Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the functions of neutrophils

Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility, and elasticity of these tissues. In recent years, glucosamine has been used widely to treat osteoarthritis in humans and animal models. Neutrophils, which usually function as the primary defenders in bacterial infections, are also implicated in the destructive, inflammatory responses in arthritis. In this study, we have evaluated the effects of glucosamine on neutrophil functions using human peripheral blood neutrophils. Glucosamine (0.01-1 mM) dose-dependently suppressed the superoxide anion generation induced by formyl-Met-Leu-Phe (fMLP) or complement-opsonized zymosan and inhibited the phagocytosis of complement-opsonized zymosan or IgG-opsonized latex particles. Furthermore, glucosamine inhibited the release of granule enzyme lysozyme from phagocytosing neutrophils and suppressed neutrophil chemotaxis toward zymosan-activated serum. In addition, glucosamine inhibited fMLP-induced up-regulation of CD11b significantly, polymerization of actin, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). In contrast, N-acetyl-glucosamine, an analogue of glucosamine, did not affect these neutrophil functions (superoxide generation, phagocytosis, granule enzyme release, chemotaxis, CD11b expression, actin polymerization, and p38 MAPK phosphorylation) at the concentrations examined (1-10 mM). Together these observations likely suggest that glucosamine suppresses the neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis.     

Differentiation by in vitro treatment of lidocaine-epinephrine and prilocaine-felypressine in neutrophils.

Neutrophils are often the first cells of the immune system to encounter an invader, such as bacteria and fungi. Lidocaine-epinephrine induced transient potentiation of the production of superoxide anion, while prilocaine-felypressine induced persistent inhibition of the production in neutrophils. Moreover, lidocaine-epinephrine inhibited the production of hydrogen peroxide in spite that it potentiated the production of superoxide anion, while prilocaine-felypressine inhibited the production of hydrogen peroxide as well as superoxide anion. By contrast, lidocaine-epinephrine and prilocaine-felypressine are both effective in significantly inhibiting adhesion and phagocytosis. Using flow cytometric analysis, both local anesthetics were found to be effective in inhibiting the expression of Mac-1 (CD11b/CD18) in neutrophils. These results suggest that lidocaine-epinephrine and prilocaine-felypressine differentially modulate the production of superoxide anion, and could similarly inhibit adhesion, phagocytosis, and the production of hydrogen peroxide by neutrophils.     

Comparison of inhibitory effects of local anesthetics on immune functions of neutrophils

Immunological effects of a variety of local anesthetics on adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils were compared. Neutrophils were isolated by peritoneal lavage from rats, 4 h after injection of 1% glycogen. Lidocaine, mepivacaine, procaine, prilocaine and tetracaine at 1 mg/ml inhibited adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide in neutrophils. Moreover, lidocaine, mepivacaine, procaine and prilocaine at 0.1 mg/ml inhibited the production of superoxide anion and hydrogen peroxide but not adhesion or phagocytosis. In contrast, tetracaine at 0.1 mg/ml inhibited phagocytosis, and the production of superoxide anion and hydrogen peroxide but not adhesion. At 0.01 mg/ml, however, tetracaine inhibited the production of superoxide anion and hydrogen peroxide; in contrast, other drugs failed to affect neutrophil function. These results suggest that the local anesthetics may affect adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils.     

Engagement of adenosine receptors inhibits hydrogen peroxide (H2O2-) release by activated human neutrophils

Adenosine and its analogs, acting at specific cell surface receptors, inhibit generation of superoxide anion by neutrophils. Since it has been suggested that hydrogen peroxide (H2O2) release may not be contingent upon superoxide anion release, we studied the effects of 2-chloroadenosine, a potent adenosine receptor agonist, on the formation of H2O2 by neutrophils exposed to various stimuli: n-formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A, phorbol myristate acetate (PMA), serum-treated zymosan particles (STZ), and immune complexes. 2-Chloroadenosine (0.01-10 microM) inhibited formation of H2O2 by neutrophils exposed to FMLP, concanavalin A, and STZ particles. As we have found with O2- generation, 2-chloroadenosine failed to inhibit H2O2 release by neutrophils stimulated by either phorbol myristate acetate or immune complexes. The data show that whereas adenosine and its analogs inhibit neutrophil release of H2O2 and superoxide anion in response to most ligands, they fail to inhibit activation of neutrophils by immune complexes. Nor do they inhibit neutrophil activation by PMA, an agent which bypasses cell surface receptors by direct activation of protein kinase C. Surprisingly, we found that adenosine deaminase activity was adsorbed onto zymosan particles during opsonization and enhanced release of H2O2 by neutrophils exposed to STZ. These studies with yeast cell walls suggest that if microorganisms adsorb adenosine deaminase from serum, then the intracellular microbicidal activity of neutrophils is enhanced.     

