Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity

The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.     

Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.     

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.     

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.     

Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum

The ability of thioglycollate (TG)-elicited mouse peritoneal macrophages to adhere to a laminin substratum has been studied. These cells do not adhere to laminin-coated (20 micrograms/ml) surfaces, but the addition of phorbol myristate acetate (PMA; 50 ng/ml) results in their rapid adherence and spreading on this substratum. TG-elicited and PMA-activated macrophages, however, can bind soluble laminin. Macrophages adhere to fibronectin-coated surfaces and tissue culture plastic without PMA stimulation, and PMA does not increase the number of cells that adhere to these surfaces. The predominant surface proteins that bind specifically to laminin-Sepharose exhibit an Mr of 67 and 36 kD, but the expression of these proteins does not increase after PMA stimulation. Laminin receptor antibodies immunoprecipitate the 67-kD protein from radiolabled surface lysates and are capable of blocking macrophage adherence to a laminin substratum. Indirect immunofluorescence microscopy indicates that PMA stimulation does not increase receptor expression, but that it may induce the aggregation of the receptor on the cell surface. PMA stimulation also promotes macrophage spreading and induces a reorganization of the actin cytoskeleton. Taken together, these data indicate the mechanism by which PMA promotes macrophage adherence to laminin does not involve increased 67-kD receptor surface expression, but that it is related to the changes in cytoskeletal and receptor surface organization that occur in response to PMA stimulation.     

Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression

The extracellular matrix component, laminin, enhances the chemotactic responsiveness of polymorphonuclear leukocytes (PMN) in vitro, and low doses of chemoattractant substances augment the expression of PMN cell surface receptors for laminin. This study determined whether laminin acts in concert with chemoattractants to activate PMN. Laminin (5 to 100 micrograms/ml) stimulated lysozyme release and superoxide production in response to the chemoattractant, FMLP by as much as 69%. These results could be explained by changes in cell surface chemoattractant receptor expression in that incubation of normal PMN with laminin (5 to 75 micrograms/ml) increased the binding of 19 nM FML[3H]P by 35 to 80%. This corresponded to as much as a 2.5-fold increase in the number of chemoattractant receptors/cells which had a lower average affinity. Laminin did not change the number or affinity of FML[3H]P receptors present on organelle-depleted PMN cytoplasts, and the laminin-induced increase in FML[3H]P receptors expressed on PMN from a patient with a specific granule deficiency was only 11 to 21% of that seen in normal PMN. These findings suggest that chemoattractants augment the expression of laminin receptors which mediate PMN attachment to basement membranes, followed by laminin-induced increases in the expression of cryptic chemoattractant receptors contained in intracellular granules, with resultant augmentation of the oxidative burst.     

Prostaglandin E1 inhibits N-formyl-methionyl-leucyl-phenylalanine-mediated depolarization responses by decreasing the proportion of responsive cells without affecting chemotaxin-induced forward light scatter changes

Hypaque-Ficoll-purified human polymorphonuclear neutrophils (PMN) equilibrated with the membrane potential-sensitive probe 3,3'dipentyloxacarbocyanine [di-O-C(5)(3)] were incubated with buffer or cytochalasin B (cyto B) followed by incubation with prostaglandin E1 (PGE1) (0 to 10(-5) M) for 5 min at 37 degrees C. The cells were then stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) (0 to 10(-5) M). Changes in forward light scatter (FWD-SC), 90 degrees scatter (90 degrees -SC), and fluorescence intensity were measured by flow cytometry to determine the effects of PGE1 on FMLP-induced shape change, secretion, and membrane potential responses, respectively. In other experiments, the effects of PGE1 preincubation on FMLP +/- cyto B and phorbol myristate acetate-induced (O2) production were measured by superoxide dismutase-inhibitable cyto c reduction. PGE1 had no direct effects on the FWD-SC, 90 degrees-SC, or resting potential fluorescence of unstimulated or cyto B-pretreated PMN. PGE1 produced a dose-dependent inhibition of the proportion of depolarizing PMN in response to FMLP, which was maximal at 10(-6) M (42.1 +/- 6.9% inhibition, p less than 0.005), but was apparent at 10(-8) M. The PGE1-induced inhibition was maximal after 30 sec of incubation at 37 degrees C and was caused by a decrease in the maximal percentage of depolarizing PMN without a significant change in the FMLP dose-response curve (Km = 2.43 vs 3.62 X 10(-8) M, control vs PGE1-treated) or an inhibition in the degree of depolarization by the responding subpopulation. PGE1 also inhibited the loss of 90 degrees-SC induced by FMLP in cyto B-pretreated cells (secretion response) (46.2 +/- 16.5% inhibition of the maximal 90 degrees-SC loss, n = 5, p less than 0.005), but did not affect the increase in FWD-SC seen with FMLP-induced PMN activation or the ability of cyto B to recruit more PMN to depolarize. PGE1 also inhibited FMLP +/- cyto B-induced O2 production in a dose-dependent fashion; phorbol myristate acetate-induced O2 production was also slightly inhibited, but only at high PGE1 concentrations. The data indicate that PGE1 inhibits FMLP-induced cell activation by a mechanism that involves a step distal to the recruitment of unresponsive PMN by cyto B, and that PGE1 is capable of inhibiting depolarization responses without affecting FMLP-induced shape change, providing more support for a dissociation between the two activation pathways.     

