Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.

Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils.

The activation of human neutrophils by monosodium urate and calcium pyrophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, respectively. In this study, we investigated the potential involvement of tyrosine phosphorylation in microcrystal-mediated activation of human neutrophils. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that triclinic monosodium urate and calcium pyrophosphate dihydrate crystals stimulated a time- and concentration-dependent tyrosine phosphorylation of at least five proteins (pp130, 118, 80, 70, and 60). While phosphoprotein (pp) 118 and pp70 were the major phosphorylated substrates, pp70 was the dominant one in reactivity with antiphosphotyrosine antibodies. When the temporal patterns, as well as the levels of tyrosine phosphorylation for both types of crystals were compared, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphorylation patterns induced by microcrystals differed from those stimulated by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unopsonized latex beads or zymosan) agonists which stimulated preferentially the tyrosine phosphorylation of pp118. The ratio of the intensities of pp118 and pp70 were specific of the stimulation with microcrystals when compared to those observed with the other soluble or particulate agonists. Colchicine, a drug used specifically in the treatment of gout and pseudogout, inhibited microcrystal-induced tyrosine phosphorylation, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosphorylation. Furthermore, while colchicine inhibited the activation of the NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these results (a) demonstrate that tyrosine phosphorylation is involved in the mechanism of activation of human neutrophils induced by microcrystals; and (b) suggest, on the basis of the characteristics of the observed patterns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.

Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo.

Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 microM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

In vitro leukotriene (LT) C4 synthesis by blood eosinophils from atopic asthmatics: predominance of eosinophil subpopulations with high potency for LTC4 generation.

We compared the leukotriene (LT) C4 generation by the eosinophil density subpopulations isolated from the blood of asthmatics and normal subjects. Using discontinuous Percoll gradients, eosinophil subpopulations with densities of 1.075, 1.078, 1.081, 1.084, 1.087 and 1.100 g/ml were isolated from the blood of six atopic asthmatics and seven normals. In normals, most eosinophils (94%) were recovered in the density fractions (1.084, 1.087, and 1.100 g/ml). In asthmatics, the eosinophil density profile was shifted towards lower cell density: the eosinophil subsets 1.078, 1.081 and 1.084 g/ml were increased 4.5, 30.3 and 8.9-fold respectively compared to normals (p less than 0.0001); the intermediate subsets, the hypodense 1.081 and normodense 1.084 g/ml fractions being the predominant subpopulations. The ability of asthmatic eosinophil subsets 1.078 to 1.087 g/ml to release LTC4 was measured by reversed-phase HPLC, LTC4 production was highest with the normodense 1.084 g/ml eosinophil subset (149 +/- 28 pmol/10(6) eosinophils, p less than 0.01 compared to the 1.081 fraction). The eosinophils from the hypodense 1.081 g/ml fraction also released more LTC4 (112 +/- 19 pmol/10(6) eosinophils) than the 1.078 and 1.087 g/ml fractions (70 +/- 16 and 67 +/- 12 respectively, p less than 0.01). These results show that, compared to normals, blood of mild atopic asthmatics contains an elevated number of eosinophils of intermediate density which have a high capacity for LTC4 production.

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils.

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.

The IL-1 and IL-1 receptor antagonist (IL-1Ra) response of human neutrophils to EBV stimulation. Preponderance of IL-Ra detection.

The present study investigated the effect of EBV on gene expression and protein synthesis of IL-1 and its natural IL-1R antagonist (IL-1Ra) in human peripheral blood neutrophils. EBV induced a rapid accumulation of IL-1 and IL-lRa mRNA in neutrophils that was associated with the later appearance of considerable amounts of IL-1alpha, IL-1beta, and IL-Ra proteins. Approximately 3200 and 610 times more IL-Ra than IL-1alpha a or IL-1beta, respectively, was secreted by neutrophils in response to EBV. The effect induced by EBV cannot reflect an overall metabolic activity of neutrophils, since EBV failed to induce granulocyte-macrophage OF synthesis. Heat-inactivated virus was unable to stimulate cytokine synthesis, whereas UV-irradiated virus retained the full IL-1- and IL-1Ra-inducing potential of the native particle. Furthermore, pretreatment of cells with cycloheximide or phosphonoacetic acid did not abrogate the effect of EBV, suggesting that EBV does not penetrate the cell, but that a virion's structural molecule is required to induce such an effect. In this respect, neutralization of the viral particles with the mAb 72A1, which is known to react with glycoprotein gp350 of the viral envelope, inhibits the production of IL-1 and IL-1Ra, suggesting that gp350 could be involved in this process. Thus, the elevated levels of IL-1Ra detected for EBV-stimulated neutrophils might be part of a mechanism used by the virus to evade the immune system.

Activation of human neutrophils by technical toxaphene.

