Two chromogranin A-derived peptides, chromofungin and catestatin, induce neutrophil activation via a store-operated channel-dependent mechanism

Introduction

New endogenous antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.

Methods

PMNs were treated with different concentrations of CgA-derived peptides in the presence of several drugs. Calcium mobilization was observed using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy were performed to analyze the intracellular localization of the peptides. The calmodulin-binding and iPLA2-activating properties of the peptides were shown by surface plasmon resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMN treatment with CgA-derived peptides was performed using HPLC and nano-LC MS-MS.

Results

Using flow cytometry we first observed that after 15 seconds, in the presence of extracellular calcium, chromofungin (CHR) or catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in the absence of extracellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes of exposure. Treatment with 2-aminoethoxydiphenyl borate, a store-operated channel blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and cmZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. See Figure 1.

Figure 1

CHR and CAT induce store-operated channel (SOC) activation.

Conclusions

For the first time, we demonstrate that CHR and CAT penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway [1]. Furthermore, new experiments show that CAT penetrates quickly into immune cells such as dendritic cells and macrophages. To conclude, this study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.