The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

Effects of piriprost (U-60, 257B) and leukotrienes on alkaline phosphatase activity of rat endometrial stromal cells in vitro.

The present study investigated the effects of piriprost (U-60,257B; an inhibitor of LT synthesis) and various LTs on alkaline phosphatase (ALP) activity of rat endometrial stromal cells in vitro. Mature ovariectomized rats were pretreated with hormones to sensitize their uteri for the decidual cell reaction. Endometrial stromal cells were isolated and cultured for up to 72 hr with various treatments. The ALP activity in all experiments was significantly (p less than 0.01) higher at 72 hr than at 24 hr, irrespective of treatment. We examined the effects of 100 microM piriprost, with or without 1 microM LTB4, 0.01 microM LTC4, 0.1 microM LTD4 or 0.001 microM LTE4 on ALP activity. At 72 hr, as indicated by analyses of variance, there were significant interactions (p less than 0.01) between the effects of piriprost and the LTs. Piriprost by itself increased (p less than 0.01) ALP activity in all experiments, and a further increase (p less than 0.01) in ALP activity was observed when either LTB4, LTC4, LTD4 or LTE4 was added with piriprost. LTB4, LTD4, or LTE4 alone had inhibitory effects (p less than 0.01) while LTC4 alone had no effect. These studies suggest LTs may be involved in decidualization which, in vitro, is accompanied by an increase in endometrial ALP activity. However the exact role of LTs is still unclear.     

Examination of the effects of piriprost (U-60,257B) on alkaline phosphatase activity of rat endometrial stromal cells in vitro.

Previously, piriprost (U-60,257B; an inhibitor of leukotriene (LT) synthesis) was shown to increase alkaline phosphatase (ALP) activity in cultured endometrial stromal cells (1). The present study investigated the mechanism of action of piriprost in this system. Sensitized rat endometrial stromal cells were isolated and cultured for up to 72 hr with various treatments. Piriprost (100 microM) was found to decrease 5-hydroxyeicosatetraenoic acid (a 5-lipoxygenase product) by 53% after 72 hr which provided evidence that 5-lipoxygenase was being inhibited by piriprost. Lactate dehydrogenase (LDH) activity confirmed that piriprost was not toxic to the cells. The possibility of piriprost acting in an analogous manner with that of PGs was examined. Three microM PGE2 or 20 microM carba-prostacyclin (CP), an analogue of PGI2, maximally increased (p less than 0.01) ALP activity at 72 hr and the further addition of 100 microM piriprost to PGE2 or CP caused an additional, additive increase in ALP activity. This indicated that the mechanism of action of piriprost was probably different from that of PGE2 or PGI2. The possibility that piriprost was shunting arachidonic acid into PG production was examined. Ten microM indomethacin (an inhibitor of PG synthesis) caused a decrease (p less than 0.01) in ALP activity and a 99% reduction in PGE2 at 72 hr. The effects of the combination of 100 microM piriprost and 10 microM IM were statistically additive, suggesting that the effects of piriprost were not due to an increase in PG production. These studies suggest that the effects piriprost on possible in vitro decidualization may be due to inhibition of 5-lipoxygenase.     

Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish "triggering" from "activation" of human neutrophils

Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses; N-formylmethionylleucylphenylalanine (fMLP) (0.1 microM) provokes brisk generation of superoxide anion; leukotriene B4 (LTB4, 0.1 microM) is a poor stimulus. In contrast, phorbol myristate acetate (PMA, 1.6 microM) acting directly via protein kinase C is a potent stimulus for O2-. We compared the kinetics of appearance of various "second messengers" with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an "early" (less than or equal to 15 s) and a "late" (greater than 15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Cytochalasin B increased the late phase of DG labeling elicited by all agonists. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Early increments of DG were also accompanied by addition of plasma membrane (ultrastructural morphometry); the ratio of surface perimeter to area increased rapidly (10 s) and persisted (60 s) in response to fMLP. Increments were more gradual in response to PMA. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 Mr protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP (early and late) and PMA (late). In contrast, LTB4 provoked only early phosphorylation of this protein. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only "triggering" (the rapid generation of a signal) but also "activation" (the maintenance of a signal) to sustain responses.     

