Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.
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.
Zhang
Dan
D
INSERM U575, Physiopathologie du Système Nerveux, Strasbourg, France.
Shooshtarizadeh
Peiman
P
Laventie
Benoît-Joseph
BJ
Colin
Didier André
DA
Chich
Jean-François
JF
Vidic
Jasmina
J
de Barry
Jean
J
Chasserot-Golaz
Sylvette
S
Delalande
François
F
Van Dorsselaer
Alain
A
Schneider
Francis
F
Helle
Karen
K
Aunis
Dominique
D
Prévost
Gilles
G
Metz-Boutigue
Marie-Hélène
MH
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
D002147:Calmodulin; D053379:Chromogranin A; D010446:Peptide Fragments; D011506:Proteins; C437333:chromofungin; C109084:chromogranin A (344-364); D054512:Phospholipases A2, Calcium-Independent