Role of Intracellular Chloride in the Reversible Activation of Neutrophil β2 Integrins: A Lesson from TNF Stimulation

Abstract

The process of β2 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 β2 integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of β2 integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin α subunits, and epitope CBRM1/5, localized to the I domain on the α-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 β2 integrin activation, 2) Cl−i decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl−-free buffers, activates β2 integrins, and 3) restoration of the original Cl−i levels is accompanied by deactivation of β2 integrins. We also show that Cl−i decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pHi does not seem to be relevant for β2 integrin activation. The results of our study emphasize the role of Cl− as a new PMN “second messenger.”

KCl Cotransport Is an Important Modulator of Human Cervical Cancer Growth and Invasion

“…The role of [Cl Ϫ ] i has been suggested as an important second messenger to regulate ␤ 2 integrin activation of polymorphonuclear leukocytes in response to tumor necrotic factor stimulation (22,23). Further work will be required to determine whether these changes in integrin and MMP expression reflect KCC-associated changes in [Cl Ϫ ] i or [K ϩ ] i or other functions of KCC possibly unrelated to ion transport.…”

“…The alteration of [Cl Ϫ ] i has been suggested as an important signal to regulate the activation of integrin in polymorphonuclear leukocytes (22,23). Although epithelial cells express a variety of cell adhesion molecules, integrins are the most important extracellular matrix receptors known to play a major role in the invasion and progression of most types of cancers (24).…”

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

“…Thrombin receptors activate chloride fluxes in megakaryocytes causing a rapid and immediate ion efflux following cell stimulation (56). Menegazzi and colleagues (33,57) have further identified a chloride efflux in polymorphonuclear lymphocytes that is directly and reversibly associated with ␤ 2 integrins and suggest a role for intracellular chloride ions as a second messenger in these cells. Interestingly, polymorphonuclear lymphocytes have been demonstrated to respond to activation with a fall in intracellular chloride concentrations and a corresponding integrin activation (33,57).…”

“…Menegazzi and colleagues (33,57) have further identified a chloride efflux in polymorphonuclear lymphocytes that is directly and reversibly associated with ␤ 2 integrins and suggest a role for intracellular chloride ions as a second messenger in these cells. Interestingly, polymorphonuclear lymphocytes have been demonstrated to respond to activation with a fall in intracellular chloride concentrations and a corresponding integrin activation (33,57). Although the authors did not identify which precise chloride channel was responsible for this alteration in chloride homeostasis, they demonstrated that its effect was specific for ␣ L and ␣ X integrins (containing KVGFFKR), not the ␣ M integrin (contains KLGFFKR).…”

“…However, indirect evidence for integrin modulation of specific chloride ion currents comes from studies that demonstrate that conformational change of ␤ 2 -integrins in polymorphonuclear leukocytes is inhibited by hypertonic saline (32) and is dependent on altered chloride fluxes in these cells (33). In addition, CLCA (chloride channel, calcium activatable) protein has been identified as ligands for ␤ 4 -integrins facilitating tumor metastasis.…”