Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.

Abstract

and cell morphology. Fluorescent images of fura 2-10aded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]~ was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]~ transients.These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2÷]i transients were abolished by chelation of extraceUular Ca 2÷. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]t activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of Using a rapid monitoring system We have described spontaneous and chemoattractant-triggered, multiple [Ca2+]~ elevations in neutrophils adherent both to albumin or fibronectin-coated surfaces (11). More recently Marks and Maxfield (19) have shown that such [Ca2÷]i transients appear to be linked to neutrophil movement during chemotaxis on poly-D-lysine-coated glass in the presence of serum. Neutrophils that extended pseudopods and assumed a polarized morphology were always observed to exhibit [Ca2÷]i transients even in the absence of chemoattractants. While [Ca2+]i transients are associated with cell spreading and locomotion, it is not clear whether transients in some way cause the movement, or if they are the result of cell movement.Spreading and locomotion are mediated by receptors that bridge the cell to the substrate. The numerically and functionally dominant adhesion receptor of neutrophils is the integrin CR3 (also known as Mac-l, Mol, CDllb/CD18, and am~2) (36). Blockade of this receptor with mAbs prevents spreading and/or chemotaxis on a variety of substrates including albumin (6), fibrinogen (38), complement protein C3bi (39), as well as on endothelial cells in vitro (9, 17) and in vivo (4,26,33). CR3 is likely to make a major contribution to cell locomotion since the chemoattractant stimuli C5a (17), tumor necrosis factor (TNF)a (17), interleukin (IL)-8 (7), and PMA (35) cause dramatic enhancement of the ca-

Relationship between Age-Dependent Changes Of Bovine Neutrophil Functions and Their Intracellular Ca2+ Concentrations.

“…[Ca 2+ ]i is a very important second messenger for neutrophil functions [14,17,19]. In the present study, the increase of [ 2+ ]i is an important factor but not sufficient to elicit an LDCL response, as was previously reported by us [12].…”

Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

“…Leukocyte integrins have been reported to signal an increase in intracellular calcium, inositol phospholipid hydrolysis (31,36,58,67,79), and tyrosine phosphorylation of a number of intracellular proteins, including Fgr (8), paxillin (23), PLC-␥ (31, 39), p130…”

“…Phosphorylation may depend on the ␣-subunit, as suggested by the observation that phosphorylation of paxillin in TNF-treated neutrophils is dependent on Mac-1 but not LFA-1 binding (26). Ligation of ␤ 2 -integrins on polymorphonuclear neutrophils (PMN) also induces an increase in the intracellular calcium concentration (31,36,58) and the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ], possibly by tyrosine phosphorylation of phospholipase C (PLC)-␥ and D-myoinositol-trisphosphate 3-kinase, respectively (31,48). Antibody cross-linking of ␤ 2 -integrins on adherent human neutrophils has been shown to trigger activation of p21 ras through tyrosine phosphorylation of the protooncogene product Vav (84).…”

Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation

“…Activation of PI3-K by integrins regulates phosphorylation of Raf-1 Ser 338 through the serine/threonine kinase Pak-1, providing a co-stimulatory signal that enhances Raf-1 activation by Ras and increases stimulation of cell growth by integrins (14). After integrin activation, increase of intracellular calcium concentration [Ca 2ϩ ] i has been observed upon cell attachment to ECM or binding of anti-integrin antibodies to platelets, macrophages, neutrophils, osteoclasts, and embryonic stem cells (15)(16)(17). Elevation of [Ca 2ϩ ] i upon activation of ␣7␤1 integrin in skeletal muscle cells results from both inositol triphosphate-evoked Ca 2ϩ release from sarcoplasmic/ endoplasmic reticulum and extracellular Ca 2ϩ influx through voltage-gated, L-type plasma membrane Ca 2ϩ channels (18).…”