Role of intracellular chloride in the reversible activation of neutrophil beta 2 integrins: a lesson from TNF stimulation.

The process of beta(2) integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes' (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl(-) content (Cl(-)(i)) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl(-)(i) in the activation of beta(2) integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of beta(2) integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin alpha subunits, and epitope CBRM1/5, localized to the I domain on the alpha-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl(-)(i) because 1) inhibition of Cl(-)(i) decrease is invariably accompanied by inhibition of beta(2) integrin activation, 2) Cl(-)(i) decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl(-)-free buffers, activates beta(2) integrins, and 3) restoration of the original Cl(-)(i) levels is accompanied by deactivation of beta(2) integrins. We also show that Cl(-)(i) decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pH(i) does not seem to be relevant for beta(2) integrin activation. The results of our study emphasize the role of Cl(-) as a new PMN "second messenger."

Triggering of chloride ion efflux from human neutrophils as a novel function of leukocyte beta 2 integrins: relationship with spreading and activation of the respiratory burst.

PMN residing on immobilized fibronectin have been shown to respond to TNF with an intense and long lasting Cl- efflux that leads to a marked decrease of the unusually high basal Cl- content of these phagocytes. The finding that this Cl- efflux depends, at least in part, on beta2 integrin engagement stimulated the present investigation, which addresses the question as to whether beta2 integrins per se, in the absence of PMN agonists, are able to generate signals triggering Cl- efflux. We induced beta2 integrin cross-linking by plating PMN onto surface-bound mAbs directed against either the common beta-chain (CD18) or the individual alpha-chains (CD11a, CD11b, CD11c) of LFA-1, CR3, and gp150/95. Anti-CD18 mAbs triggered a marked release of Cl- ions, which was accompanied by spreading and activation of the respiratory burst. Cross-linking of gp150/95 and LFA-1 generated the most powerful signals for the activation of Cl- efflux. The results of three independent experimental approaches, i.e., kinetic studies, use of Cl- transport inhibitors, and modulation of Cl- efflux with different amounts of anti-beta2 integrin mAbs, indicated that Cl- efflux regulates both spreading and respiratory burst triggered by beta2 integrin cross-linking. Cl- efflux appears to be independent on either alterations of [Ca2+]i or changes in the plasma membrane potential and shows sensitivity to a raise in pHi. This study uncovers a new signaling ability of beta2 integrins and contributes to highlight the role of Cl- efflux in the outside-in signal transduction pathway regulating adherence-dependent PMN responses.

Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (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-alpha (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 TNF-induced Cl- 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 beta 2 integrins cosignaled for an early, marked, and prolonged Cl- efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between Cl- efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced Cl- efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of Cl- efflux, such as ethacrynic acid. Moreover, Cl- 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 Cl-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.