Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.     

A role for calcium and protein kinase C in agonist-stimulated adhesion of human neutrophils.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [( Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.     

Endogenous phospholipid metabolism in stimulated neutrophils differential activation by FMLP and PMA

Endogenous phospholipid metabolism was examined during the initial 0–120 seconds of neutrophil (PMN) stimulation. When PMN were exposed to the chemotactic peptide FMLP (10^?7 M) or the tumor promotor, phorbol myristate acetate (PMA, 1 μg/ml) extensive changes in specific phospholipid (PL) classes were evident within 15 seconds. The profile and kinetics of stimulus-induced PL changes were stimulus-dependent. Five seconds after the addition of FMLP, PMN content of PC, PS and PA increased, while the level of PI decreased. Kinetic studies revealed that only PA levels remained elevated (0–120 s) while other PL decreased. In contrast, when cells were exposed to PMA (1 μg/ml), the levels of PC and PS rapidly increased (< 15 s). With PMA as stimulus, changes in PI and PA were not observed until > 60 s. Results indicate that exposure to PMN to stimuli leads to rapid changes in specific PL. In addition, they support the concept that neutrophils rapidly “remodel” endogenous PL upon stimulation.     

Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

Lack of effect of pertussis toxin on TNF-alpha-induced formation of reactive oxygen intermediates by human neutrophils

When human PMN were plated on fetal calf serum-coated polystyrene surfaces, addition of TNF-alpha, FMLP or PMA elicited adhesion and H2O2 formation. These effects of TNF-alpha and FMLP, but not of PMA, were impaired by removal of extracellular Ca2+. In addition, H2O2 formation induced by FMLP but not by TNF-alpha or PMA was inhibited by prior treatment with pertussis toxin (250-500 ng/ml). Thus, although the sequelae of TNF-alpha-receptor interaction on human PMN remain to be characterized in detail, they do not involve a pertussis toxin-sensitive GTP-binding protein.     

Upregulation of Respiratory Burst of Polymorphonuclear Leukocytes By A Bleomycin Derivative, Peplomycin

The influence of peplomycin (PLM) on the respiratory burst of peripheral blood polymorphonuclear leukocytes (PMN) was investigated. Short-term (5 min) treatment of human PMN with 0.1μg/ml to 100μg/ml of PLM increased phorbol myristate acetate (PMA)-and formyl-methionyl-leucyl-phenylalanine (FMLP)-induced luminol-dependent chemiluminescence. PMN, as well as alveolar macrophages from rabbits treated with 0.5 to 1.0 mg/kg of peplomycin per day for 5 days, generated more superoxide (O₂^-) than the cells from untreated rabbits. In both PLM-treated and untreated PMN, chemiluminescence induced by FMLP and PMA was decreased to less than 50% of the control by staurosporine, superoxide dismutase (SOD) and catalase. However, the peak intensity in PLM-untrcated PMN was decreased to about 30% of the control by genislein, while this agent induced a slight decrease in peak intensity in the PLM-treated PMN. Inositol triphosphate and diacyl glycerol levels were not clearly increased by PLM, but an increase of intracellular Ca and a shift of protein kinase C (PKC) to the membrane occurred in PMN within 1 min after PLM treatment. Western blotting revealed that the tyrosine phosphorylation of a 115 kDa protein was upregulated by 5 to 50μg/ml of PLM. While, PLM suppressed SOD activity in alveolar macrophages and PMN. These results seem to indicate that PLM increases the respiratory burst of PMN and macrophages both by way of direct PKC activation and by the upregulation of protein tyrosine phosphorylation. This increased reactive oxygen generation, together with the suppression of SOD activity seems to be tissue-impairing.     

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

Formyl-Met-Leu-Phe-dependent serine kinase for a 64,000 molecular weight protein of polymorphonuclear leukocytes in a cell-lysate system

In the Triton X-100-treated polymorphonuclear leukocytes (PMN), which were stimulated with formyl-Met-Leu-Phe (FMLP) for 1 min, a 64,000 molecular weight protein (p64) was preferentially phosphorylated by the incubation with [gamma-32P]ATP in the presence of Mg2+, but not in the presence of Ca2+. Phosphoamino acid analysis of pp64 revealed that the p64-kinase was a serine-specific protein kinase. The p64 was maximally phosphorylated in the first minute, suggesting that the rapid phosphorylation was related to the initial reaction for activation of the FMLP-stimulated PMN functions. The FMLP-stimulated phosphorylation of p64 was slightly inhibited by the addition of cGMP in the reaction mixture. However, addition of cAMP, the cyclic nucleotide-dependent kinase inhibitor (H-8), protein kinase C-inhibitor (H-7) or Ca/calmodulin-dependent kinase inhibitor (W-7), showed no effect on the phosphorylation. These data suggest that phosphorylation of p64 seems to be a novel protein kinase specific to p64.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Pig IαI appears unmodified in plasma in case of endotoxin-induced disseminated intravascular coagulation

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-α-inhibitor (IαI) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a procine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine IαI in plasmas collected from pigs after endotoxin infusion. To explain these data. IαI was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, procine cells were unable to proteolyze IαI. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.     