Endomorphins delay constitutive apoptosis and alter the innate host defense functions of neutrophils

Recent studies have shown that opioid peptides are released from cells of the immune system during inflammation and stress, and are associated with altered immune responses. Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions. The aim of this study was to evaluate immunological effects of opioid peptides endomorphins 1 and 2 on constitutive apoptosis, superoxide anion production, hydrogen peroxide production, adhesion, phagocytosis, and chemotaxis of neutrophils. Neutrophils were isolated by peritoneal lavage from rats. Endomorphins 1 and 2 significantly delayed constitutive neutrophil apoptosis. The delay of neutrophil apoptosis was markedly attenuated by LY294002, a phosphoinositide 3-kinase inhibitor. Moreover, endomorphins 1 and 2 activated the phosphoinositide 3-kinase pathway as determined by phosphorylation of BAD. In contrast, endomorphins 1 and 2 blocked the production of superoxide anion and hydrogen peroxide by PMA-stimulated neutrophils. In addition, endomorphins 1 and 2 inhibited neutrophil adhesion to fibronectin. Moreover, endomorphins 1 and 2 potentiated neutrophil chemotaxis toward zymosan-activated serum and IL-8, respectively. However, endomorphins 1 and 2 did not alter phagocytosis of Escherichia coli by neutrophils. These results suggest that endomorphins 1 and 2 may act to delay neutrophil apoptosis and alter the natural immune functions of neutrophils.     

Comparative studies of modulatory effect to the function of rat peritoneal neutrophils treated with new quinolones.

Some of the immunological effects of a variety of new quinolones on adhesion, phagocytosis, and production of reactive oxygen intermediators in neutrophils were studied. Ofloxacin, lomefloxacin, fleroxacin, and levofloxacin potentiated the phagocytosis of Escherichia coli in neutrophils. Moreover, lomefloxacin, and sparfloxacin significantly potentiated adhesion of neutrophils. In contrast, tosufloxacin was effective in significantly and persistently potentiating the production of superoxide anion, whereas the other agents markedly inhibited such production. Furthermore, tosufloxacin was effective in significantly potentiating the production of hydrogen peroxide, whereas sparfloxacin markedly inhibited such production. These results suggest that the new quinolones at a therapeutic concentration may affect functions such as phagocytosis, and production of superoxide anion in neutrophils.     

Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells

Defensins, a family of cationic, structurally related, antimicrobial peptides, contribute to host defense by disrupting the cytoplasmic membrane of microbes. Here we show that human neutrophil defensins selectively induce the migration of human CD4+/CD45RA+ naive and CD8+, but not CD4+/CD45RO+ memory, T cells. Moreover, human neutrophil defensins are chemotactic for immature human dendritic cells derived from either CD34+ progenitors or peripheral blood monocytes. Upon maturation induced by treatment with tumor necrosis factor alpha (TNF-alpha), dendritic cells lose their responsiveness to human neutrophil defensins. The chemotactic effect of human neutrophil defensins on both T and dendritic cells is pertussis toxin-sensitive, suggesting that a G(ialpha) protein-coupled receptor is responsible. Human neutrophil defensins are also chemotactic for immature murine dendritic cells. These data suggest that, in addition to their antimicrobial role, human neutrophil defensins also contribute to adaptive immunity by mobilizing T cells and dendritic cells.     

Activation of phospholipase D is an early event in integrin-mediated signalling leading to phagocytosis in human neutrophils

Integrin receptors on human neutrophils mediate adhesion and phagocytosis. These functions are linked to a signal-transduction cascade that rearranges the cytoskeleton. The intention of this study was to clarify how activation of phospholipase D (PLD) is coupled to the complement receptor three (CR3, CD18/CD11b)mediated ingestion process. Carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK) inhibited PLD activation induced by complement-opsonized yeast particles (COYP) by 39%. Phagocytosis of these particles was reduced by zLYCK to the same extent. Anti-CD18-antibodies bound to protein A-positiveStaphylococcus aureus bacteria induced a significant PLD activation. These particles were not ingested which implicates that CR-mediated ingestion per se is not required to induce PLD activity. Cytochalasin B-treatment, which blocks actin reorganization, partly reduced COYP-mediated PLD activity, but had no effect on activity caused by anti-CD 18-coated particles. This excludes activation of PLD to be a secondary event, but rather an early signal in the phagocytic uptake prior to actin reorganization. These data suggest an important and early role for PLD in integrin-mediated phagocytosis.     

