Stimulation Of Polymorphonuclear Leukocytes Research Articles

Stimulation of human polymorphonuclear leukocytes by leukotriene B4 and platelet-activating factor: an ultrastructural and pharmacological study.

Human polymorphonuclear leukocytes (PMNs) stimulated by N-formylmethionyl-leucylphenylalanine (fMLP), leukotriene B4 (LTB4) and platelet-activating factor (PAF) showed a similar sequence of ultrastructural changes. Exposure of unstirred PMNs to these soluble stimuli induced a rapid (within 10 s) plasma membrane ruffling followed by polarization of cells associated with formation of broad agranular lamellipodia (maximal by 30 s). Similar changes were observed in stirred PMN suspensions except that the polarized cells formed multicellular aggregates with the agranular lamellipodia extending outward from the clumps. In addition, these receptor-directed agonists induced concentration-dependent PMN aggregation that was rapid and reversible, and in cells pretreated with cytochalasin B the agonists induced a degranulatory response. The calcium ionophore ionomycin also activated PMNs but, in contrast to the other stimuli, ionomycin produced irreversible aggregation and evoked enzyme release in the absence of cytochalasin B. Ionomycin stimulated LTB4 release, whereas the response induced by PAF or fMLP was barely detectable. The possible role of LTB4 and PAF in mediating responses to other agonists was investigated pharmacologically using the 5-lipoxygenase inhibitor PF 5901 and a specific PAF antagonist, kadsurenone. PF 5901 blocked ionomycin-induced LTB4 release but did not inhibit aggregation or degranulation induced by ionomycin, PAF, fMLP, or LTB4. Kadsurenone blocked PAF-induced aggregation and degranulation but not responses to LTB4, ionomycin, or fMLP. We conclude that LTB4 and PAF are potent activators of human PMNs that act independently of one another and that these lipids appear not to be involved in the responses to the other agonists studied.

Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding.

Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 +/- 0.5 x 10(-7) M and 5.7 +/- 1.3 x 10(-6) M, respectively. Stimulation of PMNLs by 10(-6) M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/10(7) cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 +/- 79 pg/10(7) cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 +/- 0.5 x 10(-6) M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 +/- 0.4 x 10(-5) M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.

Complement activation and polymorphonuclear neutrophil leukocyte elastase in sepsis. Correlation with severity of disease.

Complement activation is necessary for an adequate immune and inflammatory response to infections. Activation releases anaphylatoxins that cause vasodilation, increase vascular permeability, and trigger release of polymorphonuclear neutrophil leukocyte (PMN) lysosomal enzyme and oxygen radicals. Under normal circumstances, an orderly progression of such events has a beneficial antimicrobial effect. The same mechanism, however, when uncontrolled, may damage host tissues. To provide information about the clinical importance of such events in sepsis, different complement parameters (C3, C4, and the desarginated forms of C3a [C3a(des)-Arg] and C5a [C5a(des)-Arg]), PMN elastase, and malondialdehyde (a by-product of membrane peroxidation by oxygen radicals) were measured daily in 26 septic patients and correlated with two objectively assessed and previously validated severity scores (acute physiology and chronic health evaluation [APACHE II] and Sepsis Severity Score [SSS]). Nonsurvivors (n = 12) had significantly greater and longer lasting complement activation than that in survivors, as reflected by higher levels of catabolic peptides (C3a(des)-Arg) and lower levels of native proteins (C3 and C4). C3a(des)-Arg, C3, C4, and the C3a(des)-Arg-C3 ratio were correlated with Sepsis Severity Scores. Polymorphonuclear neutrophil leukocyte elastase levels were higher in nonsurvivors and were correlated with C3a(des)-Arg and the C3a(des)-Arg-C3 ratio. Malondialdehyde levels were significantly higher in all patients than in controls, without, however, any relationship to severity of disease or clinical outcome. Since the higher and more persistent the complement activation and polymorphonuclear neutrophil leukocyte stimulation, the worse the patient's prognosis, we conclude that these mechanisms may be important in the clinical development of sepsis.

Albumin and fatty acid effects on the stimulated production of 1- O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) by human polymorphonuclear leukocytes

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 μM and at a palmitic acid concentration of 100 μM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 μM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 μM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.

Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes.