Toxaphene is a persistent organic pollutant (POP) known to be composed of numerous congeners. Toxaphene technical mixture applied as a pesticide consists of over 800 congeners. Among these, T(2) and T(12) are the two environmentally prevalent forms found in humans. Although toxaphene is known to exert some toxic effects, including potential proinflammatory properties, little is known concerning its action on cells of the human immune system, especially neutrophils. In the present study, we found that toxaphene was not necrotic for human neutrophils incubated for up to 24 h with concentrations ranging from 0.1 to 50 microg/ml. Toxaphene was found to induce neutrophil superoxide production (O(-)(2)) in a concentration-dependent manner. The potency and the kinetics of toxaphene-induced O(-)(2) by neutrophils were found to be similar to that of the classical neutrophil agonists phorbol 12-myristate 13-acetate (PMA). Furthermore, the use of various transduction signal inhibitors (genistein, pertussis toxin, staurosporine, H-7, and HA-1077), suggests that, as for PMA, toxaphene mediates its effect primarily via PKCs and, to a lesser extend, via tyrosine kinases. In this respect, staurosporine, H-7, and genistein were found to inhibit toxaphene- and PMA-induced O(-)(2) production by 52, 72, and 31% and by 63, 62, and 23%, respectively. Toxaphene was also found to significantly enhance neutrophil phagocytosis of opsonized sheep red blood cells and to induce neutrophil apoptosis. The induction of neutrophil apoptosis was paralleled with a decrease in CD16 expression. T(2) and T(12), the two prevalent congeners found in humans, were also found to significantly increase the O(-)(2) production in neutrophils at a concentration of 5 microg/ml. We conclude that neutrophils are important targets for toxaphene, as this POP can activate O(-)(2) production by a PKC- and tyrosine kinase-dependent mechanism, induce phagocytosis, and accelerate the apoptotic rate. This is the first study that focuses on toxaphene/human neutrophil interactions.

Granulocyte-macrophage colony-stimulating factor enhances EBV-induced synthesis of chemotactic factors in human neutrophils.

We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1alpha and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.

EBV induces the production and release of IL-8 and macrophage inflammatory protein-1 alpha in human neutrophils.

As the first line of defense in the immune system, neutrophils may release a variety of potent agents upon exposure to infectious agents. In this study we have investigated the ability of human neutrophils to produce chemotactic cytokines, or chemokine in response to EBV. Exposure of neutrophils to EBV led to an increase in accumulation of mRNA for IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). EBV stimulated a time-dependent production of immunoreactive IL-8 and MIP-1alpha by neutrophils. The ability of EBV to stimulate the synthesis of IL-8 and MIP-1alpha protein was reflected by both an accumulation of the protein in the intracellular compartment as well as increased secretion. A variety of control studies support the idea that infectious EBV is not required for induction of chemokine gene expression; however, the response is dependent on the interaction between the glycoprotein gp350 of the viral envelope and the neutrophil surface. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and for T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance the ability of this virus to infect B and T lymphocytes via increased recruitment to sites of infection.

Crystal-neutrophil interactions lead to interleukin-1 synthesis.

Normal human blood neutrophils were studied for their capacity to synthesize and release interleukin-1 (IL-1) species after phagocytosis of triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate crystals (CPPD). MSU crystals were more potent inducers of IL-1 generation than CPPD or unopsonized zymosan. Microcrystal-stimulated neutrophils characteristically secreted most of the newly synthesized IL-1. Colchicine partly inhibited the secretion of IL-1 by neutrophils during phagocytosis of solid particles. However, colchicine selectively inhibited IL-1 synthesis induced by microcrystals. These results suggest that neutrophil-derived IL-1 may contribute to the pathogenesis of crystal-induced arthritis.

Crystal-induced neutrophil activation. I. Initiation and modulation of calcium mobilization and superoxide production by microcrystals.

The effects of monosodium urate and calcium pyrophosphate dihydrate crystals on the levels of cytoplasmic free calcium and on the oxidative burst in normal human blood neutrophils were examined. The pattern of sensitivity to granulocyte-macrophage colony-stimulating factor, colchicine, cytochalasin B, pertussis toxin, diglyceride kinase, and protein kinase C inhibitors differentiated the mechanism(s) of neutrophil activation by the crystals from that involved in the responses to soluble chemotactic factors and indicated that individual crystals can use several activation pathways.

Crystal-induced neutrophil activation. V. Differential production of biologically active IL-1 and IL-1 receptor antagonist.

