Priming of human neutrophils with N-formyl-methionyl-leucyl- phenylalanine by a calcium-independent, pertussis toxin-insensitive pathway

Resting neutrophils may be "primed" to augmented effector function, eg, superoxide (O2-) production in the respiratory burst, upon a second stimulation with a variety of soluble agonists including formylated methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). At priming concentrations of FMLP (5 x 10(-9) mol/L) that did not initiate O2- generation, two metabolic activities were noted: (1) approximately a threefold increase in the baseline intracellular calcium (Ca++i) level, that was not dependent on extracellular Ca++, and (2) a rapid rise in intracellular pH that was blocked by 5-(N,N- dimethyl) amiloride (DA), that had no effect on the Ca++i response to priming. Furthermore, there were no significant increases in inositol metabolites in cells primed and stimulated with FMLP compared with cells receiving the stimulating dose of FMLP alone and pretreatment with pertussis toxin (PT) (before the addition of the priming -5 x 10(- 9) mol/L dose of FMLP), whereas abolishing the response to FMLP during the second stage of stimulation, had (1) no effect on FMLP-primed cells subsequently stimulated with PMA, and (2) only partially ablated the rise in Ca++i initiated with FMLP. That FMLP priming involved distinctive processes to those of the well characterized FMLP-coupled Ca++-dependent activation cascade was shown by the full priming effect attained in a Ca++-free buffer, which did not sustain an O2- response to a second-stage FMLP stimulation, but sustained a primed response to PMA. These data demonstrate that FMLP primes human neutrophils by a Ca++-independent and PT-insensitive pathway, offering a functional model for studying heterogeneous FMLP receptor-coupled reactions.     

Characterization of influenza A virus activation of the human neutrophil

Neutrophil dysfunction consequent to influenza A virus infection has been described in vivo and in vitro and may contribute to the serious bacterial sequelae which occur in influenza-infected hosts. On the premise that such dysfunction may represent a form of "deactivation," we sought to characterize neutrophil activation by the virus in comparison with other agonists. The virus induces a respiratory burst in which H2O2 (but not O2-) are formed. Preceding the respiratory burst, a rise in intracellular calcium (Ca2+i) is noted, but both responses are nearly independent of extracellular Ca2+, unlike those elicited by the other well-characterized Ca2+-dependent agonists, formyl-methyl-leucyl-phenylalanine (FMLP), or Concanavalin-A (Con-A). The Ca2+ increase is paralleled by IP3 generation, implying that it is the result of phospholipase C (PLC) activation. The virus also elicits neutrophil membrane depolarization, which is independently mediated from the Ca2+ increase and respiratory burst and may reflect protein kinase C (PK-C) activation. Virus-induced responses are insensitive to pertussis toxin (PT); cholera toxin does inhibit these responses but in a nonspecific manner. Thus, although influenza virus activates PLC in neutrophils, it does so in a PT-insensitive manner and does not elicit or require a discernible Ca2+ influx to generate a respiratory burst response. In aggregate, the data indicate that influenza A virus activates neutrophils in a manner distinct from that of other well-described neutrophil agonists. These results illustrate the diversity of neutrophil activation mechanisms and support the notion that further characterization of this pathway may facilitate understanding of neutrophil dysfunction induced by the virus.     

Interactions of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor alpha in the priming of the neutrophil respiratory burst.

Exposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% +/- 86%, n = 41, and 581% +/- 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony-stimulating factor (G-CSF) alone caused only modest priming (202% +/- 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNF alpha and GM-CSF and TNF alpha are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM-CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% +/- 50% of preinfusion response to FMLP, P less than .005, n = 8). Priming was also seen in patients receiving G-CSF (152% +/- 58%, n = 5), although this did not reach conventional significance levels (.05 less than P less than .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNF alpha, with a doubling in H2O2 production.     

Comparison of influenza A virus and formyl-methionyl-leucyl-phenylalanine activation of the human neutrophil.

