Age-related changes in the calcium homeostasis of adherent neutrophils.

The elderly are susceptible to infections and show a decline in neutrophil (PMN) functions that are regulated by cytosolic calcium [Ca2+]i. This study measures [Ca2+]i in suspended and adherent PMN of young and elderly individuals by using flow cytometry, confocal microscopy, the bacterial peptide fMLP, and the fluorescent Ca2+ indicator fluor-3/acettoxymethyl ester. PMN from both age groups show a steep and transient fMLP-induced Ca2+ increase. This increase is independent of external divalent cations and is desensitized by a subsequent exposure to the same agonist. Adherent PMN of the elderly express elevated [Ca2+]i before (basal) and after fMLP activation but show reduced ability to mobilize Ca2+ into and from the cytosol. PMN of the elderly take longer (13.7 +/- 3 s) to attain the maximal response compared to those of young adults (5.7 +/- 0.8 s). PMN from both age groups show heterogeneity in the time and magnitude of this response. However, PMN of the elderly show a decrease in the proportion of cells with prompt and effective reaction and an increase in the representation of a cell subpopulation manifesting delayed response. We conclude that age-related delayed and reduced PMN response to a bacterial peptide could hamper functional activities that are essential in host protection against infections.

Dynamic changes in neutrophil defensins during endotoxemia.

Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis. This study determines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes (PMN) in rabbits treated with LPS. PMN functional activity was assessed by measuring CD18 expression and H(2)O(2) production and by examining the lungs. NP-2 and, to a minor extent, NP-5 of circulating PMN increase during endotoxemia. This early increase is concomitant with neutrophilia and elevated CD18 expression and H(2)O(2) production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels. A decline in defensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMN and enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage. This study shows that LPS-induced changes in the defensins of circulating PMN correlate with the number and activated condition of these cells and suggests that PMN-derived products implement the inflammatory reaction that leads to lung injury and sepsis.

Flow cytometric analysis of defensins in blood and marrow neutrophils.

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.

Activation-associated changes in blood and bone marrow neutrophils.

Accelerated granulocytopoiesis and polymorphonuclear leukocyte (PMN) activation via the complement system are important events in the inflammatory response. This study tests the hypothesis that PMN of the peripheral blood and the bone marrow behave differently when stimulated with zymosan-activated plasma (ZAP). PMN were treated with ZAP and processed to determine the content and distribution of F-actin, the expression of CD18-integrins, and the cell area/shape with the use of a combination of flow cytometry, fluorescence, and light microscopy. The results show that untreated bone marrow PMN display similar F-actin content and cell area but express less CD18 than peripheral blood PMN. ZAP-activated peripheral blood PMN show a marked increase in the F-actin content, CD18 expression, cell area, and length. Highly elongated PMN develop a polar shape in which microfilaments are redistributed in cell protrusions and CD18 is located in the uropod and the lamellipodium. In comparison, activated bone marrow PMN show a lower and more transient increase in the F-actin content, express less CD18, show a reduced increase in the cell area, and display reduced polarity and microfilament redistribution. We conclude that bone marrow PMN show lower complement-activation response than peripheral blood PMN and suggest that the lower expression of surface membrane integrins and related cytoskeletal filaments may account for their reduced ability to develop the polar shape required for diapedesis and migration.

Neutrophil activation and lung injury associated with chronic endotoxemia in rabbits.

Chronic endotoxemia produces emphysematous lung destruction in several animal models. The present study was designed to examine changes in the polymorphonuclear leukocytes (PMN) and the lung parenchyma of rabbits that received either saline (control, n = 6) or Escherichia coli endotoxin (LPS, n = 6) 2-3 times weekly for 15 to 28 weeks. Peripheral blood was collected just before and after each intravenous injection and lung tissue was processed at the end of the experiment. PMN myeloperoxidase was stained with diaminobenzidine tetrahydrochloride (DAB)-H2O2, and CD11/CD18 was detected with immunogold. The changes in the PMN and the lung parenchyma were quantitatively analyzed. The results show that each dose of LPS produced an initial fall, followed by a rise in the circulating mature and immature PMN cell counts. Repeated doses of LPS induced PMN activation, degranulation, and an increase in the mean thickness of the alveolar wall (control, 4.1 +/- 0.2; LPS, 5.1 +/- 0.5; p < .05) at 28 weeks without evidence of alveolar septa destruction. Morphometric analysis of intravascular PMN showed an increase in the volume (V) of myeloperoxidase-containing azurophil granules (control, 6.1 +/- 1.3 microns3; LPS, 13.1 +/- 2.8 microns3; p < .05); a trend for a decrease in the V of specific granules (control, 15.8 +/- 3.4 microns3; LPS, 10.2 +/- 1.5 microns3; p = .09); an increase in the V of the cytoplasm (control, 37.3 +/- 6.4 microns3; LPS, 54.5 +/- 7.1 microns3; p < .05); and an increase in CD11/CD18 expressed as the number of gold particles per micrometer of cell surface membrane (G/micron) (control, 7.1 +/- 1.4 G/micron; LPS, 18.1 +/- 7.8 G/micron; p < .05). The results indicate that chronic endoxemia in rabbits, accelerates the release of PMN from the bone marrow, enhances the retention of both mature and immature activated PMN in the pulmonary microvessels, and causes alveolar wall thickening rather than emphysematous lung destruction.