Phosphatidic acid phosphohydrolase (PPH) activity was determined in human polymorphonuclear leukocytes (PMNs) by measuring the hydrolysis of [32P]phosphatidic acid (PA) added to cell sonicates. Enzyme activity was localized primarily to a soluble fraction. Soluble and particulate activities required magnesium and were inhibited by calcium, N-ethylmaleimide, sphingosine, and propranolol. The activity in unstimulated PMNs was 0.64 +/- 0.11 nmol of PA hydrolyzed.mg protein-1.min-1 in particulate and 4.20 +/- 0.42 in soluble fractions. Stimulation of PMNs with 1 microM f-Met-Leu-Phe (FMLP) for 10 min caused a slight decrease in soluble activity and a small increase in the activity of particulate fractions. Preincubation with 10 microM cytochalasin B for 5 min before FMLP stimulation markedly enhanced both of these changes. The effect of FMLP plus cytochalasin B was rapid (less than 10 s), whereas the calcium ionophore A23187 (1 microM) and phorbol myristate acetate (100 ng/ml) caused slower and smaller changes in enzyme activity. These results indicate that after chemoattractant stimulation; PPH activity decreases in the soluble fraction and increases in the particulate fraction suggesting that PPH may participate in signal transduction in the PMN.

Stimulation of human polymorphonuclear leukocytes by recombinant human interleukin-1 beta.

Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes. The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1 beta and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-1 beta elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8 x 10(-11)-8.8 x 10(-8) M. The effect could be blocked by prior treatment with the IL-1 receptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1 beta enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40%. The priming effect of rhIL-1 beta could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1 beta failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1 beta has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1 beta is a direct and specific, but probably weak stimulator of the PMN leukocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the selectivity of enzymes involved in platelet‐activating factor formation in stimulated cells

The present studies were undertaken to obtain further insight into the selectivities of the enzymes, i.e., phospholipase A2 and acetyltransferase, involved in platelet-activating factor (PAF) production upon stimulation of human polymorphonuclear leukocytes (PMN) and platelets. After appropriate stimulation of the cells in the presence of [3H]acetate the total PAF and analogs, i.e., 1-alkyl-2-acetyl-, 1-alkenyl-2-acetyl-, and 1-acyl-2-acetyl-glycero-3- phosphocholine were isolated by high performance liquid chromatography. The isolated mixture was subjected to treatment with phospholipase A1 to differentiate acetate incorporation into 1-ether linked and 1-ester linked species. The ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs amounted to 13.8 +/- 1.0 and 1.3 +/- 0.1 for PMN and platelets, respectively. When compared to the ratio of 1-ether linked and 1-ester linked species in the diradylglycerophosphocholine precursors in each cell type, i.e., 1.13 for PMN and 0.22 for platelets, these data suggested a pronounced selectivity for the phospholipase A2 and/or acetyltransferase in the process of PAF production. When the experiments were repeated with cells that had been pretreated with phenylmethanesulfonylfluoride (PMSF) to block the acetylhydrolase, the most dramatic effects were observed on acetate incorporation into 1-acyl-2-acetyl-glycero-3-phosphocholine, which increased much more than that into 1-alk(en)yl-2-acetyl-glycero-3-phosphocholine. Under these conditions, the ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs became 1.4 +/- 0.2 and 0.17 +/- 0.02 for PMN and platelets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide-induced stimulation of CD11b/CD18 expression on neutrophils. Evidence of specific receptor-based response and inhibition by lipid A-based antagonists.

Gram-negative bacterial septicemia is a common clinical syndrome resulting, in part, from the activation of phagocytic leukocytes by LPS. By using flow cytometry, we have characterized LPS-induced expression of the beta 2 integrin CD11b/CD18. After exposure to Salmonella minnesota R595 LPS, expression of neutrophil CD11b/CD18 is rapidly upregulated, beginning within 5 min and achieving a peak fluorescence (typically two- to threefold over base line) by 30 min. The increase in CD11b/CD18 expression was similar in kinetics and magnitude to that produced by FMLP, PMA, and human rTNF-alpha. Concentrations of LPS necessary to stimulate a response were as low as 1 ng/ml of R595 LPS; a maximal response was observed between 30 and 100 ng/ml. The upregulation of CD11b/CD18 due to LPS was not interrupted by protein synthesis inhibitors. A group of glucosamine disaccharide lipid A-like molecules: Rhodobacter sphaeroides lipid A, lipid IVA, KDO2IVA, and deacylated LPS were able to block the stimulatory effect of LPS. This inhibition was specific for the actions of LPS as stimulation of polymorphonuclear leukocytes (PMN) by FMLP, human rTNF alpha, PMA, and rewarming were not altered by the disaccharide inhibitors. PMN which were exposed to the specific disaccharide LPS antagonists and then washed, were refractory to stimulation by LPS. The monosaccharide lipid A precursor lipid X also blocked stimulation of neutrophils by LPS, although with a 100-fold reduction in potency. Unlike the disaccharide inhibitors, PMN exposed to lipid X were still responsive to LPS stimulation after washing. The PMN response to LPS was less sensitive in the absence of serum, although upregulation of CD11b/CD18 could still be seen using higher concentrations of LPS. Monoclonal antibody directed against CD14 (clone 3C10), also specifically inhibited LPS induced PMN CD11b/CD18 expression both in the presence and absence of serum. These findings support the hypothesis that LPS stimulates neutrophils by interacting with specific cellular receptors.

