Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2 - PubMed (original) (raw)

doi: 10.1371/journal.pone.0004501. Epub 2009 Feb 19.

Peiman Shooshtarizadeh, Benoît-Joseph Laventie, Didier André Colin, Jean-François Chich, Jasmina Vidic, Jean de Barry, Sylvette Chasserot-Golaz, François Delalande, Alain Van Dorsselaer, Francis Schneider, Karen Helle, Dominique Aunis, Gilles Prévost, Marie-Hélène Metz-Boutigue

Affiliations

Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2

Dan Zhang et al. PLoS One. 2009.

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.

Methodology/principal findings: 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/significance: 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Ca2+ influx evoked in human PMNs by CGA-derived peptides.

For flow cytometry analysis, human PMNs (5.105 cells/ml) were loaded with Fluo-3AM probe (5 µM) and its fluorescence intensity of Fluo-3 was monitored at λEm530 nm. Traces were obtained from 3000 PMNs and are averaged from triplicates at least. Peptides were added in the Ca2+-free EGTA buffer at t = 0 and 1.1 mM CaCl2 at t = 620 s. A) Time course variations of the intracellular Ca2+ in human PMNs in response to 20 µM CAT or 20 µM CHR and compared with other CgA-derived peptides (20 µM of CgA4–16, CgA17–40 or CgA65–76), tryptic digests of either 20 µM CAT, CHR or scrambled CAT. B, C) Time course variations of the intracellular Ca2+ in human PMNs in response to increasing concentrations (1–200 µM) of CHR (B), CAT (C) or a mixture of 50 µM CAT and 50 µM CHR (C).

Figure 2

Figure 2. Concentration-dependent Ca2+-influx after stimulation of human PMNs by CHR and CAT.

A) Representative diagrams of flow cytometry determination of the Ca2+ influx in the basic (M1) and peptide-activated (M2) windows, respectively. These influxes (in black) were obtained 15 s after the addition of 50 µM CHR or 50 µM CAT at t = 0 (in grey). PMNs activated by peptide-induced Ca2+ influx correspond to the M2 population. B) Flow cytometry quantification of PMNs in the M2 window in percent of the total initial population of PMNs, after 15 s exposure to increasing concentrations of CHR or CAT (1–200 µM).

Figure 3

Figure 3. Ca2+ influx in PMNs induced by CHR, and CAT and arachidonic acid (AA).

Time-lapse intracellular calcium imaging was performed on cells loaded with 1 µM Fura-2-AM and then further treated with 20 µM CHR, 20 µM CAT and 50 µM (AA) in Hepes-buffered Krebs medium in presence of 2.5 mM CaCl2. A–C) Left panels, Representative traces showing average change in [Ca2+]i (F340nm/F380nm) recorded in the ratio mode from individual PMNs loaded with Fura-2. The arrow indicates the time of addition of 20 µM CHR, 20 µM CAT and 50 µM AA. In separate assays (as indicated) either 100 µM 2-aminoethyl diphenylborinate (2-APB) or 1 µM gadolinium chloride (Gd3+) were applied 2 min before addition of peptides or AA. Traces are averaged from triplicates. Right panels, Summary of data in left panels, showing the maximal Ca2+ influx (mean ratio±S.E.) obtained with the different conditions as indicated below bars. (n), number of PMNs used for each experiment; *, indicate significant differences (p<0.05), significance for difference from effect of CHR, CAT or AA alone.

Figure 4

Figure 4. Specificity of the Ca2+ entry provoked by 50 µM CHR and 20 µM CAT.

Characterization by spectrofluorimetry of the specificity of the Ca2+ entry induced by 50 µM CHR or 20 µM CAT for comparison with peptide effects on Mn2+ entry. Simultaneous recordings of fluorescent intensity variations of PMNs loaded with Fura-2 in the presence of 1 mM CaCl2 (a, b) and 0.2 mM MnCl2 (c, d) at the corresponding wavelengths of excitation λEx340 nm and λEx360 nm. Divalent cations and peptides were added as indicated by arrows. Recordings are representative of three experiments.

Figure 5

Figure 5. Interaction of CHR and CAT with calmodulin (CaM).

A) Fluorescence confocal microscopy of PMNs after incubation during 120 s with 20 µM rhodamine-labeled peptides (Rho-CHR or Rho-CAT). Control peptide: 20 µM rhodamine labeled Hippocampal Cholinergic Neurostimulating Peptide (Rho-HCNP). The PMNs nuclei were labeled with Draq-5. B) Alignment of the CHR and CAT sequences with two CaM binding motifs 1-8-14 B and 1-5-10. The alignment has been obtained manually so that hydrophobic residues occupy invariant positions shown with boldface letters. The net positive charge is also indicated. C) (1) Interactions between CaM, CAT and CHR by Surface Plasmon Resonance (SPR) Peptides were immobilized on a CM5 Biacore chip using amine-coupling chemistry. CaM (5 µM) was added at arrow and allowed to stabilize for 10 min. Dissociation by washing was recorded for the following 10 min. All measurements were performed at 20°C. The resulting sensorgrams were analyzed using BIAevaluation Software. (2) Calmodulin-Affinity Chromatography of CHR and CAT. The retained and eluted peptides were immunodetected by dot blot with anti-CHR (monoclonal anti-CgA47–68) and anti-CAT (polyclonal anti-CgA344–364). Wash (5th wash after adsorption of peptides).