Influenza A virus (IAV) activates the human neutrophil, but induces a dysfunctional state as well. Cell activation may contribute to the containment of the virus and/or cause local tissue damage. Certain features of the neutrophil activation response elicited by IAV are distinctive when compared with that triggered by formyl-methyl-leucyl-phenylalanine (FMLP). An atypical respiratory burst response occurs in which hydrogen peroxide, but no superoxide, is formed. This unusual respiratory burst stoichiometry persists despite marked priming of the IAV-induced response. A comprehensive examination of the activation cascade initiated by these stimuli failed to show an explanation for these differences. Both IAV and FMLP comparably stimulate inositol trisphosphate and phosphatidic acid production. The subsequent increase in intracellular calcium (Ca2+i) upon FMLP stimulation was more dependent on extracellular Ca2+ than with IAV activation, but both stimuli induced Ca2+ influx. FMLP and IAV exhibited equal susceptibility to inhibition by protein kinase inhibitors in eliciting the respiratory burst, and actin polymerization occurred in response to each agonist. A possible explanation for the anomalous respiratory burst induced by IAV is that O2- is generated at an intracellular site inaccessible to assay, and/or virus binding to sialic acid constituents of the plasma membrane alters the O2- generating capacity of the respiratory burst oxidase; evidence for each mechanism is offered.     

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

OBJECTIVES--To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. METHODS--Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. RESULTS--The production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. CONCLUSION--It is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.     

Neutrophil adhesive dysfunction in thermal injury: the role of fibronectin

We examined neutrophil substrate adherence in 19 subjects with burns involving 1%-83% of their body surface area. Within 24 h of injury, neutrophils from burn patients demonstrated a 50% reduction in adhesion to both gelatin and plastic substrates when stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), phorbol myristate acetate, and calcium ionophore A23187. Neutrophil substrate adherence examined as long as two weeks after burn injury remained abnormal. Neutrophils, from burn patients, stimulated with FMLP, phorbol myristate acetate, and calcium ionophore A23187 demonstrated a 51%, 37%, and 45% decrease, respectively, in release of immunoreactive fibronectin compared with control neutrophils. In neutrophils from burn patients there was a 31% reduction in total neutrophil-associated fibronectin compared with controls. The decrement in release and total cellular content of fibronectin in neutrophils from burn patients did not change when reexamined on day 7 after injury. The magnitude and time course of alterations in the cellular content and release of fibronectin correlate with adhesive dysfunction after burn injury.     

Increased superoxide production by polymorphonuclear leukocytes in systemic lupus erythematosus.

Normal and lupus PMN show an enhancement in superoxide production in vitro when stimulated with lupus serum. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used, lupus PMN showed an O2- production of 2.1 nmol/min/10(7) cells, which is 5.2 times the response of normal PMN stimulated by FMLP. Our results show the existence of serum factors in SLE patients that can stimulate O2- production by PMN. Lupus neutrophils showed an increased response to membrane stimuli such as FMLP, capable of triggering the cell respiratory burst. Lupus neutrophils appeared more responsive to membrane stimuli. The serum and cellular factors seemed to indicate an increase rate of superoxide production by PMN in lupus patients, which could be relevant factors in the development of vasculitis and tissue damage.     

The effects of rhGM-CSF on the neutrophil respiratory burst when studied in whole blood

A simple semiquantitative cytofluorometric method has been developed for measuring neutrophil respiratory burst activity in whole blood samples. This technique avoids the introduction of laboratory artefacts which modulate neutrophil function. In addition, flow cytometric analysis allows the response to be studied in individual cells. We show here that neutrophils examined freshly ex vivo, exhibit only weak respiratory burst activity in response to stimulation with the chemotactic peptide FMLP (10(-6) M). Prior incubation with rhGM-CSF results in an increase in the number of responding cells from 13.5 +/- 2.36% (mean +/- SEM) to 46.7 +/- 6.3% (P less than 0.0001) with an increase in total respiratory burst activity of 567% (P = 0.001). The majority of neutrophils in whole blood (67.1 +/- 8.1%) exhibit respiratory burst activity in response to stimulation with phorbol ester (1 micrograms/ml of TPA), and this response is also significantly primed by rhGM-CSF (P = 0.004). The enhancement of respiratory burst activity induced by rhGM-CSF is due to both recruitment of previously unresponsive neutrophils, and to intensification of the response of the responding cells. In vivo administration of rhGM-CSF also results in priming of the respiratory burst in response to FMLP, although the enhancement of activity is not as great as that obtained when pre-infusion blood samples are incubated with rhGM-CSF in vitro.     

Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides

We have used a cytofluorometric method to study the effect of tumor necrosis factor alpha (TNF) priming on the oxidative burst, FMLP- receptor expression and actin polymerization of whole blood polymorphonuclear neutrophils (PMN). This technique permits the study of single cells and, thus, allowed us to examine the responsiveness of PMN to TNF in whole blood. We found that TNF in whole blood strongly primed a subpopulation of PMN to produce H2O2 in response to FMLP stimulation, whereas TNF and FMLP alone did not have a significant effect. Furthermore, adding TNF to whole blood increased the capacity of a subpopulation of PMN to bind N-formyl peptides at 4 degrees C, a phenomenon that could account, at least in part, for the strong H2O2 production in response to FMLP after TNF priming. Dual-color cytometric analysis showed that TNF primed actin polymerization on the same subpopulation in response to FMLP. Because the PMN subpopulation, which strongly bound N-formyl peptides at 4 degrees C, was no longer detectable after 1 minute of incubation at 37 degrees C, our data suggest that TNF treatment of PMN in whole blood primes a subpopulation that actively cycles FMLP receptors. These results suggest that PMN in the circulation may respond weakly to bacterial peptides and that TNF may play a critical role in the induction of the oxidative burst in vivo.     

Exposure of human neutrophils to exogenous nucleotides causes elevation in intracellular calcium, transmembrane calcium fluxes, and an alteration of a cytosolic factor resulting in enhanced superoxide production in response to FMLP and arachidonic acid

Exposure of human neutrophils to micromolar concentrations of both hydrolyzable and nonhydrolyzable purine nucleotides caused the generation of transient rises in intracellular calcium (Ca2+), Ca2+ fluxes across the membrane, and primed the cells for enhanced production of superoxide (O2-) when subsequently exposed to agonists such as FMLP and arachidonic acid. The neutrophils were most sensitive to adenosine triphosphate (ATP) and ATP-gamma-S, which produced Ca2+ transients and enhanced O2- production at concentrations as low as 1 to 5 mumol/L, with a doubling of O2- generation at 25 to 50 mumol/L. Adenosine diphosphate (ADP), guanosine triphosphate (GTP), and 5'-adenylylimidodiphosphate (AMP-PNP) required approximately 10-fold higher concentrations to cause similar effects. Adenosine did not cause Ca2+ fluxes or a Ca2+ transient and was inhibitory of O2- production. There was a strong correlation between a nucleotide's ability to generate a Ca2+ response and its ability to enhance O2- generation. Nitrogen cavitation and subcellular fractionation of the neutrophils after a brief exposure to ATP, ATP-gamma-S, and AMP-PNP revealed that the enhanced O2- generating capacity was stable and detectable in a cell-free assay system. By combining variously treated cytosolic and membrane fractions, it was found that the enhanced O2- production was attributable to a modification of a component(s) of the cytosol.     

Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors.

Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time-courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM-CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.     

Evidence for participation of vicinal dithiols in the activation sequence of the respiratory burst of human neutrophils

Phenylarsine oxide (PAO) specifically forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP). Pretreatment of human neutrophils with micromolar concentrations of PAO inhibited release of superoxide anion (O2-) stimulated by N- formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol 12-myristate 13- acetate (PMA); the inhibition was reversed with DMP, but not with 2- mercaptoethanol. PAO did not affect O2- release in previously stimulated cells. PAO did not affect the FMLP-induced Ca2+ response, suggesting that PAO affects a postreceptor event that does not modulate the Ca2+ transient. Treatment of isolated membrane or cytosolic fractions with PAO did not change the rates of arachidonate-stimulated O2- production in a cell-free system. Pretreatment of unstimulated neutrophils with PAO inactivated cytosolic protein kinase C (PKC); the inactivation was reversed with DMP. However, PAO did not affect PMA- induced translocation of beta-PKC protein or reduce the PKC activity translocated to the membrane. PAO had no effect on tyrosine kinase activity but inactivated phosphotyrosine phosphatase; stimulus-induced tyrosine phosphorylation of several proteins was markedly enhanced. These results suggest that vicinal dithiols play an essential role in activation of the respiratory burst oxidase. Possible sites for the activity of these essential vicinal dithiols include PKC and the regulatory balance of tyrosine phosphatase activity and tyrosine phosphorylation.     

