Complement C5a‐Induced Changes in Neutrophil Morphology During Inflammation

Abstract

The complement and neutrophil defense systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a, dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape-change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluid optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl−/HCO3−-exchanger or the Cl−-channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP–P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pre-treatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming

“…Here, we present a novel observation describing the series of MORE changes that occur in neutrophils following exposure to priming agents and demonstrate an overlap of the morphological and functional properties of neutrophils. It has been reported that polarization and reorganization of the actin cytoskeleton increase with neutrophil migration, facilitating invasion at inflammatory sites and the clearance of pathogens . We speculate that the MORE changes seen in neutrophils may allow for neutrophils to be freed from entrapment within microvasculature .…”

“…In addition to the described functional and biochemical changes, neutrophils undergoing activation exhibit morpho‐rheological (MORE) changes to their biophysical shape. It has been reported that activated neutrophils are smaller and stiffer than their unactivated and un‐primed counterparts, although there exist conflicting reports of neutrophils expanding poststimulation . Travel into and through the microcapillary system is essential for neutrophil migration to sites of infection or damage.…”

“…It has been reported that activated neutrophils are smaller and stiffer than their unactivated and un-primed counterparts, [10][11][12][13] although there exist conflicting reports of neutrophils expanding poststimulation. 14 Travel into and through the microcapillary system is essential for neutrophil migration to sites of infection or damage. Narrow capillary constrictions (5 m) force neutrophils to deform in order to pass through.…”

C5a anaphylatoxin and its role in critical illness‐induced organ dysfunction

“…[61][62][63] Recent reports by Denk and colleagues demonstrated that C5a-C5aR1 interactions lead to iondependent changes in intracellular pH and fluid content, driving neutrophil degranulation, shape change and chemotaxis. 64,65 C5aR1 ligation also prolongs neutrophil survival through potentiation of the Akt/Bad pathway and decreasing activity of the pro-apoptotic caspase 9. 42,66,67 In neutrophils, the effects of C5a on ROS production and apoptosis have been demonstrated to be dependent on phosphoinositide signalling.…”

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

“…1C, n=60, 000). Circularity feature is a parameter for evaluating the irregular cell contour in image flow cytometry system [30]. However, some LPS-treated DCs with low miR-590-5p-Cy3 fluorescence intensity had a high level of CD11c (Fig.…”