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

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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...

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.