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J L Mege

Publications and source records attributed to J L Mege.

At least 19 recordsLinked to original sources
PubMed1995

Morphological polarization of human polymorphonuclear leucocytes in response to three different chemoattractants: an effector response independent of calcium rise and tyrosine kinases.

Chemoattractants such as interleukin-8, C5a and N-formylmethionyl-leucyl-phenylalanine induce a cytosolic calcium rise involved in triggering the secretory functions of human polymorphonuclear leucocytes. We studied the possible role of calcium rise in membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization, which are all events contributing to chemotaxis. Membrane ruffling was assessed by right-angle light-scatter changes, the cellular content of polymerized actin by fluorescence of bodipy phallacidin, the intracellular distribution of filamentous actin by fluorescence microscopy and image digitization, and morphological polarization by scanning electron microscopy. Pretreatment of polymorphonuclear leucocytes with 50 microM BAPTA/AM, an intracellular calcium chelator, lowered the basal level in cell calcium and inhibited the transient calcium rise stimulated by 2 nM interleukin-8, 2 nM C5a, and 10 nM N-formylmethionyl-leucyl-phenylalanine. However, BAPTA pretreatment of polymorphonuclear leucocytes did not modify membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization stimulated by chemoattractants. Downstream effectors may be protein tyrosine kinases. However, the tyrosine kinase inhibitor tyrphostin did not affect the cytoskeletal characteristics elicited by chemoattractants. Taken together, our results suggest that the transductional pathway leading to cytoskeleton organization and morphological polarization of polymorphonuclear leucocytes is different from that leading to secretion.

Actins↗
PubMed1994

Non-endotoxinic tumour necrosis factor-alpha-inducing factors in haemodialysis.

A transmembrane passage of endotoxins may account for the dysfunction of cytokine production which has been often reported in haemodialysis. We developed an assay based on the ability of patient serum to stimulate tumour necrosis factor alpha (TNF alpha) secretion in normal peripheral blood mononuclear cells. Three groups of subjects were investigated: normal controls (n = 14), patients with chronic renal failure, CRF (n = 15), and patients dialysed with polyacrylonitrile (n = 7), polysulphone (n = 8), and cellulose acetate (n = 7). Sera from dialysed patients displayed a significantly higher TNF alpha-inducing activity than those of controls and CRF patients. The ability of serum to elicit TNF alpha secretion was neither modified during the dialysis session nor influenced by the type of haemodialysis membrane. TNF alpha-inducing activity in serum was not inhibited by polymyxin B, known to impair endotoxin-dependent cell responses, thus suggesting that it was not related to circulating endotoxins. We conclude that non-endotoxinic factors are present in serum from dialysed patients and are able to induce cytokine secretion.

Antigens, CD↗
PubMed1993

Zymosan-induced tyrosine phosphorylations in human monocytes. Role of protein kinase C.

Protein tyrosine phosphorylations are involved in the proliferation and secretory responses of immune cells, but their role in phagocytes is poorly understood. The ability of unopsonized zymosan to induce protein tyrosine phosphorylations was investigated in human monocytes. The addition of zymosan to monocytes resulted in an increase in tyrosine phosphorylation of several endogenous proteins including 28-, 33-, 38-, 42-, 47-, 55- to 60-, 62-, 68-, 90-, 105-, 116-, and 120-kDa proteins; 55- to 60-kDa proteins were the predominant phosphoproteins. Moreover, we studied the effects of tyrphostin 23, a specific tyrosine kinase inhibitor, on stimulated tyrosine phosphorylations and early secretory responses of monocytes, i.e., arachidonic acid release and oxidative metabolism. We showed that tyrphostin inhibited zymosan-stimulated tyrosine phosphorylations and arachidonic acid release, but that it did not affect superoxide generation induced by zymosan. Zymosan binds mainly to CR3 receptor on human monocytes, and CR3 is devoid of intrinsic tyrosine kinase activity. It was predictable that zymosan stimulated a tyrosine kinase distal to the receptor or associated with it. We observed that PMA mimicked zymosan-induced tyrosine phosphorylations, thus suggesting that both agonists used a common transductional pathway implicating the serine/threonine kinase, protein kinase C. The antagonists of protein kinase C, sphingosine and calphostin C, inhibited zymosan-stimulated tyrosine phosphorylations. We suggest that, in human monocytes, zymosan-induced tyrosine phosphorylations are involved in cell responses such as the release of arachidonic acid, and that they require the sequential activation of protein kinase C and cellular protein tyrosine kinases.

