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. 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.


Introduction
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 (bCGA 248-293 ) was able to inhibit the glucose-evoked insulin secretion from pancreatic betacells [2] initiated the concept of a prohormone function for this protein [3]. Numerous endogenous cleavage products of CgA have since been identified in the intragranular matrix of chromaffin cells, resulting from the proteolytic digestion at 13 sites [4] by intragranular enzymes, such as prohormone convertases PC1/3, PC2, neuroendocrine-specific carboxypeptidase E/H, Lys and Arg-aminopeptidases [5]. Among the CGA derived fragments, several induce biological activities [1] an their in vitro actions strongly suggest involvement in homeostatic processes, such as calcium and glucose metabolisms [6], cardiovascular functions [7][8][9][10][11], inflammatory reactions [12,13], pain relief, tissue repair [14], gastrointestinal motility [15,16] and in the first line of defence against invading microorganisms [17][18][19]. The possible implication of CgA and some of its derived-peptides in human diseases has also been reviewed [20,21].
We have identified a range of antimicrobial peptides deriving from the natural processing, not only of CgA but also 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]. These new antimicrobial peptides are integrated in the concept that the adrenal medulla plays an important role in innate immunity [26]. Furthermore, when polymorphonuclear neutrophils (PMNs), known to accumulate at sites of inflammation are stimulated by lipopolysaccharide or other bacterial agents, such as Panton-Valentine leucocidin (PVL) [27,28], these cells produce and secrete intact and processed forms of CgA, such as Vasostatin-I and -II (residues 1-76 and 1-113) [18] and Cateslytin (residues 344-358) [17,[29][30][31], the Nterminal fragment of Catestatin (residues 344-364) [32]. In view of the established function of PMNs as central effectors cells in innate immune responses to inflammatory stimuli, it is of a great importance to understand the implications of the production and secretion of CgA-derived peptides for the regulation of PMNs responses to external stimuli.
In the present study, we have investigated the effects of two of the potent antimicrobial CgA-derived peptides on activation of PMNs release, i.e.: Chromofungin (CHR, bovine/human CgA 47-66 ) [24] and Catestatin (CAT, bovine CgA 344-364 ) [17]. By use of biochemical techniques, confocal microscopy, flow cytometry, calcium imaging, surface plasmon resonance and proteomic analyses, these two CgA peptides have now been demonstrated to stimulate exocytosis from PMNs by provoking a transient Ca 2+ entry independent of release from internal stores. The mechanism by which CHR and CAT may induce Ca 2+ influx appears to involve calmodulin (CaM) binding. The subsequent activation of the calcium-independent phospholipase A2 (iPLA2) in the complex regulation of store-operated channels (SOCs) [33][34][35][36][37], seemingly potentiates the release of a range of PMNs factors of importance identified by use of nanoLC-MS/MS.