Stable adhesion and migration of human neutrophils requires phospholipase D-mediated activation of the integrin CD11b/CD18

The pathways regulating integrin-mediated adhesion during neutrophil migration are incompletely defined. Using a flow-based model in which human neutrophils rolling on P-selectin were activated to migrate by the chemoattractant peptide fMLP, we investigated the role of phospholipase D (PLD). fMLP-stimulated PLD generation of phosphatidate (PtdOH); while inhibition of PtdOH production with butan-1-ol had no effect on the initial immobilisation of rolling neutrophils (supported by activation of constitutively surface-expressed beta(2)-integrin CD11b/CD18) it impaired longer-term stability of adhesion and reduced the rate of migration (supported by activation of de novo-exocytosed CD11b/CD18). PtdOH regulated these processes by controlling activation of exocytosed CD11b/CD18, and appeared to act by directly stimulating phosphatidylinositol 4-phosphate 5-kinase type I to generate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Cell-permeable PtdIns(4,5)P(2) recovered migration of neutrophils after PLD inhibition; PtdIns(4,5)P(2) appeared to act by promoting talin binding to CD18 and hence activating CD11b/CD18, as migration was inhibited when neutrophils were loaded with peptides previously shown to block the interaction between PtdIns(4,5)P(2) and talin or talin and CD18. Thus, these data indicate that PLD-synthesised PtdOH stimulates the generation of PtdIns(4,5)P(2), which in turn mediates talin binding to, and activation of, CD11b/CD18 required for neutrophil stable adhesion and migration.     

Isoproterenol inhibition of isolated human neutrophil function

The human PMN can contribute to the inflammatory response. Several neutrophil responses can be inhibited by agonists that increase the cellular levels of cyclic AMP. In the following article, we compared the effects of ISO on lysosomal beta-glucuronidase release, superoxide generation, and CL in isolated human PMNs. ISO inhibited the neutrophil CL response to opsonized zymosan in a dose-dependent fashion with maximal effects at 10(-4)M. ISO inhibition of CL was not enhanced by the addition of theophylline, nor was CL inhibited by the exogenous addition cyclic AMP except at a very high concentration of 10(-3)M. ISO also suppressed beta-glucuronidase release and superoxide generation in neutrophils during an incubation with opsonized zymosan particles. For ISO to inhibit beta-glucuronidase release and superoxide generation, theophylline (5 X 10(-4)M) was necessary. ISO effectively inhibits three neutrophil functions that are capable of causing tissue inflammation. Although ISO suppressed all three neutrophil responses, the inhibitory mechanisms appear to be variable.     

Effects of leukocyte inhibitory factor (LIF) on human neutrophil function

The effects of the lymphokine, leukocyte inhibitory factor (LIF), on human neutrophil function were studied. This soluble mediator, which is defined by its specific inhibition of neutrophil locomotion, does not interfere with chemotactic factor binding and does not affect basal or stimulated superoxide generation by neutrophils. In contrast, phagocytosis of opsonized Staphylococcus aureus is markedly inhibited by LIF, and degranulation is stimulated by this lymphokine. The possible mechanisms of LIF action on neutrophils are discussed.     

Morphology of defensin-treated Staphylococcus aureus.

Defensins are a family of broad-spectrum antimicrobial peptides found abundantly in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Defensins are known to form ion channels on the membranes of target cells. These channel formations and the cytotoxicity of defensins are intimately linked. We showed the morphological effects of defensins on the cytoplasmic membranes of Staphylococcus aureus by transmission electron microscopy. S. aureus exposed to defensins developed characteristic mesosome-like structures but did not show remarkable changes in cell walls. Defensins induced such structural changes not only at high concentration but also at low concentrations that were not bactericidal. We also showed that increasing the concentration of NaCl in the reaction mixture completely inhibited the occurrence of membranous changes of target cells exposed to defensins. These findings are, to our knowledge, the first report of morphological changes in gram-positive bacteria treated with defensins. Our results indicate that the first effect of defensins in S. aureus is to damage cytoplasmic membranes directly; they also support previous reports that the cell membrane is the principal target of defensins.     

