Background: Distinguishing ingested particles from those attached to the cell surface is an essential requirement when performing quantitative studies of phagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans-human neutrophils (PMN) system. Methods: The assay is based on the observation that the vital dye trypan blue (TB), while quenching the green fluorescence of fluorescein-labeled C. albicans, causes them to fluoresce red. PMN were incubated with fluorescein-labeled yeast particles for the required time. Aliquots of the incubation mixtures were then promptly diluted with an equal volume of a TB solution at pH 4.0, and subsequently analyzed by flow cytometry for green and red fluorescence.

Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.

Abstract. Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-a (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M,A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751. We have studied TNFinduced C1-movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of [32 integrins cosignaled for an early, marked, and prolonged C1-efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between CI-efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced CI-efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of CI-efflux, such as ethacrynic acid. Moreover, CI-efflux induced by means other than TNF treatment, i.e., by using Cl--free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of CI-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2--forming NADPH oxidase. N 'EtrrRoamLtC polymorphonuclear leukocytes (PMN) 1 respond to both particulate and soluble stimuli with a vigorous respiratory burst. This leads to the release of toxic oxygen molecules that contribute to both the PMN microbicidal activity and the tissue inflammatory damage. Among the physiologically relevant soluble stimuli, cytokines such as tumor necrosis factor-a (TNF), granulocyte/macrophage-colony stimulating factor, and granulocyte-colony stimulating factor are peculiar, since they activate the respiratory burst only in PMN residing on biologic surfaces, e.g., on proteins of the extraAddress all correspondence to R. Menegazzi, Istituto di Patologia Generale, Universit~t di Trieste, via A. Fleming, 22, 34127 Trieste, Italy. Tel.: (39) 40 572012. Fax: (39) 40 567862.1. Abbreviations used in this paper: 9-AC, anthracene-9-carboxylic acid; CHC, c~-cyano-4-hydroxy-cinnamic acid; C1-1, intracellular chloride content; DIDS, 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid; EA, ethacrynic acid; FBG, fibrinogen; FMLP, N-formyl-methyonil-leucyl-phenylalanine; FN, fibronectin; FN-PMN, PMN residing on FN-coated surfaces; G-buffer, glucuronate-containing buffer; MA, o-[(3-hydroxymercuri-2-methoxypropyl)carbamoyl] phenoxyacetic acid; Oz-, superoxide anion; PMN, neutrophilic polymorphonuclear leukocytes; s-PMN, PMN in suspension; poly (HEMA), poly(2-hydroxyethyl methacrylate); TNF, tumor necrosis factor-a; TNF-R, TNF receptor. cellular matrix immobilized on a solid support, but...

The process of β2 integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes’ (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl− content (Cl−i) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl−i in the activation of β2 integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of β2 integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin α subunits, and epitope CBRM1/5, localized to the I domain on the α-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl−i because 1) inhibition of Cl−i decrease is invariably accompanied by inhibition of β2 integrin activation, 2) Cl−i decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl−-free buffers, activates β2 integrins, and 3) restoration of the original Cl−i levels is accompanied by deactivation of β2 integrins. We also show that Cl−i decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pHi does not seem to be relevant for β2 integrin activation. The results of our study emphasize the role of Cl− as a new PMN “second messenger.”

Chloride ion efflux is an early event occurring after exposure of human neutrophils to several soluble agonists. Under these circumstances, a rapid and reversible fall in the high basal intracellular chloride (Cl−i) levels is observed. This event is thought to play a crucial role in the modulation of several critical neutrophil responses including activation and up-regulation of adhesion molecules, cell attachment and spreading, cytoplasmic alkalinization, and activation of the respiratory burst. At present, however, no data are available on chloride ion movements during neutrophil phagocytosis. In this study, we provide evidence that phagocytosis of Candida albicans opsonized with either whole serum, complement-derived opsonins, or purified human IgG elicits an early and long-lasting Cl− efflux accompanied by a marked, irreversible loss of Cl−i. Simultaneous assessment of Cl− efflux and phagocytosis in cytochalasin D-treated neutrophils indicated that Cl− efflux occurs without particle ingestion. These results suggest that engagement of immune receptors is sufficient to promote chloride ion movements. Several structurally unrelated chloride channel blockers inhibited phagocytosis-induced Cl− efflux as well as the release of azurophilic—but not specific—granules. It implicates that different neutrophil secretory compartments display distinct sensitivity to Cl−i modifications. Intriguingly, inhibitors of Cl− exchange inhibited cytosolic Ca2+ elevation, whereas Cl− efflux was not impaired in Ca2+-depleted neutrophils. We also show that FcγR(s)- and CR3/CR1-mediated Cl− efflux appears to be dependent on protein tyrosine phosphorylation but independent of PI3K and phospholipase C activation.