Possible involvement of a 95-kDa protein phosphorylation in phorbol 12-myristate 13-acetate-induced suppression of zymosan phagocytosis in guinea pig macrophages

Recently, we characterized a surface antigen (Z-1) of guinea pig macrophages by monoclonal anti-Z-1 antibody. The Z-1 antigen consists of two different polypeptide chains; alpha (140 kDa) and beta (95 kDa). This antigen is closely correlated with the phagocytic activity of the cells for zymosan and presumably functions as a receptor for zymosan. In the present study, the effect of phorbol 12-myristate 13-acetate (PMA) on the function of Z-1 was examined. Incubation of ortho-[32P]phosphate-labeled macrophages with PMA greatly increased the phosphorylation of the beta subunit of Z-1 but not that of the alpha subunit. Optimal phosphorylation was observed when cells were incubated with 300 ng/ml of PMA for 60-120 min. The PMA-induced phosphorylation was markedly suppressed by treatment of the macrophages with H-7, an inhibitor of protein kinase C. A chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP) also caused phosphorylation of the beta subunit. Unlike PMA, fMLP maximized the phosphorylation within 30 s. Purified Z-1 was an excellent substrate for the exogenously added protein kinase C only in the presence of both Ca2+ and phosphatidylserine. H-7 completely inhibited the in vitro phosphorylation. These data suggest that the beta subunit of Z-1 is phosphorylated by protein kinase C. The phosphorylation of Z-1 by PMA and fMLP coincided with inhibition of zymosan phagocytosis. A linear relationship was obtained between the level of phosphorylation of Z-1 and the degree of inhibition of zymosan phagocytosis induced by PMA. Thus, the results suggest that zymosan uptake is negatively regulated by protein kinase C-mediated phosphorylation of the beta subunit of Z-1.     

Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors

We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Modulation of the expression of L-selectin by chemotactic agents and GM-CSF

L-selectin is an adhesion molecule with constitutive expression located on the membrane of granulocytes, monocytes and lymphocytes. It is involved in the early stages of migration of these cells toward either the sites of inflammation or lymphoid tissues. After the cells are activated, L-selectin is down-regulated with shedding of a soluble fragment. Flow cytometry was used to measure L-selectin expression levels on the granulocyte surface, after incubation with a phorbol esther (PMA), two chemotactic factors (fMLP and LTB4) and a cytokine (GM-CSF). Under basal conditions, the expression of L-selectin was found in a high percentage (95.0 +/- 0.7) of granulocytes; PMA stimulation led to a marked decrease in expression (3.2 +/- 0.6). Chemotactic factors also led to a significant decrease in L-selectin expression (69.9 +/- 5.0 for LTB4, and 53.70 +/- 4.3 for fMLP), whereas the incubation with GM-CSF produced no significant changes (89.1 +/- 4.8). When all the conditions were compared, the PMA effect was significantly higher than those observed with other stimuli; furthermore, the expression upon incubation with fMLP and LTB4 was statistically significant. These results suggest that the level of activation reached by granulocytes is directly related to their capacity for shedding L-selectin from the cell surface, and that these levels are lowered after the stimulation by chemotactic factors. GM-CSF activates several important functions of granulocyte cells, however it does not induce L-selectin shedding.     

Intracellular Ca2+ mobilization in immature and more mature U937 induced to differentiate by dimethyl sulfoxide or phorbol myristate acetate

Intracellular Ca2+ mobilization in U937 cells was studied. Stimulation of immature U937 cells with leukotriene B4 (LTB4) increased intracellular Ca2+ levels, whereas stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) failed to increase intracellular Ca2+ levels. U937 cells cultured with 1.5% dimethyl sulfoxide (DMSO) for 4 days (DMSO-U937 cells) responded to LTB4 and possessed the ability to respond to fMLP. U937 cells cultured with 1 ng/ml phorbol myristate acetate (PMA) for 4 days (PMA-U937 cells) lost the ability to respond to LTB4, although they responded to fMLP. Treatment of DMSO-U937 cells with 100 ng/ml PMA for 3 min suppressed intracellular Ca2+ increase induced by LTB4 and fMLP. The fMLP-induced Ca2+ rise in PMA-U937 cells was not suppressed by a further treatment with 100 ng/ml PMA. DMSO-U937 cells responded to inositol 1,4,5-trisphosphate (IP3), indicating that IP3 functions as a messenger of intracellular Ca2+ mobilization from endoplasmic reticulum in U937. The magnitude and duration of the rise in Ca2+ induced by IP3 in DMSO-U937 cells treated with 100 ng/ml PMA for 3 min were similar to those of the controls. When DMSO-U937 cells were Ca2+-depleted, addition of Ca2+ resulted in a transient overshoot of Ca2+ influx. However, the transient overshoot was not observed, when PMA-U937 cells were tested. These results indicate that Ca2+ efflux in PMA-U937 cells is increased by an activated exit pump, which may be directly or indirectly related to the functional state of PMA-U937 cells.     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