Results
Since PMNs are central effectors in innate immune response to inflammatory stimuli, we investigated the effects induced by two highly conserved antimicrobial CgA derived peptides on these cells. Chromofungin (CHR, RILSILRHQNLLKELQDLAL) corresponds to the active antifungal domain CgA 47-66 of bovine and human Vasostatin-1 CgA 1-76 [24] while Catestatin (CAT, RSMRLSFRARGYGFRGPGLQL) corresponds to a potent antimicrobial peptide CgA 344-364 conserved in both cattle and man. These two peptides are not lytic for mammalian cells and display antimicrobial activities at the micromolar range [17,24].
The inducing effects of CHR and CAT on the Ca 2+ influx were concentration-dependent in the 5-200 mM range ( Figure 1B, C). Interestingly, the simultaneous addition of CHR and CAT at equal concentrations provoked a higher Ca 2+ increase ( Figure 1C) than the sum of effects observed by separately added peptides ( Figure 1B and C), suggesting that CHR and CAT may have synergic effects on the transient Ca 2+ entry in PMNs.
An immediate increase of activated PMNs (M1/M2) was observed 15 s after the application of either CHR or CAT ( Figure 2A). With different concentrations of CHR or CAT (0-200 mM), the percentage of activated PMNs was concentration dependent, being maximal at 50 mM for CHR (45%) and CAT (60%), respectively ( Figure 2B). Ca 2+ entry evoked by CHR and CAT via Ca 2+ selective Store Operated Calcium channels (SOCs) Pretreatment of PMNs with the specific blocker of SOCs, 2aminoethyl diphenylborinate (2-APB) for 2 min before addition of peptides, completely blocked the entry of extracellular Ca 2+ by CHR or CAT ( Figure 3A and 3B), suggesting that the two peptides were involved in the activation of Store Operated Calcium Entry (SOCE) through SOCs. Ca 2+ entry in response to CHR and CAT was also compared to that induced by Arachidonic Acid (AA), acting through the Arachidonic-Regulated Channels (ARCs) (Fig 3C). The calcium influx induced by 50 mM AA was insensitive to 2-APB, while inhibited by gadolinium chloride (Gd 3+ ), in marked contrast to the influx induced by CHR or CAT ( Figure 3A-C). Thus, the Ca 2+ -entry induced by the two CgAderived peptides was pharmacologically distinct from that by AA.
Finally, the specificity for divalent cation entry into PMNs loaded with Fura-2 was assessed by comparison of the peptide induced Ca 2+ entry (traces a and b) with that of Mn 2+ (traces c and d) at the corresponding wavelengths of excitation (l ex340nm and l ex360nm ) ( Figure 4). By comparison of the traces for CAT (a, c) and CHR (b,d) there was no evidence for Mn 2+ entry in response to either peptide, consistent with activation of SOCs selective for Ca 2+ and non-permeable for Mn 2+ in PMNs [38].

Calmodulin binding of CHR and CAT
The uptake of rhodamine-labeled CHR and CAT into isolated PMNs was demonstrated by confocal microscopy ( Figure 5A). After 2 min incubation, the fluorescent rhodamine-labeled peptides were detected in the cytoplasm. In contrast, the control peptide, rhodamine-labeled Hippocampal Cholinergic Neurostimulating Peptide (HCNP), unable to induce Ca 2+ influx into PMNs (not shown), was not detected in the cytoplasm, indicating a specificity for the entry of rhodamine-labeled CHR and CAT ( Figure 5A).
The two cationic sequences CAT and CHR may be aligned with two Ca 2+ -dependent CaM binding motifs 1-8-14 B and 1-5-10 [39], as illustrated in Figure 5 B and previously demonstrated for binding of CHR to CaM [24,40]. Interactions between CaM, CAT and CHR were assessed by surface plasmon resonance (SPR) ( Figure 5 C1), revealing that not only CHR but also CAT specifically interacted with CaM, while scrambled CAT was inactive. In addition, CaM interacts with both CHR and CAT ( Figure 5C2) when assayed by CaM-binding affinity chromatography and dot blot immunodetection of CgA in the fixed material with anti-CHR (monoclonal anti-CGA 47-66 ) [14] and anti-CAT (polyclonal anti-CGA 344-364 ) [17].

Role of iPLA 2 in the Ca 2+ entry evoked by CHR and CAT
Previous studies have demonstrated that functional iPLA 2 is involved in activation of SOCs from different cell types [37] and that displacement of inhibitory CaM from iPLA 2 may activate iPLA 2 , subsequently generating of lysophospholipids for activation of SOCs [36]. Confocal microscopy confirmed the presence of immunoreactive iPLA 2 in PMNs ( Figure 6A, right panel). The membrane localization of immunoreactive iPLA 2 could be verified after subcellular fractionation, Triton X-100 treatment and western blotting ( Figure 6B, [36]). The enzymatic activity of iPLA 2 in PMNs was stimulated not only by two CaM inhibitors, i.e. calmidazolium (CMZ) and N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide (W7), but also by CHR and CAT ( Figure 6C). Interestingly, a comparison of CHR and CAT sequences with two CaM-binding peptides of iPLA 2 reveals marked homologies ( Figure 6D). Of note, the CHR sequence may be aligned with one of the iPLA 2 CaM-binding motif (iPLA2 618-635 ), while the CAT sequence aligns with another, i.e. the iPLA 2 CaM-binding motif (iPLA2 691-709 ) [41].