Leukocytic ·O 2 − and cardiac dysfunctions in isolated perfused rat hearts

Superoxide radicals are supposed to contribute to myocardial reperfusion injury. Their origin, however, is still a matter of debate. Polymorphonuclear leukocytes (PMNL) have been discussed as a major postischaemic .O2- source [Lucchesi and Mullane (1986) Ann Rev Pharmacol Toxicol 26: 201-224]. We studied the role of .O2- derived from human polymorphonuclear leukocytes in reperfused and normoxic perfused isolated rat hearts. During reperfusion PMNL exerted deleterious effects on different parameters (e.g. contractility, coronary flow) of isolated rat hearts. Under normoxic perfusion conditions stimulation of PMNL with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) caused bradycardia and a transient vasoconstriction. Superoxide dismutase (SOD) administration did not influence any of the PMNL effects mentioned, suggesting that leukocytic .O2- was not involved in PMNL-induced cardiac dysfunctions.

Release of proteinases from stimulated polymorphonuclear leukocytes

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes.

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on beta-glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.

Possible existence of leukotriene D4 receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes.

Previous work from this laboratory has demonstrated that stimulation of human polymorphonuclear leukocytes (PMN) with the peptidyl leukotriene D4 (LTD4) results in the formation of second messenger signals, i.e. mobilization of intracellular free Ca2+ ([Ca2+]i) and hydrolysis of phosphoinositides (PIP2). Based on these earlier results we have employed radioligand binding techniques to study the presence of LTD4 receptors in intact human PMN leukocytes. The binding of [3H]-LTD4 to LTD4 receptors is rapid, reversible, specific and saturable. Scatchard analysis of the binding data indicates the presence of 116-275 identical receptors per neutrophil with an apparent dissociation constant (KD) of 1,1-2,3 nM. Only one class of binding sites was identified. The LTD4 receptors are located in the plasma membrane and are specific for LTD4 since binding is unaffected by other leukotrienes. Furthermore, LTD4 induces a rapid and persistent translocation of Protein Kinase C (PKC) from the cytosol to the membrane. The LTD4 binding and PKC translocation could be blocked in a concentration dependent manner by the new and specific LTD4 receptor antagonist ICI 198,615. These observations strongly suggest that human PMN might possess specific LTD4 receptors which are coupled to the inositol trisphosphate pathway resulting in a rise in the cytoplasmic free Ca2+ and redistribution of protein kinase C. A mechanism of signal transduction for leukotriene D4 is proposed.

Stimulation of human polymorphonuclear leukocytes potentiates the uptake of diclofenac and the inhibition of chemotaxis

Diclofenac sodium, a non-steroidal anti-inflammatory drug, has been shown to impair the stimulation of human polymorphonuclear leukocytes (PMNs) by chemoattractants. To gain insight into the mechanism of action of this agent, we investigated the uptake of diclofenac by resting and activated PMNs and the effect of the drug on PMN locomotion. During incubation of resting PMNs at 37° in the presence of 78 μM (25 μg/mL) diclofenac, drug uptake reached a plateau in less than 2 min. The resulting cellular to extracellular diclofenac concentration ratio ( C E ) was 1.01 ± 0.13 (mean ± SD). Stimulation of PMNs at 37° but not at 4° with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), induced a rise in diclofenac uptake, which was dependent on incubation lime and diclofenac and stimulus concentrations. Maximal C E was 1.83 ± 0.18 and 4.40 ± 0.60 (mean ± SD) for PMNs stimulated with 10 μM fMLP and 0.16 μM PMA, respectively. The diclofenac associated with PMNs was predominantly present in the soluble fraction of disrupted cells. Interestingly, PMNs which were pretreated with diclofenac and stimulated with fMLP, exhibited impaired random and directional locomotion induced by activated serum, as compared to controls, i.e. PMNs treated with diclofenac alone or fMLP alone. Thus, stimulation of PMNs enhances diclofenac uptake and potentiates the drug impairment of chemotactic activity. These findings could explain, in part, the observed anti-inflammatory properties of this compound.