A novel syndrome of severe neutrophil dysfunction: unresponsiveness confined to chemotaxin-induced functions

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2- acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.     

Diverse Effects of Cytochalasin B on Priming and Triggering the Respiratory Burst Activity in Human Neutrophils and Monocytes

Cytochalasin B, despite its potent enhancing effect on superoxide (O2-) release triggered by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and many other agonists, significantly inhibited O2- release triggered by interleukin 8 (IL-8) and platelet-activating factor in human neutrophils. Cytochalasin B also enhanced changes in membrane potential stimulated by FMLP but inhibited those stimulated by IL-8. Using IL-8 as a triggering agonist, we found that the priming effect of tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on O2- release was slightly but significantly potentiated by cytochalasin B. O2- release triggered by TNF and GM-CSF was completely abolished by cytochalasin B. In contrast to these diverse effects of cytochalasin B on O2- release, changes in cytoplasmic pH stimulated by FMLP, IL-8, TNF, and GM-CSF were not or were only minimally affected by cytochalasin B. Unlike human neutrophils, human monocytes stimulated by FMLP showed inhibition of O2- release and changes in membrane potential in response to cytochalasin B, and the priming effect of TNF and GM-CSF on O2- release in human monocytes was completely abolished by cytochalasin B. These findings indicate the diverse effects of cytochalasin B on phagocytes and suggest distinct regulatory mechanisms according to the functions, agonists, and cell types.     

Modulation of neutrophil oxidative responses to soluble stimuli by platelet-activating factor

The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl- methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF-mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation.     

Superoxide anion production during Anaplasma phagocytophila infection

Anaplasma phagocytophila persists within neutrophils and prevents the respiratory burst by inhibiting gp91(phox). Mutations in gp91(phox) result in chronic granulomatous disease (CGD), which is diagnosed by use of the nitroblue tetrazolium (NBT) and Fc-Oxyburst assays that examine whether cells produce O2-. This study assessed whether the NBT and Fc-Oxyburst assays could detect a respiratory burst during A. phagocytophila infection. O2- production was inhibited in HL-60 cells and neutrophils infected with A. phagocytophila. In a mouse model of A. phagocytophila infection, 15%+/-4% (mean+/-SD) of polymorphonuclear leukocytes from infected mice had an ineffective respiratory burst compared with 1%+/-1% (mean+/-SD) of the neutrophils from uninfected animals. A population of neutrophils that did not produce O2- was also detected in 2 patients with A. phagocytophila infection. These data demonstrate respiratory burst inhibition by A. phagocytophila in vivo and on an individual cell basis by use of assays designed to evaluate CGD.     

Ca2+ response in neutrophils after exposure to bacterial N-formyl-methionyl-leucyl-phenylalanine: delayed response in ulcerative colitis

OBJECTIVE: In acute stages of ulcerative colitis (UC), neutrophils migrate from the circulation into inflamed colonic tissue, initiated by yet unknown stimuli. The bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is a component of the surface membrane of colonic bacteria such as Escherichia coli and stimulates Ca2+ influx into neutrophils, reflecting the fact that ionized calcium is an important secondary messenger for several neutrophil functions, including locomotion, phagocytosis and free oxygen radical production. Recent studies have revealed that Ca2+ dependent ICAM-1/beta 2-integrin mediated neutrophil migration is impaired in UC patients. The aim of the present work was to study the influx of Ca2+ into peripheral blood neutrophils of UC patients after exposure to FMLP and after binding of either beta 2-integrins or intercellular adhesion molecule-1 (ICAM-1). METHODS: The relative intracellular Ca2+ levels ([Ca2+]i ) were measured spectrofluorometrically in neutrophils isolated from eight UC patients and eight controls. The cells were exposed to 1 nm FMLP, 5 pm free ICAM-1, or antibodies binding ICAM-1 or the beta 2-integrins CD11a, CD11b, CD11c and CD18. RESULTS: A pronounced increase in [Ca2+]i was observed by exposure of cells to FMLP, and neutrophils from UC patients showed a consistent and significant delayed response as compared to cells from control subjects (P < 0.01). Antibody mediated cross-linking of CD18 triggered a small but detectable increase in [Ca2+]i, which did not differ between patients and controls. CONCLUSION: A delayed response to bacterial peptides appears to be a phenotypic trait for neutrophils of UC patients. A connection between FMLP stimulated Ca2+ influx and CD11/CD18 upregulation is discussed.     