CHR and CAT directly activate SOCs via iPLA 2
Two inhibitors of iPLA 2 were compared with 2-APB for effects on peptide inducing Ca 2+ influx ( Figure 6C, 7A, B). Bromoenol lactone (BEL), a specific inhibitor of iPLA 2 and bromophenacyl bromide (BPB), an inhibitor of PLA 2 , abolished the transient Ca 2+ influx induced by CHR or CAT similarly to 2-APB. In addition, the two CaM antagonists W7 and CMZ induced longer lasting rises in influx ( Figure 7C, D), that were significantly suppressed by the PLA 2 inhibitors BEL and BPB and the specific SOC blocker 2-APB.
Interestingly, CMZ and some CaM inhibitory peptides may directly activate SOCs without the release of intracellular Ca 2+ stores [42][43][44]. Hence, these findings indicate that CAT and CHR, being unable to evoke a Ca 2+ mobilization from intracellular stores in absence of extracellular Ca 2+ , by their 2-APB sensitive induction of Ca 2+ entry via SOCs may converge on iPLA 2 in a manner similar to the two CaM inhibitors W7 and CMZ.

Proteomic analysis of the PMNs secretions released by CHR and CAT
PMNs secretions obtained after treatment with CHR and CAT were chromatographied by HPLC ( Figure 8A, B) and the isolated fractions were identified by NanoLC-MS/MS analysis (supplementary data). Factors involved in innate immunity were identified among these proteins, i.e. lactotransferrin, lysozyme, neutrophil gelatinase associated lipocalin, S100 calcium binding protein A8, S100 calcium binding protein A9, heat shock 70 kDa protein and leukotriene A4 hydrolase ( Figure 8C and complete data in the annexe with Figures S1 and S2).
CgA-derived fragments in the PMN secretions obtained after treatment with CHR and CAT were detected by western blotting, revealing traces of Vasostatin-1 and a CAT-including fragment (CgA 340-395 ) (data not shown), consistent with the previously report of several CAT-including peptides with molecular weights in the range 70-15 kDa [17]. The major fragment corresponds to CgA 340-395 (15-17 kDa) after stimulation of PMNs by CAT.