Neutrophils produce IL-1 when stimulated by monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals. Neutrophils also generate the IL-1R antagonist (IL-1Ra), especially when incubated with granulocyte-macrophage CSF (GM-CSF) or TNF-alpha. We studied the simultaneous expression of IL-1 and IL-1Ra by GM-CSF- or TNF-alpha-treated neutrophils activated by MSU or CPPD. Neutrophils incubated with GM-CSF or TNF-alpha produced approximately 300 or 200 times more IL-1Ra than IL-1, respectively. Suboptimal concentrations of MSU or CPPD induced low amounts of IL-1 without affecting IL-1Ra. Interaction of GM-CSF- and TNF-alpha-treated neutrophils with MSU or CPPD up-regulated IL-1 while simultaneously down-regulating IL-1Ra. As a result, the bioactivity of IL-1 secreted was enhanced. Synergistic increases of IL-1 (but not IL-1Ra) mRNA levels were noted in GM-CSF- or TNF-alpha-treated neutrophils exposed to CPPD. Treatment of neutrophils with colchicine before incubation with GM-CSF or TNF alpha, inhibited crystal-induced IL-1 by 50 to 55%, but failed to significantly affect IL-1Ra. The IL-1Ra to IL-1 ratio was significantly increased by 185 to 220%. These results demonstrate that IL-1 and IL-1Ra production by human neutrophils are differentially regulated, that the combined presence of GM-CSF or TNF-alpha and microcrystals favor the production of biologically active IL-1 over that of IL-1Ra, and that colchicine selectively inhibits IL-1 without affecting IL-1Ra production.

Crystal-induced neutrophil activation. II. Evidence for the activation of a phosphatidylcholine-specific phospholipase D.

OBJECTIVE: To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD). METHODS: Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl-phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1-0-3H-alkyl-2-acetyl-sn-glycero-3-phosphocholine. The cells were stimulated with microcrystals of triclinic morphology. RESULTS: Both MSU and CPPD crystals induced a time- and concentration-dependent accumulation of phosphatidic acid mass and elevation in levels of alkyl-phosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal-stimulated production of superoxide anions. CONCLUSION: These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil-microcrystal interactions may be dependent on this biochemical pathway.

Inhibition of tyrosine phosphorylation by wortmannin in human neutrophils. Dissociation from its inhibitory effects on phospholipase D.

BACKGROUND: Recent studies have indicated that the regulation of the activation of human neutrophils depends on tyrosine phosphorylation and on phospholipase D. Furthermore, a tentative causal relationship between these two signalling pathways has been indirectly implied derived through the use of inhibitors of tyrosine kinases. The fungal metabolite, wortmannin is at present the only compound known to inhibit the receptor-mediated activation of phospholipase D in human neutrophils. Its mechanism of action is presently unknown. EXPERIMENTAL DESIGN: The ability of peripheral blood neutrophils to respond to various agonists with an increase in activity of phospholipase D and an enhancement of tyrosine phosphorylation in the absence or presence of wortmannin was monitored. RESULTS: Wortmannin was found to inhibit the stimulation of tyrosine phosphorylation by fMet-Leu-Phe, and by the inflammatory microcrystals monosodium urate and calcium pyrophosphate dihydrate. This effect of wortmannin was not secondary to inhibition of phospholipase D as U73122, a previously described phospholipase C inhibitor, was also found to inhibit phospholipase D without affecting tyrosine phosphorylation. CONCLUSIONS: The results make it likely that one of the earliest sites of action of wortmannin in human neutrophils is at the level of tyrosine phosphorylation which then exerts a modulatory influence on the activation of phospholipase D.

Tyrosine phosphorylation in activated human neutrophils. Comparison of the effects of different classes of agonists and identification of the signaling pathways involved.

The tyrosine phosphorylation responses initiated in human neutrophils by soluble and particulate agonists were characterized. Chemotactic factors, hematopoietic growth factors, and inflammatory microcrystals stimulated in a time- and concentration-dependent manner the tyrosine phosphorylation of distinct patterns of substrates: pp120, pp85, pp70, and pp60 in the case of chemotactic factors; pp155, pp130, pp120, pp85, pp60, and pp40 in the case of granulocyte macrophage-CSF; and pp130, pp120, pp70, and pp60 in the case of monosodium urate (MSU) crystals. Several of the single bands on one-dimensional blots (including pp40, pp70, and pp120) could be resolved into multiple spots on two-dimensional gels. The responses of several other chemotactic factors resembled those of FMLP. Cytokineplasts retained the capacity to respond to FMLP, granulocyte-macrophage-CSF, or MSU crystals with a stimulation of tyrosine phosphorylation, and contained the major substrates detected in intact neutrophils. Several unrelated tyrosine kinase inhibitors (herbimycin A, genistein, and erbstatin) strongly diminished the tyrosine phosphorylation response to chemotactic factors. Pertussis toxin abrogated the tyrosine phosphorylation response to FMLP, whereas protein kinase C (Ro 21-8220, chelerithryn) inhibitors were without effect. Chelation of intracellular calcium attenuated the tyrosine phosphorylation response to FMLP. These results indicate that G proteins play a crucial role in the coupling of chemotactic factor receptors to tyrosine phosphorylation and that this coupling occurs in parallel to that of phospholipase C. These results also underline the complexity of the transduction pathways implicated in the initiation of tyrosine phosphorylation.