Flavonoid modulation of human neutrophil function

Flavonoids are naturally occurring plant compounds that have been demonstrated to possess a variety of anti-inflammatory effects. We studied the effects of flavonoids on three aspects of neutrophil function that are commonly considered to be associated with inflammation: the release of lysosomal enzymes, the chemiluminescence (CL) response, and the production of superoxide anion. Quercetin and eight other flavonoids at a 10(-5)M concentration inhibited the neutrophil CL response to opsonized zymosan particles by approximately 60% or more. In contrast, the release of lysosomal beta-glucuronidase from neutrophils stimulated with opsonized zymosan was only inhibited by two flavonoids, quercetin and chalcone, and only at concentrations of 1.5 X 10(-4)M to 2 X 10(-4)M. Quercetin also inhibited the generation of superoxide anion by neutrophils but to a lesser degree than its effect on CL. The present studies demonstrated that certain flavonoids are not uniformly active in inhibiting neutrophil CL, beta-glucuronidase release, or superoxide generation. The effects of flavonoids on neutrophil functions probably depend on many variables including the response measured, the activating stimulus, and specific flavonoid structural features.     

The role of CD18-mediated adhesion in neutrophil sequestration induced by infusion of activated plasma in rabbits.

Infusion of activated plasma induces neutropenia and sequestration of neutrophils within the microvasculature. This study examined the role of the adhesion glycoprotein complex, CD11/CD18, in this sequestration. Rabbits pretreated with either the anti-CD18 monoclonal antibody (mAb) 60.3 or saline were given infusions of zymosan-activated plasma (ZAP) or saline. The effect of mAb 60.3 on the changes in circulating neutrophil counts, radiolabeled neutrophil kinetics in the lung, and the pulmonary microvascular accumulation of neutrophils induced by ZAP infusion was determined. The data show that pretreatment with mAb 60.3 did not inhibit either the rate of onset or the severity of the neutropenia but prevented the sustained neutropenia. In addition, mAb 60.3 completely prevented the ZAP-induced changes in radiolabeled neutrophil kinetics and largely inhibited the accumulation of neutrophils within the capillaries and the small vessels when evaluated after 15 min of ZAP infusion. We conclude that neutrophil accumulation is a two-step process, the first occurring through a CD18-independent mechanism that may involve a stimulus-induced decrease in neutrophil deformability and acts to slow neutrophil transit through the lung. The second step requires CD18-dependent adhesion and is needed for prolonged accumulation of neutrophils within the pulmonary microvasculature.     

Phagocytosis of heat‐killed Staphylococcus aureus by eosinophils: comparison with neutrophils

Eosinophils are characterized by several functional properties, such as chemotaxis, adhesion, superoxide anion production, and degranulation. In this article, we have studied the role of bacterial ingestion by eosinophils in comparison with that by neutrophils. Eosinophils and neutrophils were purified by using the Percoll gradient method followed by selection with CD16‐coated immunomagnetic beads and centrifugation through a Ficoll‐Hypaque gradient combined with dextran sedimentation, respectively. Both cells were preincubated with anti‐FcγRIIa mAb (CD32 mAb), anti‐FcγRIIIb mAb (CD16 mAb), anti‐CR3 (CD11b mAb), or anti‐CR1 (CD35 mAb) before being examined for phagocytosis of opsonized heat‐killed Staphylococcus aureus (S. aureus). Phagocytosis and production of hydrogen peroxide were simultaneously measured by flow cytometry using S. aureus labeled with propidium iodide and stained with 2′,7′‐dichlorofluorescein diacetate. Eosinophils showed significantly lower activity than neutrophils in both phagocytosis and hydrogen peroxide production. Phagocytosis by both cells was decreased by heat‐inactivated serum. Phagocytosis by neutrophils was significantly inhibited by CD16 mAb and CD32 mAb, whereas that by eosinophils was only inhibited by CD35 mAb. Whereas the mechanism of phagocytosis by neutrophils was mediated by CD16 and CD32, that of eosinophils was modulated by complement receptor 1 (CD35).     

Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion.

