Complement fragment C3a stimulates Ca²⁺ influx in neutrophils via a pertussis‐toxin‐sensitive G protein

Abstract

The signal pathways of neutrophils following stimulation with the complement fragment C3a (C3a) were studied in neutrophils and compared to the pathways activated by complement fragment C5a (C5a). Analysis of polyphosphoinositol lipid turnover showed that C5a, but not C3a, activated phosphatidylinositol‐bisphosphate‐3‐kinase (PtdInsP2 3‐kinase) indicating that different signal pathways are activated by the two anaphylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosolic free Ca2+ levels were analyzed in Fluo‐3‐labelled neutrophils by flow cytometry. C3a stimulated a fast and concentration‐dependent increase of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a‐triggered Ca2+ rise. Addition of EGTA to the extracellular buffer prior to stimulation did not significantly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In marked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo‐1/AM or Fluo‐3. Preincubation of neutrophils with pertussis toxin inhibited both C3a‐ and C5a‐stimulated Ca2+ transients, indicating the involvement of guanine‐nucleotide‐binding proteins (G proteins) in these processes. In order to examine whether the C3a receptor is coupled to G proteins, binding of guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTP[S]) to purified neutrophil plasma membranes was studied. Both C3a and C5a stimulated high‐affinity binding of [35S]GTP[S] up to 1.5‐fold and 3‐fold, respectively. These data suggest that the two anaphylatoxins activate pertussis‐toxin‐sensitive G proteins, which then trigger different signal transduction pathways. C3a specifically stimulated Ca2+ influx from the extracellular medium, whereas C5a additionally activated the PtdInsP2 3‐kinase and stimulated Ca2+ mobilization from intracellular stores.

C3a and C5a Are Chemotactic Factors for Human Mesenchymal Stem Cells, Which Cause Prolonged ERK1/2 Phosphorylation

“…Although carboxypeptidase N also converts C5a to C5a(desArg), this modified polypeptide still retains a reduced functional ability and affinity for the C5aR (9), whereas C3a(desArg) loses the complete ability to bind and activate the Ca3R (31). However, even in serum-free conditions, activation of the C3aR causes a transient response, and certain pathways that necessitate a more sustained response such as phosphatidylinositide production are not invoked by C3a in neutrophils (32). In comparison, activation of the C5aR on leukocytes shows a more robust respiratory burst, calcium mobilization, and chemotaxis (9,29,32).…”

“…However, even in serum-free conditions, activation of the C3aR causes a transient response, and certain pathways that necessitate a more sustained response such as phosphatidylinositide production are not invoked by C3a in neutrophils (32). In comparison, activation of the C5aR on leukocytes shows a more robust respiratory burst, calcium mobilization, and chemotaxis (9,29,32).…”

“…C3aR internalization in MSC, monocytes, and HEK293 cells was determined by confocal microscopy. Monocytes were used for comparison because C3aR activation is best documented for myeloid cells (11,14), and HEK293 cells were used because they are among the most commonly used cells to determine the function of transfected receptors. The different cell types were stimulated with 300 nM C3a or 100 nM C5a for the indicated times and stained as described for each panel.…”

The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization

“…Previous studies have shown that C3a can readily degranulate eosinophils, basophils, and mast cells (11,12) while also activating and modulating cytokine release from monocytes/macrophages (13). However, despite high expression of C3aR on neutrophils and a clear Ca 2+ mobilization response in response to C3a (or other C3aR ligands) (9,14), a clear role for C3a has not yet been ascribed to neutrophils (15), which form the vast majority of the granulocytic population.…”

Characterization of C3a and C5a Receptors in Rat Cerebellar Granule Neurons during Maturation

“…C3a and C5a exert their biological effects through specific binding to membrane receptors, named, respectively, C3aR 1 and C5aR (CD88). These two receptors belong to the seven-transmembrane receptor superfamily and are coupled to a G i protein (2,3). C3aR and C5aR are expressed in myeloid cells (4 -8) and, more surprisingly, in non-myeloid cells and tissues (7)(8)(9)(10).…”