Figure 6

Figure 6. CHR and CAT stimulate iPLA2 activity.

A) Fluorescence confocal microscopy of PMNs: Left, PMNs treated only with the secondary antibody (Alexa Fluo 488-conjugated donkey anti-rabbit IgG dilution 1∶2000). Right, PMNs stained with polyclonal anti-iPLA2 as primary antibody before Alexa Fluo 488 conjugated donkey anti-rabbit IgG. B) Western-blot analysis: The presence of a 82 kDa protein corresponding to iPLA2 was immunodetected in the membrane fraction obtained from PMNs. A comparable signal was obtained from the supernatant fraction of membranes treated with 0.1% Triton X-100. C) iPLA2 activity assay: Stimulation of iPLA2 activity after treatment by 20 µM CHR or 20 µM CAT for 30 min at 37°C and comparison with the effects of two CaM-binding factors (1 µM CMZ, 50 µM W7) and one iPLA2 inhibitor (25 µM BEL). Activity of iPLA2 is expressed as the absorbance (Abs/mg of protein) at λ405 nm. Results from two similar experiments, each performed in triplicate are presented as mean±S.E. * p<0.05, significance for difference from controls. D) Alignment of CHR and CAT sequences with two CaM-binding peptides of iPLA2. CHR sequence has been aligned with the iPLA2 CaM-binding motif aa 618 to aa 635 while the CAT sequence has been aligned with the iPLA2 CaM-binding motif (691–709). Corresponding homologous residues are in bold and underlined fonts.

Figure 7

Figure 7. Involvement of iPLA2 in Ca2+ entry evoked by CHR and CAT in PMNs.

A–D) Left panels: Effects of the SOC blocker (100 µM 2-APB) and inhibitors of iPLA2 (25 µM BEL) and PLA2 (10 µM BPB) on Ca2+ entry evoked by 20 µM of CHR or CAT, and two CaM antagonists (30 µM W7 and 1 µM CMZ). The representative traces show the average change in intracellular Ca2+ concentration (Fura-2 ratio, F340/F380) recorded simultaneously in a number of individual PMNs at 37°C. The following inhibitors were added: BEL (30 min), BPB (10 min) and 100 µM 2-APB (2 min) before addition of peptides or CaM antagonists (at arrows). Results are obtained from at least three independent experiments. Right panels: Bars represent the maximum Ca2+ influx (Ratio±S.E.) in different treatments (indicated below each bar); (n) number of PMNs; * p<0.05, significance for difference from peptide or CaM antagonist alone.

Figure 8

Figure 8. HPLC of proteins in PMNs secretions induced by CHR and CAT and implication for innate immunity.

A, B) Secretion from PMNs (1.108 cells) was induced during 30 min stimulation by (A) 20 µM CHR or (B) 20 µM CAT. The secreted proteins >3 kDa were purified by RP-HPLC on a Macherey Nagel reverse-phase C18 column (4×250 mm; particle size 5 µM and pore size 100 nm). Numbered peaks in the chromatograms indicate protein fractions subjected to proteomic analyses. (C) Proteomic identification by NanoLC-MS/MS analysis of protein fractions involved in innate immunity (protein identification probability >93%). The numbered HPLC fractions correspond to the peaks of secreted protein in the chromatograms presented in A and B, respectively.

Figure 9

Figure 9. Model for the action of CHR and CAT on PMNs activation.

Stress and infection lead to two different pathways for stimulation of PMN secretion, by release of CgA and CgA-derived peptides from the adrenal medulla, as indicated by 1a–6a (black), and by PVL leucocidin stimulation by S. aureus infections, as indicated by 1b–3b (grey), respectively. Abbreviated symbols: P (CHR, CAT), P' (CAT), CPP (Cell Penetrating Peptide), CaM (calmodulin), iPLA2 (calcium independent phospholipase A2) LysoPL (lysophospholipids); GC (Golgi complex); PVL (Panton-Valentine leucocidin), R (receptor). The stress-stimulated pathway leads to penetration of P into the cytoplasm (2a), resulting in removal of inhibitory CaM, activation of iPLA2 to produce LysoPL (3a) and activation of Ca2+ influx through SOC (4a), converging on activated docking of secretory granules (5a) and subsequent release of proteins of relevance for innate immunity (6a). The negative feedback induced by P' on nicotinic cholinergic receptor of chromaffin cell is also indicated (7a). The infective route leads to activation of the putative PVL receptor coupled to opening of Ca2+-channels and a rise in intracellular Ca2+ (3b) that converges on docking of secretory granules and subsequent secretion of proteins of relevance for innate immunity (6a). Transcription of CgA in response to stress and infection is also indicated. Insert.The alignment of the two P sequences (CHR and CAT) with that of Penetratin (Cell penetrating peptide, CPP).

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