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We have defined major differences between events elicited during activation of human neutrophils by opsonized Candida hyphae (noningestible) or zymosan (ingestible) and by unopsonized Candida hyphae (also noningestible). In response to opsonized particles, a transient increase in cytosolic free calcium was initially observed, followed by depolarization in the transmembrane potential and a respiratory burst. Without opsonins, zymosan did not elicit a response from neutrophils. In contrast, unopsonized Candida hyphae elicited a transient increase in cytosolic calcium, as well as triggered a respiratory burst; they did not, however, elicit a depolarization of the neutrophil membrane. Furthermore, the rate of superoxide generation decreased after a prolonged lag period. The calcium flux was also delayed and coincided with the onset of superoxide generation. Thus, our data demonstrate that Candida albicans hyphae activate neutrophils in the absence of added serum components and initiate a neutrophil respiratory burst without an antecedent membrane depolarization.

Clostridium difficile, a common enteric pathogen, mediates tissue damage and intestinal fluid secretion by release of two protein exotoxins: toxin A, an enterotoxin, and toxin B, a cytotoxin. Because toxin A elicits an intense inflammatory reaction in vivo, we studied the effects of highly purified C. difficile toxins on activation of human granulocytes. Toxin A at concentrations of 10(-7) to 10(-6) M, but not toxin B, elicited a significant chemotactic and chemokinetic response by granulocytes that was comparable with that induced by the chemotactic factor N-FMLP (10(-7) M). Neither toxin stimulated release of superoxide anion from granulocytes. Toxin A produced a rapid, transient rise in cytosolic [Ca2+]i, as measured by quin 2 fluorescence. Pertussis toxin and depletion of intra- and extracellular calcium blocked the toxin A effect on cytosolic [Ca2+]i. These findings suggest that the inflammatory effects of C. difficile toxin A in the intestine may be related to its ability to mobilize intracellular Ca2+ and elicit a chemotactic response by granulocytes.

In granulocytes harvested from human blood, an elevation of the cytosolic concentration of Ca2+ ions is by itself insufficient to activate the cell's respiratory burst. We report herein that, when granulocytes are "primed" by a 90-min preincubation with the recombinant human hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSFrh), elevation of the concentration of cytosolic Ca2+ ions ([Ca2+]i) becomes a more effective transduction signal capable of triggering the generation of substantial quantities of superoxide (O2-) anions by the cell. In these studies, we used four separate and independent maneuvers to induce elevation of [Ca2+]i: 1) depolarization of the cell's electrical potential through obliteration of the transmembrane Na+ and K+ gradients; 2) acidification of the cytoplasm using propionic acid; 3) addition of the calcium ionophore ionomycin; and 4) treatment of the cells with the monoclonal antibody to the C3bi receptor, PMN7C3. In all cases, elevation of [Ca2+]i through these manipulations resulted in the release of substantially greater quantities of O2- by GM-CSFrh-primed granulocytes than by unprimed, control cells. The generation of O2- was in all cases markedly reduced by chelation of either intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. We conclude that during the process of GM-CSFrh priming, the metabolic assembly responsible for O2- anion production in the granulocyte becomes altered in such a way that a subsequent elevation in [Ca2+]i provides a potent signal for its activation.

We have investigated the involvement of phospholipase C mediated polyphosphoinositide turnover in activation of polymorphonuclear leukocytes by particulate stimuli. Results showed that stimulation of leukocytes with serum opsonized zymosan (ingestible particle), serum opsonized Candida albicans hyphae (noningestible particle), or nonopsonized hyphae was followed by a transient rise in cellular inositol phosphates as has been described for neutrophil activation via the formyl peptide receptor. The kinetics of inositol trisphosphate generation paralleled both the time course of changes in cytosolic calcium concentration and the onset of superoxide anion generation, suggesting that these may be related events.

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.

Binding of murine monoclonal antibody PMN7C3 to human neutrophils results in a large rapid, dose dependent transient increase in intracellular free calcium as measured by QUIN-2 fluorescence. Unlike other calcium mobilizing agents PMN7C3 does not induce any increase in respiratory burst activity over basal level. The PMN7C3 effect requires multivalent binding. Chelation of extracellular calcium does not significantly decrease the fluorescence transient generated by exposure to PMN7C3. Lowering of basal levels of intracellular free calcium concentration by maintaining QUIN-2-loaded PMN in calcium free medium eliminates the fluorescence transient. The observations demonstrate that a cell surface receptor mediated intracellular free calcium transient may be generated without any associated respiratory burst activation.