Metabolism of arachidonic acid by peripheral and elicited rat polymorphonuclear leukocytes. Formation of 18- and 19-oxygenated dihydro metabolites of leukotriene B4.

Previous studies have shown that leukotriene B4 is metabolized by polymorphonuclear leukocytes (PMNL) by a 20-hydroxylase, a 19-hydroxylase, and a reductase. We have now identified for the first time LTB4 metabolites formed by a combination of the reductase and omega-oxidation pathways. We have also discovered that rat PMNL metabolize LTB4 by a novel pathway to 18-hydroxy products. Dihydro metabolites of LTB4 have formerly been reported only after incubation of exogenous LTB4 with PMNL, but we have now shown that they are formed to the same extent from endogenous arachidonic acid after stimulation of PMNL with the ionophore, A23187. The following metabolites have been identified after incubation of either LTB4 or arachidonic acid with rat PMNL: 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, 10,11-dihydro-12-oxo-LTB4, 19-hydroxy-LTB4, 19-hydroxy-10,11-dihydro-LTB4, 19-oxo-10,11-dihydro-LTB4, 18-hydroxy-LTB4, 18-hydroxy-10,11-dihydro-LTB4, and 18-hydroxy-10,11-dihydro-12-oxo-LTB4. Negligible amounts of 20-hydroxylated products were formed. Incubation of PMNL with 10,11-dihydro-LTB4 resulted in the formation of all of the above dihydro metabolites. However, none of the omega-oxidized metabolites of LTB4 was further metabolized to a significant extent when incubated with PMNL, possibly at least partially because they were not substrates for a specific LTB4 uptake mechanism. We found that the biosynthesis and metabolism of LTB4 is considerably enhanced in PMNL from an inflammatory site (carrageenan-induced pleurisy) compared with peripheral PMNL. When arachidonic acid was the substrate, the greatest increase was observed for products formed by the reductase pathway, which were about eight times higher in pleural PMNL. The rates of formation of both LTA hydrolase and omega-hydroxylase products were about three times higher, whereas the total amounts of 5-lipoxygenase products were about twice as high in pleural PMNL. The amounts of products formed by the above enzymatic pathways reached maximal levels about 4-6 h after injection of carrageenan and then declined.

The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes.

Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.

Changes in superoxide dismutase activity during stimulation of peripheral blood polymorphonuclear leukocytes

Changes in superoxide dismutase activity during stimulation of peripheral blood polymorphonuclear leukocytes

Involvement of calcium in the thimerosal-stimulated formation of leukotriene by fMLF in human polymorphonuclear leukocytes

Only small amounts of leukotrienes could be detected by reverse-phase HPLC analysis after stimulation of human polymorphonuclear leukocytes (PMN) by the receptor agonist N-formymethionyl-leucyl-phenylalanine (fMLP). Preincubation of the cells with the organomercury compound thimerosal prior to fMLP-addition, however, resulted in the formation of significant amounts of 5-lipoxygenase derived metabolites. This effect was dose-dependent with respect both to fMLP and thimerosal. Thimerosal alone did neither lead to the formation of HPLC-detectable leukotrienes nor to the release of arachidonic acid in [1− 14C]arachidonic acid prelabelled cells. The formation of leukotrienes by fMLP/thimerosal required extracellular Ca 2+. Measurements of intracellular Ca 2+-levels revealed that (i) thimerosal alone is able to release Ca 2+ from internal stores and (ii) thimerosal causes a persistent accumulation of Ca 2+ within the cells after stimulation by fMLP. We conclude that by the synergistic action of fMLP and thimerosal the Ca 2+-levels exceed the threshold for phospholipase A 2 activation resulting in the liberation of arachidonic acid and subsequently in the formation of 5-lipoxygenase products. Our results suggest that thimerosal may provide a model for leukotriene formation under pathophysiological conditions when SH-group oxidation leads to increased intracellular Ca 2+-levels. k]abr|PMN, polymorphonuclear leukocytes; fMLP, N-formyl-methionyl-leucyl-phenylalanine; DMSO, dimethylsulfoxide; HO-Δ 4-Ach, hydroxyicosatetraenoic acid; HPLC, high performance liquid chromatography; LT, leukotriene. Enzymes: Acyl-CoA:lysophosphatidylacyltransferase (EC 2.3.1.23); Ca 2+-ATPase (EC 3.6.1.8); lactate dehydrogenase (EC 1.1.1.27); 5-lipoxygenase (EC 1.13.11.34); phospholipase A 2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); proteinkinase C (EC 2.7.1.37).