Chemotactic peptide-induced changes in neutrophil actin conformation

The effect of the chemotatic peptide, N- formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t- BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.     

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.     

A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst.

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.     

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.     

Tumor necrosis factor as an activator of human granulocytes. Potentiation of the metabolisms triggered by the Ca2+-mobilizing agonists

TNF stimulated superoxide (O2-) release directly in human granulocytes in a dose-dependent manner (1 to 1000 U/ml), although its potency was weak. TNF-induced O2- release was inhibited by cAMP agonists or ionomycin, and was not accompanied with an increase in cytoplasmic free Ca2+ [( Ca2+]i) and membrane potential changes (depolarization). These findings indicate that neither Ca2+ mobilization nor membrane depolarization is required for TNF-receptor-mediated cell activation. The pretreatment of human granulocytes with TNF enhanced O2- release and membrane depolarization in parallel stimulated by the receptor-mediated Ca2+-mobilizing agonists (FMLP, Con A, and wheat germ agglutinin) or the Ca2+ ionophore ionomycin, but not by PMA, a direct activator of protein kinase C. The optimal effect was obtained by pretreatment of cells with 100 U/ml TNF for 5 to 10 min at 37 degrees C, although the magnitude of enhancement varied according to the agonists used as subsequent stimuli. TNF did not affect an increase in [Ca2+]i stimulated by the Ca2+-mobilizing agonists, except Con A. Con A-induced increase in [Ca2+]i was enhanced by TNF in a dose-dependent manner. These diverse effects of TNF could be partly explained by the exclusive potentiation by TNF of the metabolic events triggered by an increase in [Ca2+]i.     

CDw17: a neutrophil glycolipid antigen regulated by activation

The plasma membrane and intracellular granules of human polymorphonuclear neutrophils (PMN) contain large amounts of the glycolipid, lactosylceramide (LacCer; Gal beta 1----4Glc beta 1----1Cer). Despite its abundance, novel subcellular distribution, and lineage-restricted expression, nothing of PMN LacCer function is known. We examined the relationship between LacCer and PMN activation by assessing binding of anti-LacCer mAb (T5A7; anti-CDw17) to PMN during and after cell stimulation. CDw17 expression markedly decreased after treatment with PMA, dioctanoylglycerol, calcium ionophore, FMLP (with or without cytochalasin B or added Ca2+), TNF-alpha, or lymphotoxin. Depending on the stimulus, CDw17 declined to levels ranging from 70% (TNF, lymphotoxin) to less than 5% (phorbol ester, dioctanoylglycerol) of levels detected on untreated PMN. Loss of CDw17 from PMA-treated PMN followed dose- and temperature-dependent kinetics, with loss being detected after PMA treatment for 1 min. Membrane internalization explained PMA-induced loss of CDw17, as cell-associated 125I-anti-CDw17 became inaccessible to fluorescent anti-Ig after PMA treatment. CDw17 on PMN cytoplasts or retinoic acid-induced HL-60 cells was only slightly affected by stimulation, suggesting that down-regulation of the epitope is associated with granule exocytosis rather than superoxide production. Results with PMN from a patient with chronic granulomatous disease confirmed that normal superoxide production is not required for CDw17 loss induced by PMA or FMLP treatment. The data collectively demonstrate that reduced levels of cell-surface CDw17 are associated with granule exocytosis after PMN activation.     

Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin

Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca2+ concentration ([ Ca2+]i). Due to quenching of Fura-2, the addition of Mn2+ enabled us to compute the cytosolic concentration of total manganese ([Mn]i). The agents used were the novel Ca2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation ionophore, A23187. The agents caused transient rises of [Ca2+]i and monotonous rises of [Mn]i, suggesting influx but no efflux of Mn2+. The rise time of [Ca2+]i and the time constants and magnitude of the apparent Mn2+ influx were strongly dependent on the sequence of addition of the agonist and Ca2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca2+]i and on the influx of Mn2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not induce Ca2+ uptake in ATP-depleted red cells but increased the Ca2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca2+ flux into the cytosol and Ca2+ flux of the plasma membrane pump are involved in the transient [Ca2+]i response.