title: Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  PMNs 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
  For 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.
authors.affiliation: INSERM U575, Physiopathologie du Système Nerveux, Strasbourg, France.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Peiman
authors.initials: P
authors.lastname: Shooshtarizadeh
authors.forename: Benoît-Joseph
authors.initials: BJ
authors.lastname: Laventie
authors.forename: Didier André
authors.initials: DA
authors.lastname: Colin
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Chich
authors.forename: Jasmina
authors.initials: J
authors.lastname: Vidic
authors.forename: Jean
authors.initials: J
authors.lastname: de Barry
authors.forename: Sylvette
authors.initials: S
authors.lastname: Chasserot-Golaz
authors.forename: François
authors.initials: F
authors.lastname: Delalande
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Francis
authors.initials: F
authors.lastname: Schneider
authors.forename: Karen
authors.initials: K
authors.lastname: Helle
authors.forename: Dominique
authors.initials: D
authors.lastname: Aunis
authors.forename: Gilles
authors.initials: G
authors.lastname: Prévost
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Metz-Boutigue
mesh_terms: D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D053379:Chromogranin A / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D054512:Phospholipases A2, Calcium-Independent / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics
chemical_list: D002147:Calmodulin; D053379:Chromogranin A; D010446:Peptide Fragments; D011506:Proteins; C437333:chromofungin; C109084:chromogranin A (344-364); D054512:Phospholipases A2, Calcium-Independent