Discussion
The present experiments are the first to demonstrate that not only CAT but also the distinctly different sequence CHR may play  a role in Ca 2+ signaling outside the chromaffin cells, i.e. serving as immunomodulators for activation of PMNs secretion.
A rapid and effective response to challenge with pathogens is essential for the survival of all living organisms. Natural cationic host defense peptides represent lead molecules that boost innate immune responses and selectively modulate pathogen-induced inflammatory responses [45]. Among the different mechanisms that have evolved to this effect, the production of a large variety of natural antimicrobial peptides has gained increasing attention.
CHR and CAT are two antimicrobial peptides derived from CgA. They are stored in secretory granules of chromaffin cells and PMNs in a larger form, which may be processed extracellularly to the mature active peptides [17,18]. In the intragranular matrix, they result to the processing of CgA by numerous enzymes such as prohormone convertases (PC1 and 2), aminopeptidases and carboxypeptidases. In the extracellular medium, larger forms may be processed by kallikrein located at the plasmic membrane level and by circulatory proteolytic enzymes such as plasmin and thrombin [1]. In addition, some bacterial virulence factors are indeed proteolytic enzymes, i.e. Glu-C protease from Staphylococcus aureus, and might continue the natural processing of CgA to generate the CHR and CAT fragments during infections [17,18,46].
To date only CAT has been reported to act via a classical surface receptor, i.e. the nicotinic acetylcholine receptor [32]. On the other hand, several 70-80 kDa membrane binding proteins have been reported for Vasostatin-I (CgA 1-76 ) [47,48]. Furthermore, the sequences of CHR and CAT may also be aligned with the cell penetrating peptide (CPP), Penetratin, i.e. (Figure 9 insert), pointing the possibility for CHR and CAT, as new members of the family of CPPs [49]. Calcium is an universal secondary messenger involved in many cellular signal transduction pathways, regulating crucial functions such as secretion, cell motility, proliferation and cell death. Increased in intracellular Ca 2+ derives mainly from two sources: internal stores releasing Ca 2+ into the cytosol and channels in the plasma membrane that open to allow external Ca 2+ to flow into the cell. In PMNs, calcium signaling has been reported to be involved in oxidase activation, cell degranulation and priming response to a wide variety of proinflammatory molecules [35]. The present experiments have demonstrated that the two CgA peptides, CHR and CAT, penetrate rapidly into PMNs ( Figure 5A) and subsequently interact with CaM ( Figure 5C) to activate iPLA 2 ( Figure 6C). Displacement of CaM leads to iPLA 2 activation and production of lysophospholipids inducing the opening of SOCs [36], the entry of Ca 2+ and the release of innate immune factors among a long list of proteins ( Figure 8C and Figures S1 and S2).
The SOCE pathway remains one of the most intriguing and long lasting mysteries of Ca 2+ signaling [43]. The main question concerns the origin of the signal [43,50]: how does depletion of the internal Ca 2+ store activate Ca 2+ entry through the SOC in the plasma membrane?. The Calcium Influx Factor (CIF) is a specific factor produced by the cells following depletion of Ca 2+ stores [51] and corresponds to the diffusible messenger that is produced by Ca 2+ stores upon their depletion traveling to the plasma membrane to activate SOCs and Ca 2+ entry [43]. The mechanism by CIF may activate SOCs with a displacement of CaM from the inactive complex with iPLA 2 . The subsequent formation of lysophospholipids and the opening of SOCs, consistent with an activation of SOCs by displacement of inhibitory CAM from the inactive complex with iPLA 2 [36,37,43]. Thus, CaM inhibitors such as chemical substances and peptides, may activate SOCs by interfering with the CaM-iPLA 2 complex [36,43]. The present experiments have made evident that CHR and CAT induce a 2-APB sensitive Ca 2+ entry without depletion of Ca 2+ stores in a similar manner to CaM inhibitors, pointing to iPLA 2 , as the most likely target for these two peptides, once penetrated into the cytoplasm.
Of note, CAT is an inhibitory peptide controlling catecholamine release from chromaffin cells and noradrenergic neurons by an autocrine regulatory mechanism, non-competitively blocking the influx of Ca 2+ coupled to activation of the nicotinic acetylcholine receptor, as illustrated in Figure 9, but not when triggered by membrane depolarization [32].
Several proteins involved in innate immunity (lactotransferrin, heat shock 70 kDa protein, lysosyme, neutrophil gelatinase associated lipocalin, protein S100-A8, protein S100-A9 and leukotriene A-4 hydrolase were identified by using proteomic analysis in the secretions of neutrophils stimulated by CHR and CAT ( Figure 8C). In neutrophils, azurophil, specific and gelatinase granules contain host defense proteins in their luminal spaces. The extent of mobilization of the three types of granules depends on the stimulus intensity: gelatinase granules are most easily mobilized followed by specific granules and then azurophil granules.
Lactotransferrin is an iron-binding protein that is released from activated neutrophils at sites of inflammation by the three types of granules [52] and has antimicrobial, as well as anti-inflammatory properties [53]. Lactotransferrin serves as an autoregulator to retain PMNs at inflammatory sites [54] and it was able to delay spontaneous apoptosis of neutrophils [55]. The anti-inflammatory activity of lactotransferrin is due to binding and neutralization of proinflammatory molecules and its capacity to promote activation of neutrophils and macrophages [56]. Lysozyme is also released by three types of granules [52]. In addition to enzymatic lysis of bacterial cell wall, it can also kill bacteria by a non-enzymatic mechanisms [57]. Neutrophil gelatinase associated lipocalin (NGAL) was found in specific granules of neutrophils [58,59]. It is involved in the allosteric activation of matrix-metalloproteinase (MMP)-9 and protection from degradation. It may function as an effector molecule of innate immune system [58,60].
Proteins S100-A8 and A9, two Ca 2+ -binding proteins of the S100 family are secreted from neutrophils by the three types of granules as a heterodimeric complex (S100-A8/S100-A9) [61][62][63]. High levels of the proteins have been found in the extra-cellular milieu during inflammatory conditions [64,65]. The S100-A8/A9 heterodimer is an antimicrobial complex that exhibits cytokine-like functions in the inflammatory sites via activation of NFkappa B and p38MAPK [66]. Hsp70, an ubiquitous family of protein chaperones that assist in folding of newly synthesized peptides and translocation of proteins across biological membranes, plays regulatory roles in signal transduction, cell cycle and apoptosis [67]. Hsp70 is known to contribute to the mechanisms of cell protection against a variety of stress and cytotoxic factors, providing an increase of cell survival [68]. Leukotriene A-4 hydrolase is released by the specific granules of neutrophils. It catalyzes the final and commited step in the biosynthesis of leukotriene B4, a potent chemotactic agent for neutrophils, eosinophils, monocyted and T cells that play key roles in the innate immune response [69].
A considerable number of studies have demonstrated that the neuroendocrine and immune systems communicate to promote reciprocal regulation in the host. Not only may immune cells sense pathogens and secrete proteins that modify cells of the neuroendocrine system but factors secreted by the neuroendocrine system may also bring about changes in immune cell activity [26].
In addition to CAT, substance P is another neuro-immunomodulator that may inhibit nicotinic cholinergic-stimulated catecholamine release, but displays a lower potency [70]. PACAP displays numerous functions in cells of neuronal and non neuronal origin and functional VIP/PACAP receptors have been demonstrated on PMNs, mediating Ca 2+ -dependent pro-inflammatory activities through the activation of multiple regulatory pathways [71]. Thus, it was demonstrated that the extracellular and intracellular Ca 2+ play key roles in PACAP mediated proinflammatory activities [72]. In addition, it has been reported that, in contrast to the inhibitory effect of CAT, PACAP may induce an increase of Ca 2+ entry and catecholamine release in bovine chromaffin cells by a a unique pathway distinct from nAChR, VOCs and SOCs [73]. Hence, in contrast to the action of PACAP, CAT may control the proinflammatory activity via a . 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 mM) 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 20uC. The resulting sensorgrams were analyzed using BIAevaluation Software. To conclude, the data reported here are important for our understanding of the role for CgA in innate immunity and for its crucial role as a mediator in the cross-talk between the neuroendocrine and immune systems. All together, the immunomodulatory activities of CHR and CAT may be important for the design of pharmacological strategies to maintain homeostasis during recruitment of PMNs in microbial and systemic infections.