Catechols↗
PubMed1992

Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.

Antigens, CD↗
PubMed1992

Chronic and intradialytic effects of high-flux hemodialysis on tumor necrosis factor-alpha production: relationship to endotoxins.

Tumor necrosis factor-alpha (TNF alpha) likely plays a role in hemodialysis-associated complications. As TNF alpha is mainly produced by monocytes in response to endotoxins, we studied its production and the presence of circulating endotoxins in patients dialyzed on polyacrylonitrile (PAN) membrane. Spontaneous production of TNF alpha was observed in patients before the dialysis session and increased during the session. Endotoxins were present in serum from patients chronically dialyzed with PAN and increased during hemodialysis session. In addition, intradialytic decrease in CD14 antigen expression on circulating monocytes, which could be caused by endotoxins, was found. The continuous presence of low amounts of circulating endotoxins between sessions may explain the chronic increase in TNF alpha secretion, while high amounts of circulating endotoxins may account for intradialytic oversecretion of TNF alpha and downmodulation of CD14. We suggest that endotoxin-free dialysates should be a prerequisite for the use of high-flux membranes.

Acrylic Resins↗
PubMed1992

Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.

Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably resulted from local filament assembly and put forward the hypothesis that microfilament reorganization in two centres drives the morphological polarization of PMNs.

Actin Cytoskeleton↗
PubMed1991

Role of tumor necrosis factor-alpha in Sneddon-Wilkinson subcorneal pustular dermatosis. A model of neutrophil priming in vivo.

A patient with IgG-kappa-associated subcorneal pustular dermatosis (Sneddon-Wilkinson disease) refractory to dapsone, etretinate, and plasma exchange was successfully treated with corticosteroids. A study of neutrophils from both blood and pustules was carried out before and during treatment. Levels of tumor necrosis factor-alpha were measured in serum, pustules, content, and supernatant of monocytes. The results suggest that a hyperactivation of neutrophils in the skin is due at least partly to excessive production of tumor necrosis factor-alpha.

Aged↗
PubMed1991

Haemodialysis membranes modulate chronically the production of TNF alpha, IL1 beta and IL6.

As cytokines may play a role in the adverse effects of haemodialysis, TNF alpha, IL1 beta and IL6 were investigated before the haemodialysis session (chronic effect) and after 30 and 60 min (session effect). We found that haemodialysis exerts a chronic effect on cytokines but the type of haemodialysis membrane, Cuprophan or Hemophan, specifically influences each cytokine. Circulating levels of TNF and unstimulated production of TNF and IL1 by monocytes were increased in patients dialysed with Hemophan, whereas a greater LPS-stimulated production of TNF was observed in patients dialysed with Cuprophan. Both types of membrane induced a higher production of IL6 as compared to controls. The alternate use of Cuprophan and Hemophan demonstrated that the production of TNF and IL1 was dependent on the type of haemodialysis membrane. We also found that Cuprophan induced a reversible decrease of spontaneous and LPS-stimulated production of TNF, IL1 and IL6 during the haemodialysis session. Taken together, these results suggest that Hemophan induced a sustained production of cytokines whereas Cuprophan primed monocytes, probably through the activation of the complement pathway.

Aged↗
PubMed1990

Skin necrosis after injection of aminosides. Arthus reaction, local toxicity, thrombotic process or pathergy?

