Assembly dynamics of actin in adherent human neutrophils.

We have extended our previous studies of adherent neutrophils and compared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F-actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treatment effectively inhibited the depolymerization of F-actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G-actin levels 75-80% in suspended cells, 35-73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fixed, there were highly significant differences in G-actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G-actin with a corresponding decrease in F-actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization occurred by 1 min and, thereafter, G-actin decreased and F-actin increased reaching a steady state at 5 min. Adhesion to both laminin- and fibronectin-coated surfaces was accompanied by an increase of [Ca2+]i with a peak at 3 min, followed by a decrease from 3-5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+]i in laminin-adherent cells was about twice that in fibronectin-adherent cells at 3 min (P < 0.02). Pertussis toxin, H-7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G-protein and Protein Kinase C independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.

Specific binding of haptoglobin to human neutrophils and its functional consequences.

Haptoglobin, an acute phase reactant protein, has been shown to modulate various facets of immune responses. In this paper we examined the effect of haptoglobin on human neutrophils at the molecular level. First, we found that native haptoglobin binds at two distinct sites on neutrophils. We then examined the effects of this binding at normal and pathophysiological concentrations of haptoglobin found in human serum. Of the various functional parameters assessed, neutrophil respiratory burst activity, as assessed by superoxide (O2-) production, was inhibited by native haptoglobin when the cells were stimulated with formylmethionyl-leucylphenylalanine (FMLP), arachidonic acid (AA), and opsonized zymosan. The rise in intracellular calcium induced by FMLP stimulation was also inhibited by native haptoglobin. Since the generation of O2- was unaffected by native haptoglobin in phorbol myristate acetate (PMA)-stimulated neutrophils, the likely site of haptoglobin inhibition on neutrophil function is at a point of receptor-ligand interaction in the activation cascade. The role of haptoglobin as a modifier of the immune response has here been extended to altered neutrophil function stimulated by diverse agonists.

Inhibition of human neutrophil NADPH oxidase by Chlamydia serovars E, K, and L2.

The effects of Chlamydia trachomatis (serovars E, K, and L2) on human neutrophil activation were examined with respect to the organisms both as primary agonists and as agents that modulate cell responses to a second stimulus. Unopsonized chlamydiae alone, at ratios of 1.5 to 100 organisms per cell, failed to elicit changes in intracellular calcium or membrane depolarization or to stimulate the respiratory burst or degranulation during 60 min of incubation. Each of these functions except the respiratory burst was also normally activated when chlamydia-infected neutrophils were subsequently stimulated with formylmethionyl leucine phenylalanine or phorbol myristate acetate; the respiratory burst was inhibited 30 to 65%. Inhibition was dependent on live organisms and was maximal within 5 min of incubation. The organisms had no effect on the superoxide (O2-) assay, and the site of chlamydial inhibition was determined at the level of the NADPH oxidase itself, not at an intermediate step in the activation cascade. The mechanism of enzyme inactivation could not be determined. These results show that unopsonized chlamydiae do not elicit responses from infected neutrophils and suggest that microbicidal mechanisms other than those dependent on elaboration of toxic oxygen-derived species are required to inactivate chlamydiae.

Effects of influenza A virus on human neutrophil calcium metabolism.

Bacterial superinfection in influenza A virus-related illness may in part be explained by virus-induced neutrophil dysfunction. We here provide evidence that this effect is related to abnormal calcium metabolism of virus-infected cells. Neutrophils exposed to influenza virus for 0.5 h at 37 degrees C showed depressed O2- generation and release of radiolabeled arachidonic acid upon stimulation with FMLP. The peak cytosolic Ca2+ level achieved by virus-infected neutrophils after FMLP stimulation was significantly depressed as is efflux of 45Ca2+. This deficient Ca2+ mobilization could not be attributed to alterations of inositol phosphate production or Ca2+ influx in response to FMLP, both of which were unaffected by prior virus infection. Given these findings, the immediate effects of influenza virus on neutrophil Ca2+ metabolism were examined. The virus itself caused a rise in cytosolic Ca2+ and an efflux of 45Ca2+ without any corresponding 45Ca2+ influx. Total cell Ca2+ however was not depleted as measured by atomic absorption. Influenza virus, therefore, causes neutrophil activation leading to significant perturbations in Ca2+ metabolism and later to impaired mobilization of Ca2+ stores. This system offers a model for phagocyte deactivation and an opportunity to define control mechanisms of signal transduction.

Flavonoid impairment of neutrophil response.

Flavonoids are a class of phenolic plant pigments which impair the oxidative burst of neutrophils to an extent dependent on their hydrophobicity. The distribution of quercetin and of morin in nitrogen-cavitated neutrophils paralleled their respective hydrophobic characteristics and respiratory burst inhibition. While both flavonoids were localized primarily in the specific granule membrane of neutrophils, the amount of quercetin was considerably greater than that of morin. We here demonstrate inhibition of the initial stimulation response, depolarization of the membrane potential as monitored by fluorescence of the membrane probe diS-C3-(5), and of the respiratory burst, monitored by following the destruction of diS-C3-(5), a reaction mediated by the H2O2 produced in the burst. The flavonoids kaempferol, morin, quercetin, or fisetin were preincubated with human neutrophils at a concentration of 100 microM per 2 X 10(6) cells/ml for 2-3 min and subsequently stimulated with 1 microgram/ml of the tumor promoter phorbol myristate acetate (PMA) or with 60 micrograms/ml of immune complex. The effect of each compound differed, i.e. depolarization was enhanced by some and inhibited by others, while H2O2 generation was inhibited by each, supporting our previous findings that membrane potential depolarization and the respiratory burst are dissociable events. Concentration-response experiments, performed at flavonoid concentrations between 12.5 and 500 microM to determine the IC50 values of these compounds for depolarization and burst activation, indicated that none of the flavonoids affected the resting potential, while all perturbed the stimulus-coupled response, the direction and extent of the perturbation depending upon the stimulus, and the function assessed. These data show that the effects of flavonoids on human neutrophils are complex and suggest several sites of action depending upon the flavonoid's subcellular distribution and pathway of stimulation.

Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase.

The human neutrophil respiratory burst, activated by phorbol 12-myristate 13-acetate (PMA), results from specific receptor-ligand binding and activation of the NADPH-oxidase in the plasma membrane. The role of granule membrane constituents has been elucidated with neutrophils disrupted by nitrogen cavitation and then fractionated in Percoll gradients to resolve four postnuclear fractions: cytoplasm, light membranes or gamma fraction (site of the NADPH-oxidase), a light granule (beta) fraction containing putative constituents of the NADPH-oxidase (cytochrome b-245 and an associated flavoprotein), and a fraction of heavy granules. Cytochrome b-245 is localized to two pools of specific granules within the beta fraction as assessed by differing sedimentation in narrow Percoll gradients and translocates upon PMA-stimulation from one of these specific granule sub-pools to the plasma membrane where it exhibits no change in its midpoint redox potential. Translocation of cytochrome b-245 parallels O2-production initiated by PMA stimulation as assessed in the time course of each activity. The finding of increased amounts of the b cytochrome in cytoplast membranes relative to plasma membranes of unstimulated cells suggests that the cytoplasts, devoid of granules yet capable of O2-generation upon PMA-stimulation, are useful in assessing post-translocation events in the activation pathway of the NADPH-oxidase. These data support the hypothesis that translocation of NADPH-oxidase components from an intracellular granular pool contributes to respiratory burst expression.