It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to mediate the initial complement-dependent adhesion of metacyclic promastigotes to human monocyte-derived macrophages and that Mac-1 is the predominant complement receptor responsible for the phagocytosis of complement-opsonized metacyclic promastigotes.     

Phagocytic activation of human neutrophils by the detergent component of fluosol.

Fluosol (Alpha Therapeutic Corporation, Los Angeles, CA) an emulsion of perfluorocarbons with a high oxygen-carrying capacity, was approved as an adjunct to alleviate myocardial ischemia during coronary angioplasty. This drug also significantly enhances myocardial salvage presumably related to an action on the neutrophil. The mechanism by which fluosol and its individual components, including the detergent Pluronic F-68, affected neutrophil function was examined. During the incubation of neutrophils with fluosol, a rapid stimulation of superoxide anion production and degranulation which progressively increased over a 30-minute period was detected. Neutrophils incubated with only Pluronic F-68 produced similar amounts of superoxide anion. Cytochalasin B, an inhibitor of phagocytosis, significantly inhibited this superoxide anion generation. As shown previously, neutrophils incubated with fluosol for 30 minutes and then subsequently stimulated manifested a reduction in lysozyme release as compared with untreated cells. Results of an electron microscopic examination confirmed the cellular uptake of the fluosol within phagocytic vacuoles. Neutrophil viability determined by trypan blue was unaffected after fluosol treatment. These observations show that the fluosol emulsion, primarily through micelles formed by the detergent Pluronic F-68, activates human neutrophils by serving as a phagocytic stimulus, which produces a cell refractory to subsequent stimulation.     

Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and beta-glucan, and transmits outside-in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)-enriched lipid rafts are required for CD11b/CD18-mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti-CD11b and anti-LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer-enriched lipid rafts during phagocytosis of NOZs. DMSO-treated, neutrophil-like HL-60 cells (D-HL-60 cells) lacking Lyn-coupled, LacCer-mediated signaling showed little phagocytosis of NOZs. However, loading of D-HL-60 cells with C24 fatty acid chain-containing LacCer (C24-LacCer) reconstructed functional Lyn-associated, LacCer-enriched lipid rafts, and restored D-HL-60 cell NOZ phagocytic activity, which was inhibited by anti-LacCer and anti-CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1-LacCer loading on D-HL-60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer-associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn-coupled, LacCer-enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.     

Effects of defensin and lactoferrin on functional activity of endothelial cells <Emphasis Type="Italic">in vitro</Emphasis>

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated. α-Defensin (NP-2) from rabbit neutrophils, total α-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1–10 μg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 306–309, September, 2007     

Calcium pyrophosphate dihydrate crystals activate MAP kinase in human neutrophils: inhibition of MAP kinase, oxidase activation and degranulation responses of neutrophils by taxol.

The activation of MAP kinase in human neutrophils stimulated by both uncoated and plasma-opsonized crystals of triclinic calcium pyrophosphate dihydrate (CPPD) was investigated. The effect of taxol on MAP kinase activation and on the responses of neutrophils stimulated by plasma-opsonized crystals was determined. MAP kinase activation was identified and quantified in Mono Q chromatography separated fractions of neutrophils that had been incubated with CPPD crystals by measuring [gamma-32P]adenosine triphosphate (ATP) phosphorylation of myelin basic protein and using immunoblotting techniques. Human neutrophils were incubated with taxol (0-50 microM), added to plasma-opsonized CPPD (50 mg/ml) and MAP kinase activation, chemiluminescence, superoxide anion generation, lysozyme and myeloperoxidase release were monitored. Both uncoated and plasma coated CPPD crystals induced a large increase in MAP kinase activity in neutrophils over control levels within 1 min of incubation. Pretreatment of neutrophils with taxol was able to suppress this activation of MAP kinase. Taxol produced a concentration-dependent inhibition of opsonized CPPD-induced neutrophil chemiluminescence, superoxide anion production and myeloperoxide release. Taxol at 28 microM also significantly inhibited chemiluminescence, superoxide anion production and myeloperoxidase release from neutrophils stimulated by opsonized zymosan. This is the first report of crystal-induced activation of MAP kinase in neutrophils. Microtubule-associated processes, such as signal transduction, secretion and phagocytosis are involved in particulate-induced neutrophil responses. We have suggested that the inhibitory effect of taxol observed in this work is due to its stabilizing effect on microtubules and disruption of MAP kinase activation associated with microtubules.