Independent regulation of human neutrophil chemotactic receptors after activation

The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) over the range of PMA concentrations from 0.5 to 50 ng/ml. In contrast, activation of PMN with PMA resulted in a dose-dependent decrease in FL-C5a binding, and activation with concentrations above 5 ng/ml resulted in a complete loss of binding. This loss of binding was not caused by inactivation of the ligand or prevented by the addition of superoxide dismutase and catalase or protease inhibitors. Furthermore, incubation of PMN with supernatants from PMN stimulated to degranulate did not reduce the availability of C5a receptors. This pattern of increased FMLPL and casein binding with decreased C5a binding was also observed with cytochalasin B-pretreated PMN that were stimulated with chemotactic factors. Parallel studies of superoxide anion generation demonstrated that PMA-treated PMN were still responsive to formyl-methionyl-leucyl-phenylalanine, but not to C5a. These data demonstrate that the activation of PMN up-regulates formyl peptide and casein receptors whereas C5a receptors are down-regulated under similar conditions.     

Alterations in cell protein phosphorylation in human neutrophils exposed to influenza A virus. A possible mechanism for depressed cellular end-stage functions

When polymorphonuclear leukocytes (PMN) are exposed to most harvests of influenza A virus (depressing virus, DV) for 20 min, chemotactic, secretory, and oxidative functions are depressed upon subsequent exposure to soluble or particulate stimuli. Other harvests of influenza A virus (non-DV) do not alter these activities. The DV-induced changes in multiple functions suggest the virus may interfere with steps involved in PMN activation. Because some of these steps may be regulated by protein phosphorylation, we examined the effect of non-DV and DV on cellular protein phosphorylation. PMN loaded with 32P-labeled inorganic orthophosphate were exposed to non-DV, DV, or buffer for 30 min; cells were then treated with buffer, FMLP (10(-6) M), or PMA (100 ng/ml) for 30 s. Samples were sonicated and centrifuged; cytosolic and particulate fractions were analyzed by SDS-PAGE and autoradiography. Exposure of PMN to either non-DV or DV caused phosphorylation of several cell proteins. However, when DV-treated PMN were then stimulated with FMLP or PMA, further phosphorylation was inhibited compared to non-DV- or buffer-treated cells. This suggests that DV-induced depression of PMN end-stage functions may be due to changes in cell protein phosphorylation. DV could interfere with phosphorylation of PMN proteins by altering protein kinase activity. We therefore examined the influence of non-DV and DV on some parameters that could affect kinase function. PMN intracellular [Ca2+] was monitored by using the fluorescent Ca2+ indicator, Indo 1, and cAMP levels were measured by RIA. PMN treated with DV alone or DV plus FMLP had higher intracellular [CA2+] than PMN similarly treated with non-DV or buffer. Exposure of PMN to non-DV, DV, or buffer caused minimal changes in cAMP levels, and similar increases occurred in cAMP levels upon FMLP stimulation. To determine whether DV interferes with transmembrane signaling, the effect of influenza virus on PMN transmembrane potential was studied by using a fluorescent cyanine dye. Transmembrane potential changes were greater in PMN exposed to DV than to non-DV or buffer; however, subsequent stimulation with FMLP caused equivalent changes in transmembrane potential. Our data show that protein phosphorylation in PMN is induced by DV and non-DV infection; upon subsequent stimulation with FMLP or PMA, there is inhibited cellular phosphorylation only in PMN previously exposed to DV.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Effects of pepstatin A on neutrophils; cross-deactivation with FMLP

The ability of pepstatin A, a protease inhibitor produced by Streptomyces testaceus, to elicit a number of responses by the human PMN has been studied. In lysozyme and beta-glucuronidase release, pepstatin A 10(-5)M is equivalent to the synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 10(-7)M. In superoxide release, pepstatin A 10(-5)M produces 80% of that originated by FMLP 10(-7). After two minutes of incubation the superoxide release is important, there being no further increase after 10 minutes. Preincubation of the cells with cytochalasin B before stimulation with pepstatin A elicits a noticeable increase in O2- release. In chemotaxis, pepstatin A 10(-6) originates the same cell motility as FMLP 10(-9). Pepstatin A produces a cross deactivation with FMLP which adds further evidence to the hypothesis that both stimuli compete for the same receptor in the PMN.     

Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes

Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases to levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for by alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity (deactivation). In contrast with its effects on C3 receptors, PMA causes only a reduction in the capacity of Fc receptors to bind and ingest IgG-coated erythrocytes. Deactivation of Fc receptors also appears to involve a qualitative alteration in receptors, because the binding affinity of soluble immune complexes is sharply reduced upon stimulation of PMN with PMA. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, we loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc receptors, suggesting that the deactivation is the result of a dephosphorylation event.     

Human C5a modulates monocyte Fc and C3 receptor expression

FcIgG and C3 (CR1 and CR3) receptors are responsible for binding opsonized particles, phagocytosis, and immune adherence reactions by circulating and tissue-fixed mononuclear phagocytes. Alterations in the expression of these receptors may thus significantly influence the function of these cells. Because chemoattractants have been shown to both recruit and modulate the function of monocytes, this study specifically examines the effects of human C5a and N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) on human peripheral blood monocyte FcIgG and C3 receptor expression in vitro. Adherent, elutriator-purified monocytes were incubated with C5a (10(-7) to 10(-10) M) or FMLP (10(-5) to 10(-10) M) for 30 min at 37 degrees C, and FcIgG receptor expression was assessed by rosetting with sheep erythrocytes sensitized with limiting dilutions of IgG. Human C5a caused dose-related increases in Fc rosettes of 28% at 10(-9) M, 63% at 10(-8) M, and 167% at 10(-7) M (p less than 0.01). In contrast, no significant increases in monocyte Fc receptor expression were induced by FMLP. Similar rosetting experiments were performed with sheep erythrocytes opsonized with limiting amounts of human C3b to assess C3b receptor expression on adherent human monocytes stimulated with C5a (10(-7) to 10(-10) M) or FMLP (10(-6) to 10(-9) M) for 30 min at 37 degrees C. Again, human C5a caused dose-related increases in monocyte C3b rosette formation; at 10(-8) M and 10(-7) M concentrations of C5a, these increases equaled 119% and 196%, respectively (p less than 0.05). In these experiments, 10(-6) M FMLP also caused a significant increase of 110% in monocyte C3b rosette formation (p less than 0.05). Modulation of monocyte cell surface receptors by human C5a or FMLP was also examined by measuring cell fluorescence and side scatter by dual channel flow cytometry after staining normal leukocytes in citrated venous blood with receptor-specific monoclonal antibodies. These flow cytometric studies demonstrated that both C5a and FMLP induce dose-related increases in CR1 (C3b receptor) and CR3 (iC3b receptor) expression in both monocytes and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.     

Chemiluminescence properties of human,canine and rat polymorphonuclear cells

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct different densities were used. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/I), phorbol myristate acetate (PMA, 2.8 × 10^?6 mol/I), calcium ionophore A 23187 (10^?5 mol/l) and N-formylmethionyl-leucyl-phenylalanine (FMLP, 3.5 × 10^?6 mol/l). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity could be ordered: human > canine > rat. Response to the various agents in the human cells can be ranked: PMA ? A 23187 > zymosan > FMLP; for the dog: A 23187 > PMA > zymosan > FMLP; and for the rat: zymosan ? PMA > FMLP ? A 23187. Time course and peak maximum response were different upon stimulation in the absence and presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.     

Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils

Lipoxins (LX) are trihydroxytetraene metabolites derived from arachidonic acid via an interaction between the 5- and 15-lipoxygenases. Preincubation of [3H] myo-inositol labeled PMN with 10-7M and 10-5M LXA4 for 1 minute at 37 degrees C resulted in a concentration dependent inhibition of the generation of [3H] IP3 and [3H] IP in cells subsequently stimulated by increasing doses of LTB4 or FMLP for 1 minute at 37 degrees C. Preincubation of PMN with LXA4 did not inhibit specific binding of [3H] LTB4 to PMN. These results indicate that LXA4 inhibits chemotactic factor-induced phosphoinositide hydrolysis at a post-receptor level.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.