Activation Of Human Polymorphonuclear Neutrophils Research Articles

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.

Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases

Cystatin C, a cysteine proteinase inhibitor has recently been suggested to be a potent regulator of inflammatory processes and may act in defense against viral and bacterial infections. Two common forms of the protein were purified from the urine of a patient having received a renal transplant. The slow form of cystatin C possessed the N-terminal tetrapeptide Lys Pro Pro Arg, which was cleaved in the fast form. This peptide sequence, called postin, was synthesized. The three molecules, slow and fast forms of cystatin and the synthetic peptide, were tested for their effects on the migration activity of human polymorphonuclear neutrophils (PMNs). The slow form was found to display both chemotactic and chemokinetic activities, while the fast form and postin were only chemokinetic. Nevertheless, all the substances could induce a "motile" morphology. In addition, the two forms of cystatin C were powerful inhibitors of PMN chemotaxis induced by complement-derived chemotactic factors. This suggests that cystatin C in its two different cleaved forms and the N-terminal tetrapeptide can modulate PMN locomotion. Cysteine proteases may therefore play a role in neutrophil migration activity.

Neutrophil cytoplasts: relationships of superoxide release and calcium pools

Activation of human polymorphonuclear neutrophils (PMNs) by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) leads to a transient increase in intracellular level of ionized calcium and an alteration of the plasma membrane permeability. Calcium has been proposed as a second messenger for activation of the PMN. Modulation of intracellular pools of calcium is of importance in the regulation of PMN activation. We have studied the changes in membrane-bound and cytoplasmic calcium in PMN and PMN devoid of granules and nucleus by quantifying changes in chlorotetracycline (CTC) and Quin 2 fluorescence and comparing their relation to O(2) release. Similar to PMN, PMN cytoplasts (PMN-CPs) produce equivalent amounts of O(2) in response to 10(-7) mol/L fMLP. The decrease in CTC fluorescence following fMLP stimulation is not significantly different in PMN-CP (-9.9% +/- 3.7%) from that observed in PMN (-12.7% +/- 2.33%), suggesting that the trigger pool of Ca++ is present in PMN-CPs. Although PMNs show a net increase in free Ca++ as measured by Quin 2, PMN-CPs display a lower sustained rise, which is totally abolished in the absence of external Ca++. PMN-CPs release O(2) efficiently in the absence of external Ca++ when stimulated with 10(-7) mol/L fMLP, whereas PMNs release significantly less O(2) under the same conditions. Our results suggest that a rapid rise in free Ca++, as monitored by Quin 2 fluorescence, is not required for expression of full activation of the oxidase system and release of O(2) from PMN-CPs.

Neutrophil Cytoplasts: Relationships of Superoxide Release and Calcium Pools

Activation of human polymorphonuclear neutrophils (PMNs) by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) leads to a transient increase in intracellular level of ionized calcium and an alteration of the plasma membrane permeability. Calcium has been proposed as a second messenger for activation of the PMN. Modulation of intracellular pools of calcium is of importance in the regulation of PMN activation. We have studied the changes in membrane-bound and cytoplasmic calcium in PMN and PMN devoid of granules and nucleus by quantifying changes in chlorotetracycline (CTC) and Quin 2 fluorescence and comparing their relation to O(2) release. Similar to PMN, PMN cytoplasts (PMN-CPs) produce equivalent amounts of O(2) in response to 10(-7) mol/L fMLP. The decrease in CTC fluorescence following fMLP stimulation is not significantly different in PMN-CP (-9.9% +/- 3.7%) from that observed in PMN (-12.7% +/- 2.33%), suggesting that the trigger pool of Ca++ is present in PMN-CPs. Although PMNs show a net increase in free Ca++ as measured by Quin 2, PMN-CPs display a lower sustained rise, which is totally abolished in the absence of external Ca++. PMN-CPs release O(2) efficiently in the absence of external Ca++ when stimulated with 10(-7) mol/L fMLP, whereas PMNs release significantly less O(2) under the same conditions. Our results suggest that a rapid rise in free Ca++, as monitored by Quin 2 fluorescence, is not required for expression of full activation of the oxidase system and release of O(2) from PMN-CPs.

Trichomonacidal activity of human polymorphonuclear neutrophils: killing by disruption and fragmentation.

Polymorphonuclear neutrophils (PMNs) were shown to kill Trichomonas vaginalis in vitro; 10(2)-10(3) trichomonads were incubated with 3 x 10(6) PMNs on tissue culture plates, and surviving organisms were enumerated in pour plates. After 60 min of aerobic incubation at 37 C, 100% (+/- 0) of the trichomonads had been killed, and nitroblue tetrazolium was reduced at the interface between the PMNs and trichomonads. The importance of oxidative microbicidal systems was confirmed by the observations that only 12% +/- 12% of trichomonads were killed under anaerobic conditions and that aerobic killing was eliminated by the addition of catalase or superoxide dismutase. PMNs killed trichomonads in fresh or absorbed serum but not in bovine serum albumin, in heat-inactivated serum, or in the presence of 1 mM trypan blue; this finding suggested a role for alternative pathway activation of complement. Phase-contrast cinemicrography and electron microscopy revealed the pursuit and surrounding of individual trichomonads by groups of PMNs that were able to fragment the large protozoa and to phagocytize the pieces.

Influence of type of ingested particle on human leukocyte metabolism.

SummaryThe metabolic activity of human polymorphonuclear neutrophils (PMN) was studied following the ingestion of polystyrene latex spheres (1.08 μ), heat-killed S. aureus (0.8 μ) or heat-killed Micrococcus sp. (1.2 μ). Phagocytsois of heat-killed bacteria resulted in a greater postphagocytic increase in oxygen consumption and hydrogen peroxide production than did phagocytosis of latex particles. These data suggest that the nature of the ingested particles has an influence on postphagocytic metabolic events.