Stimulation Of Human Neutrophils Research Articles

Inhibition of FMLP-stimulated neutrophil chemiluminescence by blood platelets increased in the presence of the serotonin-liberating drug chloroquine

Introduction: Previously, we reported that human blood platelets significantly decreased the concentration of reactive oxygen species (chemiluminescence) produced by Ca 2+-ionophore-stimulated neutrophils and that the reduction was partially mediated by serotonin liberated from platelets during their activation. The aim of the present study was to investigate whether platelet inhibition can occur independently of serotonin liberation and whether it can be pharmacologically enhanced. Materials and methods: Chemiluminescence was measured after stimulation of human neutrophils with N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) in the presence of luminophore luminol. Concentration of platelet serotonin was estimated fluorometrically. Results: Platelets, added to neutrophils in the physiological cell ratio 50:1, decreased neutrophil chemiluminescence by 47%. The inhibition was not accompanied with liberation of platelet serotonin and rose after addition of chloroquine to platelet–neutrophil samples. In the absence of platelets, this drug did not affect neutrophil chemiluminescence. Chloroquine actively liberated serotonin; amine concentrations found in platelet supernatants were sufficient to inhibit neutrophil chemiluminescence. Conclusions: The presented results indicate that unstimulated platelets decreased neutrophil chemiluminescence by a serotonin-independent mechanism, yet their inhibitory effect could be enhanced pharmacologically through chloroquine-induced serotonin liberation.

ＩＬ‐１βによるヒト好中球の活性化とｐ３８ＭＡＰキナーゼ

Interleukin-1 (IL-1) plays a central role in the immune and inflammatory responses, and activates various types of cells, including human neutrophils. IL-1β exerts its effects through IL-1 receptor type I, and activates NF-κB and mitogen-activated protein kinase (MAPK) cascades. In human neutrophils, it was found that the MKK3/6-p38 MAPK cascade was selectively activated by IL-1β, and p38 MAPK activation mediated IL-1β-induced superoxide (O2-) release and up-regulation of CD11b (β2 integrin) and CD15 (a ligand for P-selectin). Our recent studies also show that stimulation of human neutrophils with various cytokines, including granulocyte colonystimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-α, results in cytokine-specific activation of distinct MAPK subtype cascades, and that extracellular signal-regulated kinase as well as p38 MAPK mediates cytokine-induced O2- release and adherence. In this review, we discuss the signaling pathways activated by IL-1β and the role of MAPK subtype cascades and JAK-STAT pathway in activation of human neutrophils by inflammatory cytokines.

Immune complex stimulation of human neutrophils involves a novel Ca2+/H+ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca2+/pH responses by subpopulations

The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described. The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca(2)(+)/H(+) voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

Production of Macrophage Inflammatory Protein (MIP)-1α and MIP-1β by Human Polymorphonuclear Neutrophils Stimulated with Porphyromonas endodontalis Lipopolysaccharide

This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS). Escherichia coli LPS was used as a positive control. Venous blood was collected and PMNs were isolated from healthy volunteers. Cells were cultured with various concentrations of LPS for different periods of time. Cell supernatants were assayed by enzyme-linked immunosorbent assay. The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p < 0.05), and this expression occurred in a time- and dose-dependent manner. E. coli LPS induced higher levels of cytokines than P. endodontalis LPS. These findings demonstrated that P. endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P. endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

Migratory Responses of Polymorphonuclear Leukocytes to Kinin Peptides

The present study examines the influence of kinins on the migratory capacity of human polymorphonuclear leukocytes under in vitro conditions using the Boyden chamber technique. By means of checkerboard analysis the migration of neutrophils induced by bradykinin could be characterized as true chemotaxis. The stimulation of human neutrophils with bradykinin, with the nonpeptide B₂ receptor agonist FR190997 as well as with des-Arg⁹-bradykinin and des-Arg¹⁰-kallidin results in a concentration-dependent migration. Pretreatment of the neutrophils with the B₂ receptor antagonist HOE-140 (icatibant) inhibited the bradykinin-induced migration but not that induced by B₁ receptor agonists, whereas the B₁receptor antagonist des-Arg¹⁰HOE-140 abolished the migration elicited by des-Arg⁹-bradykinin or des-Arg¹⁰-kallidin but not that evoked by bradykinin. Pretreatment of the neutrophils with the leukotriene B₄ (LTB₄) antagonist ZK158252 inhibited the LTB₄-induced chemotaxis as well as the chemotaxis produced by bradykinin and des-Arg¹⁰-kallidin. An involvement of interleukin-1β and of the chemokine IL-8 in the bradykinin-induced migration in vitro could be excluded during the migration time of the neutrophils. In conclusion, the present study provides pharmacological evidence showing that B₁ and B₂ kinin receptors are involved in the migration of human neutrophils in vitro, that LTB₄ participates in the downstream pathway and that the B₁ kinin receptor seems to be expressed already under physiological conditions.

Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori.

Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines. This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils. H. pylori strains (1.5 x 108 CFU/ml) were cocultured with 5 x 104 neutrophils isolated from healthy subjects. Samples were incubated with human serum with or without IgG antibodies to H. pylori. ROS production was measured using luminol-dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL-8, RANTES, MIP-1alpha and MCP-1) were measured by ELISA. The mean of the highest LmCL (peak height; PH) value stimulated by H. pylori was 3318 in the absence of serum. PH increased to 4687 when incubated with anti-H. pylori antibody-positive sera (p <.001) but antibody-negative sera did not affect LmCL response. The mean final concentration of IL-8 produced in the absence of serum was 142.6 pg/ml. Increased IL-8 production was seen by addition of antibody positive serum (p <.01). IL-8 production was not significantly correlated with production of ROS. On the other hand, H. pylori stimulation did not induce neutrophil production of RANTES, MIP-1alpha or MCP-1. H. pylori was capable of inducing IL-8 production by human neutrophils, but not C-C chemokines. Production of C-X-C dominant chemokine by neutrophils is consistent with the pathological characteristics of H. pylori-induced gastritis, where persistent neutrophil infiltration is present.

Cholesterol-modulating Agents Selectively Inhibit Calcium Influx Induced by Chemoattractants in Human Neutrophils

The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-beta-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn(2+) induced by fMet-Leu-Phe or thapsigargin. Methyl-beta-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10(-10) m platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10(-7) m platelet-activating factor. On the other hand, methyl-beta-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of FcgammaRIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-beta-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.

Cartilage Degradation by Stimulated Human Neutrophils: Elastase Is Mainly Responsible for Cartilage Damage

Although neutrophilic granulocytes are assumed to contribute to cartilage degradation during rheumatic diseases, there is still a discussion whether reactive oxygen species (ROS) or proteolytic enzymes that are both released by the neutrophils are most relevant to cartilage degradation. To gain further insight into these processes, an in vitro approach to study the interaction between the products of stimulated neutrophilic granulocytes and cartilage was used: Neutrophils from the blood of healthy volunteers were treated with different stimulators (e.g., Ca2+ ionophores) in order to induce degranulation. Supernatants of neutrophils were afterward incubated with thin slices of pig articular cartilage. Some experiments were also performed in the presence of selected enzyme inhibitors. Supernatants of cartilage were subsequently assayed by one- and two-dimensional high-resolution proton NMR spectroscopy, and the content of soluble carbohydrates in the supernatant was additionally determined by biochemical methods. The selective inhibition of elastase decreased most significantly the extent of cartilage degradation, whereas all other inhibitors had much smaller effects. These results were additionally confirmed by measuring the effect of isolated elastase on articular cartilage in the absence and presence of different inhibitors. It is concluded that elastase released [EC 3.4.21.37] by neutrophils is the most relevant enzyme for cartilage degradation.

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C.

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.

Activation of cytosolic phospholipase A2 by opsonized zymosan in human neutrophils requires both ERK and p38 MAP-kinase.

The present study demonstrates that stimulation of human neutrophils with opsonized zymosan (OZ), which binds to Fc gamma receptors (Fc gamma Rs) and C3b receptors, activates both ERK and p38 MAP-kinase. Thus, the relative role of both types of MAP-kinase, ERK and p38, in activation of cPLA2 by OZ was studied. cPLA2 activation by OZ was detected 15 sec after stimulation, maintained a plateau for 10 min and decreased thereafter. p38 MAP-kinase activation exhibited kinetics similar to that of cPLA2, while ERK activation was detected within 15 sec but decreased significantly in less than 5 min after stimulation. Pretreatment of the cells with the MEK inhibitor, PD-098059, or the p38 MAP-kinase inhibitor, SB-203580 resulted in total inhibition of ERK or p38 MAP-kinase activity, respectively. Each inhibitor caused a partial inhibition during the time course of cPLA2 activity, while their combination caused a total inhibition. Compared to OZ, inactivated OZ, which does not contain the complement proteins, induced an identical time-dependent stimulation of ERK and p38 MAP-kinase as well as a similar cPLA2 activity, suggesting that the role of the C3b receptors in this system is negligible. It is concluded that OZ activates both ERK and p38 MAP-kinase and that the two isotypes are required for the onset and maintenance of cPLA2 activity.

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.

Fibrinogen induces IL-8 synthesis in human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine or leukotriene B(4).

