Abstract
BackgroundAntimicrobial 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.Methodology/Principal FindingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown 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 PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) 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.Conclusions/SignificanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.
Highlights
Chromogranin A (CgA) is a well-studied member of the chromogranin/secretogranin family, present in secretory cells of the nervous, endocrine and immune systems [1]
We have identified a range of antimicrobial peptides deriving from the natural processing, of chromogranin A (CgA) and Chromogranin B, Proenkephalin-A and Ubiquitin co-secreted with catecholamines upon stimulation of chromaffin cells from the adrenal medulla [17,18,22,23,24,25]
CHR and CAT provoked Ca2+ entry in polymorphonuclear neutrophils (PMNs) In PMNs loaded with Fluo-3, a transient Ca2+ influx was induced by 20 mM of either CHR or CAT (20 mM) in the presence, but not in the absence, of 1 mM free extracellular Ca2+ (Figure 1A)
Summary
Chromogranin A (CgA) is a well-studied member of the chromogranin/secretogranin family, present in secretory cells of the nervous, endocrine and immune systems [1]. CgA was the first chromogranin to be characterized as an acidic protein co-stored and co-released with the catecholamine hormones from the chromaffin cells of the adrenal medulla. The discovery that Pancreastatin, a CgA-derived peptide (bCGA248–293) was able to inhibit the glucose-evoked insulin secretion from pancreatic betacells [2] initiated the concept of a prohormone function for this protein [3]. 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
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