Difference in Changes of Membrane Fluidity of Polymorphonuclear Leukocytes Stimulated With Phorbol Myristate Acetate and Formyl-Methionyl-Leucyl-Phenylalanine: Role of Excited Oxygen Species

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time- and dose-dependent. FMLP at 10(-5) M induced a decrease, while FMLP at 10(-7) M induced a rapid increase. On stimulation with 10(-7) M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10(-5) M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.

Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL)

Activation of polymorphonuclear neutrophils (PMNL) leads to the release of arachidonate from cellular phospholipids via a phospholipase A 2, and conversion of products of the 5-lipoxygenase pathway. Evidence to date indicates the dietary vitamin E (( R,R,R)-α-tocopherol) can influence both cyclooxygenase and phospholipase A 2 activities and that the effect of this vitamin is cell/tissue specific. The present study was undertaken in order to examine the effects of varying dietary tocopherol on PMNL tocopherol content and 5-lipoxygenase product profile using the ionophore A23187 as stimulant in the presence and absence of exogenous arachidonate. Feeding semi-purified diets containing 0, 30 or 3000 ppm of( R,R,R)-α-tocopherol acetate to weanling rats for 17 weeks resulted in a dose-related enrichment of PMNL tocopherol. Stimulation of PMNL elicited a significant and rapid loss of tocopherol. When PMNL were stimulated with A23187 alone, the synthesis of 5-HETE, LTB 4 and 19-hydroxy-LTB 4 was decreased in proportion to increasing dietary tocopherol concentrations. However, when exogenous arachidonate was provided with A23187, intermediate amounts of dietary tocopherol (30 ppm) still suppressed the formation of 5-lipoxygenase products, but high doses (3000 ppm) did not have any additional inhibitory effect. This differential response to high concentrations of vitamin E in the presence and absence of exogenous arachidonate highly suggest that at these concentrations, tocopherol may act principally at the level of substrate release wherease at lower concentrations, 5-lipoxygenase is inhibited. Data from this study demonstrated that attenuation of the formation of 5-lipoxygenase products in PMNL can be achieved by dietary vitamin E enrichment.

Platelet-dependent induction and amplification of polymorphonuclear leucocyte lysosomal enzyme release.

Degranulation of human polymorphonuclear leucocytes induced by opsonized zymosan (OpZ) was studied in the presence or absence of platelets, either resting or stimulated by thrombin. Lysozyme secretion from polymorphonuclear leucocytes stimulated with OpZ increased in the presence of platelets. A further increase was observed when platelets had been stimulated with thrombin. The effect was dependent on platelet concentration (10-80 platelets/polymorphonuclear leucocytes). Resting polymorphonuclear leucocytes could be activated by platelets that had been stimulated with thrombin with an optimal effect observed at 0.1 U/ml of thrombin. Substitution of platelets with platelet-released products only resulted in significant stimulation of polymorphonuclear leucocytes at concentrations above those used in the coincubations experiments. Zymosan coated with various opsonins showed a gradient in the relative intensity of polymorphonuclear leucocytes degranulation. Platelet-dependent enhancement of polymorphonuclear leucocytes degranulation was higher with opsonins that were poorer at inducing lysosomal secretion. The role of platelets as helper cells in the activation of polymorphonuclear leucocytes appears to be dominant when polymorphonuclear leucocytes are challenged in suboptimal conditions. Positive interactions between platelets and polymorphonuclear leucocytes may be relevant in vivo at the site of inflammation where platelets could enhance effector functions of polymorphonuclear leucocytes.