Extension of chemotactic pseudopods by nonadherent human neutrophils does not require or cause calcium bursts

Abstract

Global bursts in free intracellular calcium (Ca) are among the most conspicuous signaling events in immune cells. To test the common view that Ca bursts mediate rearrangement of the actin cytoskeleton in response to the activation of G protein-coupled receptors, we combined single-cell manipulation with fluorescence imaging and monitored the Ca concentration in individual human neutrophils during complement-mediated chemotaxis. By decoupling purely chemotactic pseudopod formation from cell-substrate adhesion, we showed that physiological concentrations of anaphylatoxins, such as C5a, induced nonadherent human neutrophils to form chemotactic pseudopods but did not elicit Ca bursts. By contrast, pathological or supraphysiological concentrations of C5a often triggered Ca bursts, but pseudopod protrusion stalled or reversed in such cases, effectively halting chemotaxis, similar to sepsis-associated neutrophil paralysis. The maximum increase in cell surface area during pseudopod extension in pure chemotaxis was much smaller-by a factor of 8-than the known capacity of adherent human neutrophils to expand their surface. Because the measured rise in cortical tension was not sufficient to account for this difference, we attribute the limited deformability to a reduced ability of the cytoskeleton to generate protrusive force in the absence of cell adhesion. Thus, we hypothesize that Ca bursts in neutrophils control a mechanistic switch between two distinct modes of cytoskeletal organization and dynamics. A key element of this switch appears to be the expedient coordination of adhesion-dependent lock or release events of cytoskeletal membrane anchors.

TRPM2 ion channels steer neutrophils towards a source of hydrogen peroxide

“…Some studies have found no evidence for calcium gradients in neutrophils during chemotaxis 44 , 52 , though others have obtained evidence that leading-edge calcium signals play a critical role in chemotaxis of many different cell types 53 , 54 . In the present work, we observed leading-edge calcium signals in all motile neutrophils in vitro.…”

TRPM2 ion channels steer neutrophils towards a source of hydrogen peroxide

“…Some studies have found no evidence for calcium gradients in neutrophils during chemotaxis 38,46 , though others have obtained evidence that leading-edge calcium signals play a critical role in chemotaxis of many different cell types 47,48 . In the present work, we observed leading-edge calcium signals in all motile neutrophils in vitro.…”

“…The intracellular signalling molecules PI3K and Rac, which have been proposed as drivers of chemotaxis, do not appear to play a direct role in chemotaxis, but instead fine-tune motility to all chemoattractants by regulating a step common to all chemoattractants, for example refilling of the intracellular calcium stores, and thus modulate the amplitude of the leading-edge calcium pulses. Some studies have found no evidence for calcium gradients in neutrophils during chemotaxis 38,46 , though others have obtained evidence that leading-edge calcium signals play a critical role in chemotaxis of many different cell types 47,48 . In the present work, we observed leading-edge calcium signals in all motile neutrophils in vitro.…”

Neutrophil Cell Shape Change: Mechanism and Signalling during Cell Spreading and Phagocytosis

“…Lymphocyte spreading on β 2 -integrin adhesion has also been shown to depend on µ-calpain activity [33,34]. By close observation of neutrophil pseudopod extension around a particle for phagocytosis, the timing of the Ca 2+ signal is seen to proceed immediately before the acceleration phase of engulfment, [28,35,36] and the link between Ca 2+ and acceleration is broken by inhibition of µ-calpain activity [28]. In µ-calpain null mice, neutrophils also fail to spread effectively and are aberrant in transendothelial migration [37].…”