No abstract

IntroductionWe have examined the mechanisms involved in the adherence of normal peripheral blood eosinophils to cultured human umbilical vein endothelial cells (HEC) under three conditions: (a) adherence in the absence of treatment of HEC or eosinophils with activating agents (basal adherence); (b) adherence induced by stimulation ofeosinophils with phorbol ester (eosinophil-dependent adherence); and (c) adherence induced by pretreatment of HEC with LPS, tumor necrosis factor (TNF), or IL-I (endothelial-dependent adherence). A mechanism was identified that was equally active in basal, eosinophil-dependent, and endothelial-dependent adherence. This mechanism was optimally active in the presence of both Ca"+ and Mg", and reduced in the presence of Ca"+ only or Mg"+ only. Furthermore, like the other mechanisms of eosinophil adherence, it was active at 370C but not at 4VC. A second mechanism of adherence was involved in eosinophil-and in endothelial-dependent adherence. This mechanism was dependent on the CD11/CD18 adhesion complex of eosinophils (i.e., inhibited by anti-CD18

Background: Distinguishing ingested particles from those attached to the cell surface is an essential requirement when performing quantitative studies of phagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans-human neutrophils (PMN) system. Methods: The assay is based on the observation that the vital dye trypan blue (TB), while quenching the green fluorescence of fluorescein-labeled C. albicans, causes them to fluoresce red. PMN were incubated with fluorescein-labeled yeast particles for the required time. Aliquots of the incubation mixtures were then promptly diluted with an equal volume of a TB solution at pH 4.0, and subsequently analyzed by flow cytometry for green and red fluorescence.

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

Abstract. Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-a (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M,A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751. We have studied TNFinduced C1-movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of [32 integrins cosignaled for an early, marked, and prolonged C1-efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between CI-efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced CI-efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of CI-efflux, such as ethacrynic acid. Moreover, CI-efflux induced by means other than TNF treatment, i.e., by using Cl--free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of CI-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2--forming NADPH oxidase. N 'EtrrRoamLtC polymorphonuclear leukocytes (PMN) 1 respond to both particulate and soluble stimuli with a vigorous respiratory burst. This leads to the release of toxic oxygen molecules that contribute to both the PMN microbicidal activity and the tissue inflammatory damage. Among the physiologically relevant soluble stimuli, cytokines such as tumor necrosis factor-a (TNF), granulocyte/macrophage-colony stimulating factor, and granulocyte-colony stimulating factor are peculiar, since they activate the respiratory burst only in PMN residing on biologic surfaces, e.g., on proteins of the extraAddress all correspondence to R. Menegazzi, Istituto di Patologia Generale, Universit~t di Trieste, via A. Fleming, 22, 34127 Trieste, Italy. Tel.: (39) 40 572012. Fax: (39) 40 567862.1. Abbreviations used in this paper: 9-AC, anthracene-9-carboxylic acid; CHC, c~-cyano-4-hydroxy-cinnamic acid; C1-1, intracellular chloride content; DIDS, 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid; EA, ethacrynic acid; FBG, fibrinogen; FMLP, N-formyl-methyonil-leucyl-phenylalanine; FN, fibronectin; FN-PMN, PMN residing on FN-coated surfaces; G-buffer, glucuronate-containing buffer; MA, o-[(3-hydroxymercuri-2-methoxypropyl)carbamoyl] phenoxyacetic acid; Oz-, superoxide anion; PMN, neutrophilic polymorphonuclear leukocytes; s-PMN, PMN in suspension; poly (HEMA), poly(2-hydroxyethyl methacrylate); TNF, tumor necrosis factor-a; TNF-R, TNF receptor. cellular matrix immobilized on a solid support, but...