-1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085-1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID-PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating "activated" from "resting" PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.

MeSH Terms (18)

Animals Ascitic Fluid Cell Aggregation Cell Fractionation Centrifugation, Density Gradient Chemotactic Factors Chemotaxis, Leukocyte Dose-Response Relationship, Immunologic Female Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mice, Inbred Strains N-Formylmethionine N-Formylmethionine Leucyl-Phenylalanine Neutrophils Oligopeptides

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