Human exudative neutrophils have greatly increased stores of the neutrophil chemoattractant IL-8 compared with peripheral blood cells, but the mechanism for the increase is not defined. In this report, we show that treatment of peripheral blood neutrophils with the chemotactic peptide fMLP or with leukotriene B(4) or fibrinogen results in little increase in the production of IL-8 by peripheral blood neutrophils. However, a chemotactically active dose of fMLP (5 x 10(-9) M) or leukotriene B(4) (1 x 10(-7) M) in the presence of a physiological concentration (2 mg/ml) of fibrinogen results in a receptor-mediated, pertussis toxin-sensitive, synergistic 30-fold increase in IL-8 synthesis. The levels of IL-8 attained are comparable to those observed in exudative cells. Higher concentrations of fMLP (1 x 10(-7) M) are associated with reduced IL-8 protein synthesis without IL-8 degradation, indicating a sensitive regulatory mechanism for IL-8 production. Treatment of neutrophils with fibrinogen and fMLP resulted in minimal changes in the steady state levels of mRNA for macrophage inflammatory protein-1alpha and -1beta and monocyte chemoattractant protein-1. In contrast, in the presence of fibrinogen, the steady-state level of neutrophil IL-8 mRNA increased 8-fold with 5 x 10(-9) M fMLP but was not decreased with 1 x 10(-7) M fMLP, suggesting that neutrophils are specifically adapted to modulate neutrophil IL-8 synthesis through transcriptional and posttranscriptional mechanisms. The data indicate that fibrinogen can function not only as a substrate in the clotting cascade, but also as an important effector during the evolution of the innate immune response.

Stimulation of human neutrophils and monocytes by staphylococcal phenol-soluble modulin

Modulins represent microbial products that stimulate cytokine production in host cells. The modulins responsible for gram-positive sepsis remain poorly understood. Staphylococci release a factor (or factors) that activates nuclear factor-kappa B and stimulates cytokine production in cells of macrophage lineage. This factor, termed phenol-soluble modulin (PSM), has been recently isolated from culture supernatant of Staphylococcus epidermidis. We examined the effects of PSM on proinflammatory properties of human neutrophils and monocytes in vitro. PSM activated the respiratory (oxidative) burst in neutrophils and primed neutrophils for enhanced respiratory burst activity in response to formyl-methionyl-leucyl-phenylalanine. PSM also stimulated neutrophil degranulation as reflected by increased surface expression of CD11b and CD18, which was accompanied by rapid shedding of L-selectin. Spontaneous apoptosis of both neutrophils and monocytes was inhibited by PSM. Furthermore, PSM also functioned as a chemoattractant factor for both neutrophils and monocytes. Thus, the proinflammatory properties of PSM resemble those of both lipopolysaccharide and bacterial chemotactic peptides. These findings suggest that PSM may play a role in the pathogenesis and systemic manifestations of sepsis caused by staphylococci.

Interleukin-8-positive neutrophils in psoriasis

We performed an immunohistochemical study to try to determine the cellular source of interleukin-8 (IL-8) in psoriatic skin lesions. IL-8 was positively stained in the vast majority of neutrophils but not in the mononuclear cells, macrophages, or keratinocytes. IL-8-positive neutrophils were seen both in Munro's microabcesses in cases of psoriasis vulgaris and in a small spongiform pustule and much larger macropustules of Kogoj in cases of pustular psoriasis. Some IL-8-positive neutrophils were observed in the upper dermis of pustular psoriasis. The staining was considered to be specific because it could be completely blocked by preabsorption with recombinant IL-8. In addition, stimulation of human neutrophils with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) for 18 h induced IL-8 production in vitro. In our study, IL-8 was expressed in the neutrophils of psoriasis, suggesting that neutrophils are one of the sources of IL-8 in psoriasis. The expression of IL-8 and the influx of neutrophils led us to speculate that the IL-8 autocrine and/or paracrine system functions in the formation of the microabcesses and pustules in proriasis.

Gene expression and production of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and gamma interferon-inducible protein 10 by human neutrophils stimulated with group B meningococcal outer membrane vesicles.

Accumulation of polymorphonuclear neutrophils (PMN) into the subarachnoidal space is one of the hallmarks of Neisseria meningitidis infection. In this study, we evaluated the ability of outer membrane vesicles (OMV) from N. meningitidis B to stimulate cytokine production by neutrophils. We found that PMN stimulated in vitro by OMV produce proinflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. A considerable induction of gamma interferon (IFN-gamma)-inducible protein 10 (IP-10) mRNA transcripts, as well as extracellular IP-10 release, was also observed when neutrophils were stimulated by OMV in combination with IFN-gamma. Furthermore, PMN stimulated by OMV in the presence of IFN-gamma demonstrated an enhanced capacity to release TNF-alpha, IL-1beta, IL-8, and MIP-1beta compared to stimulation with OMV alone. In line with its downregulatory effects on neutrophil-derived proinflammatory cytokines, IL-10 potently inhibited TNF-alpha, IL-1beta, IL-8, and MIP-1beta production triggered by OMV. Finally, a neutralizing anti-TNF-alpha monoclonal antibody (MAb) did not influence the release of IL-8 and MIP-1beta induced by OMV, therefore excluding a role for endogenous TNF-alpha in mediating the induction of chemokine release by OMV. In contrast, the ability of lipopolysaccharide from N. meningitidis B to induce the production of IL-8 and MIP-1beta was significantly inhibited by anti-TNF-alpha MAb. Our results establish that, in response to OMV, neutrophils produce a proinflammatory profile of cytokines and chemokines which may not only play a role in the pathogenesis of meningitis but may also contribute to the development of protective immunity to serogroup B meningococci.

