Removal of human polymorphonuclear leukocyte surface sialic acid inhibits reexpression (or recycling) of formyl peptide receptors. A possible explanation for its effect on formyl peptide-induced polymorphonuclear leukocyte chemotaxis

Removal of surface sialic acid specifically inhibits human polymorphonuclear leukocyte (PMN) chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neuraminidase-treated (NT)-PMN bound and internalized [3H]FMLP (used as receptor marker) as well as normal PMN. NT-PMN, however, retained more [3H]FMLP-associated radioactivity than normal PMN. Subcellular fractionation studies demonstrated that NT-PMN retained more sedimentable (100,000 X G for 180 min) [3H]FMLP-associated radioactivity within light Golgi-containing fractions than normal PMN. Furthermore, NT-PMN exhibited a defect in their ability to reexpress (or recycle) a population of FMLP receptors. Abnormal receptor recycling was associated with inhibition of FMLP-induced PMN chemotaxis. Thus, it appears that recycling of formyl peptide receptors may be necessary for optimal PMN chemotactic responses to FMLP. We postulate that removal of PMN surface sialic acid inhibits FMLP-induced PMN chemotaxis by blocking the reexpression (or recycling) of a population of formyl peptide receptors, perhaps by preventing trafficking of desialated receptors through a light Golgi pathway.     

Preparation and characterization of a derivative of wheat germ agglutinin which specifically inhibits polymorphonuclear leukocyte chemotaxis to the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine

A nonagglutinating derivative of wheat germ agglutinin (WGA), prepared by treating the native lectin with cyanogen bromide and formic acid and purified by affinity chromatography on an N-acetyl-D-glucosamine column, inhibited human polymorphonuclear leukocyte (PMN) chemotaxis to the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The WGA derivative (WGA-D) did not influence either the ability of PMN to migrate randomly or their chemotactic response to the complement-derived peptide C5a. Similarly, WGA-D had no effect on either FMLP-induced PMN polarization or other FMLP-induced PMN functions (i.e., selective discharge of lysosomal enzymes from cytochalasin B-treated cells, generation of superoxide anion). The inhibition of FMLP-induced PMN chemotaxis by WGA-D could not be reversed by washing the cells, or by incubating lectin-treated PMN at 37 degrees C for 20 min. The inhibitory effect of WGA-D was mediated by its specific binding to N-acetyl-D-glucosamine residues on the cell surface. WGA-D did not alter the specific binding of [3H]-FMLP to its receptor(s) on the PMN membrane. The data presented here suggest that WGA-D inhibits FMLP-induced PMN chemotaxis at a step distal to stimulus recognition.     

A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.     

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.     

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.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Mechanisms of increased pulmonary microvascular permeability induced by FMLP in isolated rabbit lungs.

We observed that the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L- phenylalanine (FMLP) induced pulmonary edema when polymorphonuclear leukocytes (PMNs) were added to isolated constant-flow buffer-perfused rabbit lungs. This study was designed to test the hypothesis that PMNs activated by FMLP induced lung injury by the modulation of reactive oxygen species (ROS), cyclooxygenase products, or cysteinyl leukotrienes (LTs). Addition of FMLP alone did not increase microvascular permeability (Kf). When PMNs were added to the isolated lung, FMLP caused an 80% increase in Kf. Wet-to-dry weight ratio was also significantly increased with PMNs + FMLP compared with FMLP only. There was a significant positive correlation between total myeloperoxidase activity in lung tissue and Kf values after FMLP (30 min). Pretreatment with two dissimilar cyclooxygenase inhibitors, meclofenamate or ibuprofen, had no effect on the PMN + FMLP-induced increase in Kf. However, the ROS inhibitor catalase and the nonantioxidant LT synthesis blocker MK 886 inhibited the PMN + FMLP increase in Kf. Perfusate levels of LTs (LTC4, -D4, and -E4) were significantly increased from baseline values 30 min after FMLP. Both MK 886 and catalase suppressed the elevation of LTs after PMN + FMLP. These results indicate that FMLP increased a pulmonary microvascular permeability in isolated buffer-perfused rabbit lungs that is PMN dependent and mediated by LT produced possibly by a result of ROS production.     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

Evidence for a direct role for sialic acid in the attachment of encephalomyocarditis virus to human erythrocytes

Sialic acid residues are required in cellular receptors for many different mammalian viruses. Sialic acid could have a direct role, being an integral part of the virus binding site on the receptor. Alternatively, negatively charged sialic acid could have an indirect role, being responsible for holding the receptor in the required configuration for virus recognition, for instance, by interacting with positively charged amino acid residues found in the polypeptide chain of receptors. We have investigated the role of sialic acid in virus attachment by studying the interaction of the small RNA virus encephalomyocarditis (EMC) with glycophorin A, its receptor on human erythrocytes. In several experiments, influenza virus A was used for control purposes. Blocking positive charges on glycophorin either in lysine residues by acetylation or in arginine residues with butanedione did not affect its interaction with EMC virus. In contrast, blocking negatively charged carboxyl groups in sialic acid residues by amidation destroyed the ability of glycophorin to inhibit EMC virus attachment suggesting an important role for this part of sialic acid in EMC virus attachment. Removal of the polyhydroxy side chain in sialic acid residues of glycophorin by mild oxidation with periodate followed by reduction with borohydride had little effect on its interaction with EMC virus. Further, sialic acid species with either an acetyl or glycolyl group attached to the amino group on position 5 interacted equally well with EMC virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils.

We studied the action of iodinated thyronines on the superoxide (O2-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced O2- production with IC50 values of about 10(-6) M and 7.10(-6) M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the O2- production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O2- production stimulated with either the fifth component of the complement (C5a) or phorbol 12-myristate 13-acetate. L-Thyroxine and L-triiodothyronine were found to block [3H]FMLP binding to its own receptor with IC50 values similar to those for the inhibition of the O2- production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O2- production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.     

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.     

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.     

Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.     

Effects of cytochalasin B, N-formyl peptide and plasma on polarisation, zeiosis (blebbing) and degranulation of polymorphonuclear leukocytes in suspension

The effects were studied of cytochalasin B and N-formyl peptide (FMLP) in various concentrations on the morphology and degranulation (release of the granule contents lysozyme and beta-glucuronidase) of polymorphonuclear leukocytes (PMN) suspended in either Hanks' solution or 100% fresh heparinised plasma. PMN in low concentrations of FMLP in Hanks' solution or in plasma alone showed "long" polarisation and did not degranulate. Cytochalasin B caused the PMN in low concentrations of FMLP or in plasma to become spherical, but no degranulation of the cells occurred. High concentrations of FMLP in Hanks' solution induced "short" polarisation of PMN with slight degranulation of the cells. Cytochalasin B together with high concentrations of FMLP in Hanks' solution induced zeiosis ("blebbing") and marked degranulation of the cells. However, cytochalasin B and high concentrations of FMLP in plasma caused PMN to exhibit "short" polarised morphology and markedly degranulate. These results suggest that degranulation of PMN can be associated with either the "short" polarised shape of the cells or zeiosis, but not the "long" polarised form. Furthermore, the results indicate that plasma, although capable of causing "long" polarisation of the cells, inhibits zeiosis without affecting the degranulation of the cells induced by cytochalasin B.     

Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation.

Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ ([Ca2+]i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in [Ca2+]i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in [Ca2+]i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML [3H]P to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.