A case of extensive skin necrosis following intramuscular injections of sisomycin is described. Eight cases of necrosis following subcutaneous injections of aminoside antibiotics have been reported in elderly females under anticoagulant theray, but the pathogenic mechanisms of these lesions have not been investigated. Defective fibrinolysis and abnormalities of oxidative metabolism of neutrophils were noted in our patient. Four major mechanisms are discussed: an Arthus reaction, a thrombotic process, a direct toxicity of aminosides for subcutaneous tissue, or a pyoderma-like neutrophilic reaction. Subjects with a tendency to thrombosis and/or abnormalities of neutrophil functions may be predisposed to these skin necroses.

Arthus Reaction↗
PubMed1990

[Function of polynuclear neutrophils in patients with juvenile periodontitis and rapidly progressing periodontitis].

It is generally assumed that early onset forms of periodontal disease (including Juvenile and Rapidly Progressive Periodontitis) are associated with a defect in neutrophil behaviour. We have investigated neutrophil functions in patients with Juvenile (J.P.) and Rapidly Progressive Periodontitis (R.P.P.). In the group of J.P. patients the directed mobility (FMLP and zymosan activated plasma) is significantly decreased. The superoxide generation in response to FMLP decreases while the PMA response is normal. In the group of R.P.P. patients no significant abnormality has been reported.

Aggressive Periodontitis↗
PubMed1990

[Impairment of neutrophil functions: study of a family with a case of juvenile periodontitis].

Neutrophil function was assessed in family in which only one member suffered from Juvenile Periodontitis (JP). Directed mobility (fMet-Leu-Phe and zymosan-activated plasma) was decreased in all siblings without involving a seric inhibitor. Adhesion was studied by a new method which allows for the evaluation of both adhesive rate and binding strengths. The latter parameters were decreased in the parents neutrophils, but remained increased in a set of twin sisters. The specific receptor induced phagocytosis was altered in all members of the family (FC receptor: IgG-SCR, C3b and mannosylfucosyl receptor: zymosan). The superoxide generation in response to fMet-Leu-Phe was decreased while the PMA response was almost normal. These results suggest an overall abnormality of ligand-receptor interactions (C5a, fMet-Leu-Phe, Fc and C4b receptors), this defect seems to involve some membrane characteristics and underlines the absence of correlation between PMN deficiency and the clinical expression of J.P.

Adult↗
PubMed1989

Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: role of protein kinase and G-proteins.

Granulocyte-macrophage colony-stimulating factor, GM-CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet-Leu-Phe and platelet-activating factor, PAF, but not by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan. The potentiation is greatest in fMet-Leu-Phe-stimulated cells. This indicates that the actions of only certain receptors are potentiated by GM-CSF. Incubation of the cells with the protein kinase inhibitor H-7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM-CSF does not affect the observed potentiation. The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM-CSF. The data suggest that neither protein kinase C nor protein synthesis is necessary for GM-CSF action. On the other hand, no potentiation can be seen in the presence of cytochalasin B. Unlike intact cells, GM-CSF does not enhance superoxide production by cytoplasts stimulated with fMet-Leu-Phe. The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM-CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM-CSF. The amount of actin associated with the cytoskeleton under control of fMet-Leu-Phe-stimulated condition is the same in normal and GM-CSF-treated human neutrophils. Botulinum D toxin ADP-ribosylates a protein with a molecular weight of 22 kDa. This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM-CSF. Botulinum D toxin does not affect the basal or the fMet-Leu-Phe-induced rise in the intracellular concentration of free calcium in human neutrophils. GM-CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet-Leu-Phe. The increases are inhibited by pertussis toxin. Several important conclusion can be drawn from these data. 1) GM-CSF potentiates the rise in Ca2+i produced by PAF and fMet-Leu-Phe, and these potentiations are inhibited in pertussis-toxin-treated cells. 2) GM-CSF does not prime cytoplasts to stimulation by fMet-Leu-Phe. This suggests that the granules and/or nucleus are necessary for the priming action. 3) The priming by GM-CSF is not mediated by the H-7-sensitive protein kinase C, botulinum D-sensitive G-protein, or protein synthesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Phagocyte-pathogen in the infected host.

