Chloride Ion Efflux Regulates Adherence, Spreading, and Respiratory Burst of Neutrophils Stimulated by Tumor Necrosis Factor-tx (TNF) on Biologic Surfaces

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 C1movements 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 C1efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between CIefflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced CIefflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of CIefflux, such as ethacrynic acid. Moreover, CIefflux 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 CIi 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. Abbreviat ions 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 not in PMN in suspension (34, 38, 39, 40). TNF is the most extensively studied in this respect. It has been shown that PMN exposed to TNF on several biologic surfaces, e.g., FBS, fibronectin (FN), and fibrinogen (FBG), adhere, spread, and activate their oxidative metabolism (18, 34, 37, 41). Several lines of evidence indicate that this series of responses is cosignaled by ligation of TNF receptors (TNF-R), more specifically the 55-kD TNF-R (TNF-R55) (34), and binding of activated proadhesive molecules to receptors on the biologic substrates. CDll/CD18 (132) integrins have been shown to be relevant in this respect, since: (a) PMN of leukocyte adhesion deficiency patients, which lack 132 integrin expression (29), fail to respond to TNF with a metabolic burst (38), and (b) anti-132 mAbs inhibit both adherence and respiratory burst of PMN exposed to TNF on biologic surfaces (32, 34, 37, 38). The relative role of TNFand 132 integrin-derived intracellular signals has not been clearly defined. It has been hypothesized that engagement of the TNF receptor leads to a modification, often called "activation," of the 132 integrins, which results in an increased affinity for the appropriate surface ligand and in an adhesive PMN response © The Rockefeller University Press, 0021-95251961101511/12 $2.00 The Journal of Cell Biology, Volume 135, Number 2, October 1996 511-522 511 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom (33, 34, 37, 46, 59). Once adherent, the cells would rely on the signaling ability of 132 integrins to initiate the process of spreading with the ongoing activation of the respiratory burst (5, 37, 39, 41). The nature of the intracellular signals involved in TNFinduced activation of PMN oxidative metabolism on biologic surfaces has been a matter of recent studies. The release of toxic oxygen molecules by TNF-stimulated PMN has been shown to be independent of a pertussis toxin-sensitive GTP-binding protein (31), weakly sensitive to protein kinase C inhibitors (31), and strongly impaired by drugs that interfere with the assembly of the cytoskeleton network (31, 34, 37, 39). Furthermore, it is not accompanied by hydrolysis of phosphoinositides nor by the release of arachidonic acid (31). It has been also shown that occupation of TNF receptors and engagement of CD18 (132) integrins interact synergistically to promote a sustained fall in cAMP in PMN plated on FBSor FBG-coated surfaces (37). This drop in cAMP is permissive for TNF-induced spreading and metabolic activation, since cAMP-elevating agents prevent both responses. Also, tyrosine phosphorylation has been demonstrated to be essential for PMN spreading and metabolic activation induced by TNF, since inhibitors of tyrosine kinases prevent spreading and metabolic activation of these cells on FBSand FBG-coated surfaces (18, 19, 32). The participation of 132 integrin--derived signals in mediating protein tyrosine phosphorylation is formally proved by the observation that TNF does not trigger this response in leukocyte adhesion deficiency PMN (6, 19). Studies on other possible intracellular signals evoked in PMN by TNF include the analysis of inorganic ion movements. Elevations in cytosolic free Ca 2+ levels are still a matter of debate. In fact, while some authors obtained evidence for TNF-induced intracellular Ca 2+ changes (44, 48), others did not (42, 49). Other groups demonstrated that TNF causes a decrease of intracellular H ÷ concentration that leads to cytoplasmic alkalinization (49, 64). Recently, attention has been focused on chloride ion movements in TNF-stimulated PMN (48, 49). The intracellular C1concentration of resting PMN is unusually high, being fourfold higher than that predicted on the basis of Nernst equation (3, 51). It has been shown that TNF (49), as well as other soluble stimuli (36, 49), causes an elflux of chloride ions from PMN in suspension, with the concomitant decrease of the intracellular chloride content (Cl-i). The relevance of such a change to effector functions of PMN, e.g., adherence, spreading, and activation of the metabolic burst, has not been investigated. In this paper, we show that TNF-stimulated PMN residing on FN-coated surfaces (FN-PMN) exhibit a massive C1efflux accompanied by a concomitant decrease of C1i that precedes adhesion, spreading, and activation of the respiratory burst. Inhibition of CIefflux also inhibits adhesion, spreading, and activation of the burst, whereas induction of C1fluxes by means other than TNF treatment (i.e., the use of Cl--free media) also causes PMN to adhere, spread, and mount a respiratory burst on FN. We therefore suggest that C1efflux plays a role in the elicitation of these three functions of FN-PMN. We also show that the TNF-dependent cascade of C1efflux, adhesion, spreading, and respiratory burst of FN-PMN is initiated by occupancy of the TNF-R55 receptor. Materials and Methods