CDNA Representational Difference Analysis of Human Neutrophils Stimulated by GM-CSF

Neutrophils are the first cell type to migrate out of the vascular space and into the inflammatory site during an acute inflammation. However, in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), a lack of clearance of neutrophils, imbalance between inflammatory mediators produced by neutrophils and their natural inhibitors make these cells a potential cause of tissue destruction in lung disease. Neutrophilic inflammation is generally characterised by high levels of local expression of activating cytokines (e.g., GM-CSF). Only a few studies have been published so far that have investigated the expression of genes preferentially expressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identification of potential novel therapeutic targets or markers of the disease. We performed representational difference analysis of cDNA, a sensitive PCR-based subtractive enrichment procedure, and isolated 12 genes, 1 EST clone, and 3 sequences not represented in the public databases. Differential expression for 9 of these clones was confirmed by Northern hybridisation. Of the above nine transcripts three were chosen and shown to be up-regulated in neutrophils cocultured with stimulated primary human bronchial epithelial cells using a semiquantitative RT-PCR approach. Among the known genes identified were HM-74, CIS1, Cathepsin C, α-enolase, CD44, and the gene Translocation Three Four (TTF), most of them previously not known to be involved in GM-CSF induced neutrophil activation. Along with its tissue and cellular distribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initial characterisation of a novel gene, P1-89 that is primarily expressed in granulocytes and is up-regulated in activated cells. Our results identify several important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflammations.

Effects of the protein tyrosine phosphatase CD45 on FcgammaRIIa signaling and neutrophil function.

Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.

Stimulation of Human Neutrophils by Chemotactic Factors Is Associated with the Activation of Phosphatidylinositol 3-Kinase γ

The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110gamma, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110gamma in neutrophils was first established both at the protein and the mRNA level. Stimulation of the cells with fMet-Leu-Phe or interleukin-8 increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. The time course of this effect (threshold within less than 5 s, maximal activation at 10-15 s) was consistent with that of the generation of PtdIns(3,4,5)P(3). Wortmannin, a PI3K inhibitor, abrogated the effects of fMet-Leu-Phe, which were also significantly inhibited by pertussis toxin. Finally, fMet-Leu-Phe also induced a significant translocation of p110gamma to a particulate fraction derived from these cells. These data indicate that p110gamma represent the major PI3K activated by fMet-Leu-Phe and interleukin-8 at very early time points following the stimulation of human neutrophils.

Differences Arising in Human Neutrophil Activation Passing from N-Formyl to N-Acetyl-oligopeptides

N-formyl- and N-acetyl-peptides were synthesized and compared in order to understand which features can best elicit biological responses. The behavior of N-formyl-peptides confirms the previously found sequential obligations in the residues, while acetyl-derivatives do not seem suitable for an efficacious stimulation of human neutrophils.

Nitric Oxide Regulates the Aggregation of Stimulated Human Neutrophils

Neutrophil aggregation is mediated by both CD18 integrin and L-selectin. Nitric oxide attenuates the integrin-mediated adhesion of neutrophils to collagen and to endothelium and may therefore affect aggregation as well. FMLP-stimulated neutrophils exposed to l-arginine showed increased and prolonged aggregation, whereas cells pretreated with L-NAME did not differ from FMLP-stimulated controls. Nitric oxide is known to induce ADP ribosylation of G-actin, which inhibits polymerization. We detected equivalent levels of total F-actin in cells pretreated with l-arginine or L-NAME and non-pretreated controls. However, neutrophils pretreated with l-arginine and stimulated by CD18 integrin cross-linking exhibited a more limited increase in total F-actin, compared to control and L-NAME-pretreated cells. Thus at least two signaling pathways may be involved FMLP-stimulated aggregation, mediated by CD18 integrins. More specifically, it is plausible that FMLP-receptor signaling upregulates CD18 integrins and endogenous NO subsequently modulates CD18-mediated signaling to prolong aggregation, possibly through ADP-ribosylation of actin.