Mg.ATP primes superoxide-generating responses in electropermeabilized neutrophils

The present study utilizes an electropermeabilized cell system to determine the effect of Mg.ATP on neutrophil superoxide (O2-)-generating responses stimulated by suboptimal concentrations of fMLP, GTP gamma S and PMA. Permeabilization in the presence of exogenously added Mg.ATP was neither sufficient to initiate O2- release nor necessary for stimulated O2- production. However, the inclusion of Mg.ATP in the permeabilization medium primed the O2(-)-generating responses mediated by suboptimal concentrations of these stimuli. The site of action of Mg.ATP is intracellular. Moreover, the fact that Mg.ATP primes responses stimulated by fMLP, GTP gamma S and PMA suggests that the modulatory effect is at the level of protein kinase C.     

Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids

The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Role of extracellular signal-regulated kinase and phosphatidylinositol-3 kinase in chemoattractant and LPS delay of constitutive neutrophil apoptosis

The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.     

Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.     

Calcium-dependent eicosanoid metabolism by concanavalin A-stimulated human monocytes in vitro. Synergism with phorbol ester indicates separate regulation of leukotriene B4 synthesis and release.

Human monocytes obtained by counter-current centrifugal elutriation released arachidonic acid when challenged in vitro with Con A, as well as with other soluble (PMA or ionomycin) or particulate stimuli (serum-treated zymosan). Cyclo-oxygenase metabolites were the principal eicosanoids detected in the supernatants of Con A-stimulated, [3H]arachidonate-labeled monocytes, 5-Lipoxygenase (5-LO) products, such as leukotriene B4 (LTB4), were conspicuously absent. Release of arachidonate and its metabolites in response to Con A was dependent on the presence of extracellular Ca2+, but not Mg2+. In contrast to serum-treated zymosan challenge, which resulted in increased inositol trisphosphate and LTB4 release, Con A-induced inositol phospholipid hydrolysis in monocytes was limited to phosphatidylinositol or phosphatidylinositol monophosphate. Despite an inability to augment LTB4 release, Con A or PMA induced a loss of 5-lipoxygenase from a cytosolic compartment that was similar to that achieved with a calcium ionophore (ionomycin), a potent stimulus for LTB4 generation. When cell-associated LTB4 was evaluated, evidence for increased LTB4 production was obtained in response to either stimulus (PMA greater than Con A). In combination, however, PMA and Con A treatment resulted in monocyte LTB4 release comparable with that observed with the calcium ionophore or STZ. LTB4 release in response to all stimuli tested was inhibited by MK-886, a drug that binds to 5-lipoxygenase-activating protein. These results indicate the following: 1) Phospholipase A2 activation and attendant arachidonic acid release induced by agents that increase intracellular Ca2+ and/or generate diacylglycerol results in increased synthesis and release of PG and increased synthesis of leukotrienes, but not necessarily leukotriene release. 2) 5-LO translocation, which may occur independently of increased intracellular Ca2+, may be necessary for LTB4 generation but is insufficient for its release. 3) 5-Lipoxygenase-activating protein activity is necessary for 5-LO activation and LTB4 release in response to all stimuli investigated here. 4) Phorbol ester, an activator of protein kinase C, may synergize with agents such as Con A (which by themselves induce a minimal intracellular Ca2+ rise), so as to result in the release of LTB4. Thus, Con A may represent a class of surface receptor-aggregating agents that initiates inflammatory changes or immunomodulation associated with liberation of PG and might predispose to release of other inflammatory mediators, such as leukotrienes, in the presence of additional signals including protein kinase activation.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.