Phagocytosis is a major mechanism of defense against bacterial infections. The ingestion of different microorganisms by blood granulocytes or monocytes may involve a variety of cell membrane recognition structures (e.g., immunoglobulin or complement receptors, lectin-like structures or other nonspecific binding sites). It is of interest to know which mechanism plays a prominent role in the management of a particular type of infection. Forty-three pathogenic bacterial suspensions were obtained from patients under mechanical ventilation at the onset of nosocomial lower respiratory tract infections. They were coincubated with blood granulocytes from the same or other patients in the presence or absence of the corresponding serum. Phagocytosis and the oxidative burst were then assayed. We conclude the following: a) Substantial phagocytosis was found under serum-free conditions and the patients' sera did not dramatically enhance bacterial uptake during the first days after the onset of clinical symptoms. b) The phagocytes from an infected patient did not display any peculiar inability to bind to the bacteria that grew successfully in this subject. Hence, the occurrence of a particular infection might be dependent on a defect of intracellular killing of ingested pathogens or on the conditions of infection development.

Adult↗
PubMed1989

Actions of the protease inhibitor phenylmethylsulfonyl fluoride on neutrophil granule enzyme secretion and superoxide production induced by fMet-Leu-Phe and phorbol 12-myristate-13-acetate.

The protease inhibitor, phenylmethylsulfonyl fluoride inhibits granule enzyme release and, above 1 mM, superoxide production from rabbit peritoneal neutrophils induced by the chemotactic peptide, fMet-Leu-Phe. At concentrations below 1 mM, it enhances superoxide production. Superoxide generation stimulated by phorbol 12-myristate-13-acetate is increased by phenylmethylsulfonyl fluoride at all concentrations studied. Phenylmethylsulfonyl fluoride has no effect on the rise in intracellular calcium or the depolarization induced by fMet-Leu-Phe but does decrease the extent of repolarization and abolishes hyperpolarization. It depresses actin polymerization and abolishes cytoplasmic alkalinization caused by fMet-Leu-Phe. The increased phosphorylation induced by phorbol 12-myristate-13-acetate in four of the five proteins studied was not affected by phenylmethylsulfonyl fluoride, but the increased phosphorylation of the fifth, a 21-kD protein was enhanced. We conclude that phenylmethylsulfonyl fluoride acts on inhibitory and enhancing processes or steps induced by fMet-Leu-Phe which are subsequent to or independent of calcium mobilization and protein kinase C activity.

Actins↗
PubMed1988

Effect of botulinum D toxin on neutrophils.

Activated botulinum D toxin ADP-ribosylates a 22 kDa molecular weight protein in homogenates obtained by sonication of a suspension of rabbit peritoneal neutrophils. The ADP-ribosylation catalyzed by activated botulinum D toxin is inhibited in homogenates obtained from cells pretreated with the toxin, suggesting that it is able to enter into these cells and be activated by them. The rise in intracellular concentration of free calcium in toxin treated cells stimulated by fMet-Leu-Phe is similar to that found in control cells. The basal concentration of intracellular free calcium is significantly elevated in neutrophils treated with the intact but not with the activated form of the botulinum D toxin. Superoxide generation in control and native toxin treated cells stimulated with fMet-leu-Phe, phorbol 12-myristate 13-acetate or opsonized zymosan is the same. The release of beta-glucosaminidase produced by fMet-Leu-Phe or Concanavalin A in botulinum D toxin treated neutrophils was slightly higher than the corresponding release in control cells. Furthermore, the fMet-Leu-Phe-induced increase in the amount of actin associated with the cytoskeleton is not inhibited by botulinum D toxin. These results suggest that the 22 kDa protein which can be ADP-ribosylated by botulinum D toxin is not involved in these stimulated neutrophil responses.

Adenosine Diphosphate Ribose↗