Effects of oxygen tension and pH on the respiratory burst of human neutrophils.

The respiratory burst of human neutrophils was measured under conditions of hypoxia and low pH. O2 -- production by neutrophils activated with opsonized zymosan fell slowly as the oxygen concentration declined to 1%, then dropped more sharply, reaching negligible levels at oxygen concentrations less than 0.25%. Production was half maximal at an oxygen concentration of 0.35% (equivalent to approximately 10-microM dissolved oxygen). O2- production by the cell-free O2- -forming system prepared from zymosan-activated neutrophils showed a similar dependence on oxygen concentration. A drop in pH caused decreases in both oxygen consumption and O2-- production by zymosan-treated neutrophils, values at PH 6.0 being 10%--20% of those observed at pH 7.5. Experiments with the cell-free O2-- -forming system suggested that this decline in respiratory burst activity at low pH was due to inefficient activation of the O2-- -forming enzyme under acidic conditions.     

Studies on defense effects of recombinant human granulocyte colony-stimulating factor (G-CSF) to infections. II. Priming effect for superoxide production by human neutrophil]

In present study, we have investigated superoxide (O2-) production from human neutrophils by recombinant human granulocyte colony-stimulating factor (G-CSF) using the microtiter plate for the purpose of being close to the inflammatory site. G-CSF by itself did not induce the release of O2- in human neutrophil on either Fetal Bovine Serum (FBS)-coated plate or plate uncoated with FBS, even if neutrophils were exposed for maximum 3 hr. However, the optimal concentration of G-CSF (50 ng/ml) was able to prime human neutrophils with enhance of O2- release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) from 10(-6) to 10(-8) M, but not by the non chemoattractant such as phorbol myristate acetate (PMA), concanavalin A, and ionomycin. These findings indicate that G-CSF might enhance bactericidal activity of neutrophils by priming them penetrating into the inflammatory site.     

Chemotactic factor enhancement of superoxide release from fluoride and phorbol myristate acetate stimulated neutrophils

Human neutrophils exposed to chemotactic concentrations of zymosan- activated serum (ZAS) and a formylated chemotactic peptide (FMLP, 10(- 7)--10(-9) M) were markedly enhanced in their ability to generate superoxide (O2-) upon stimulation with either sodium fluoride or phorbol myristate acetate (PMA). For both fluoride and PMA, enhancement was characterized by a decrease in the lag from stimulation to initiation of superoxide release and by an increase in the rate of superoxide generation--representing faster activation and increased activity of O2- generating enzyme, respectively. Chemotactic concentrations of casein, normal serum, and casein-treated serum enhanced the activity, but not the rate of activation, of the fluoride- stimulated superoxide generating system. This effect on activity was not so impressive as that obtained with FMLP or ZAS. The mechanisms by which FMLP enhanced responsiveness to fluoride and PMA were found to be different. Optimal enhancement for fluoride-stimulated responses required extracellular Ca++. Extracellular glucose, but not extracellular Ca++, was required for enhancement of FMLP of PMA- stimulated responses. A similar glucose requirement could not be demonstrated for chemotactic peptide enhancement of the superoxide- generating system stimulated by fluoride. Fluoride and PMA apparently activate the neutrophil O2- generating enzyme by pathways that are not identical. However, responsiveness of the enzyme to both agents is susceptible to modulation by cellular responses to chemotactic peptides.