Synergy between adenosine and C5a induced signaling in mast cells

Abstract

Mast cells are important effector cells in asthma and atopic disease. Upon IgE receptor cross‐linking, they release potent inflammatory mediators, cytokines, and chemokines. Mast cells can also be activated by IgE‐independent signals, including the anaphylatoxin C5a, adenosine, and other agents. The human mast cell line, HMC‐1, expresses the Gαi‐coupled C5a receptor, along with Gαi‐coupled adenosine receptors A1R (low levels) and A3R, the Gαs coupled A2aR, and the Gαs/Gαq coupled A2bR. We found that HMC‐1 cells exhibited a potent synergistic induction of chemokine release, coupled to NFAT activation, when exposed to C5a in combination with the adenosine analog, NECA. To elucidate the receptor(s) mediating this response, two strategies were employed. First, a panel of specific adenosine receptor agonists was tested in combination with C5a for synergy of chemokine release. Agents selective for A1R or A2aR did not stimulate chemokine production, while an A3R selective agonist induced low levels of chemokine production only in combination with C5a. To investigate the role of A2bR, for which no selective agonist was available, an siRNA approach was employed. Knockdown of A2bR, but not A2aR, significantly reduced NECA‐induced second messenger accumulation (cAMP and IP3) and chemokine production, and inhibited the synergy with C5a. To identify downstream signaling molecules important for this response, a broad‐based phospho‐protein screen was performed, which revealed signaling to SRC phosphorylation is enhanced upon C5a and NECA costimulation. These data suggest a cooperative signaling node in adenosine and C5a costimulation of mast cells resulting from the activation of their respective GPCRs coupled to Gαq and Gαi.