Peptide synthesis
All the synthetic peptides were prepared on an Applied Biosystems 433A peptide synthesizer (Foster City, USA), using the stepwise solid-phase approach with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Then, the synthetic peptides were purified by RP-HPLC on a Macherey Nagel Nucleosil RP 300-7C18 column (106250 mm; particle size 7 mm and pore size 100 nm). Rhodamine fluorophore 5(6)-carboxytetramethyl rhodamine was conjugated with peptides at the N-terminal extremity, as previously described [24]. Synthetic peptides were analysed by mass spectrometry and automated Edman sequencing on an Applied Sequencing System (Applied Biosystems, Foster City, USA) [5]. MALDI mass measurements were carried out on an Ultraflex TM TOF/TOF (Bruker Daltonics, USA) to perform a rapid control of synthetic peptides according to the procedure previously reported [74].

Cells and flow cytometry measurements
Human PMNs were prepared from buffy coats purchased at the Etablissement Français du Sang -Strasbourg, France from anonymous and healthy donors from either sex. Briefly, 40 mL of a 1/3 (vol/vol) dilution of blood cells in 0.9% (wt/vol) NaCl was layered on 12 mL of 15 J Prep (Techgen International, Voisins le Bretonneux, France). After centrifugation at 8006 g for 20 min, the pellet was suspended in 30 mL of 0.9% NaCl, added to 10 mL of 6% (wt/vol) dextran, and sedimented for 30 min. Thirty mL of the supernatant was centrifuged for 10 min at 8006 g. The pellet was suspended in Hepes buffer (140 mM NaCl, 5 mM KCl, 10 mM glucose, 0.1 mM EGTA, 10 mM Hepes, 3 mM Tris; pH 7.3) and the contaminating erythrocytes were removed by hypotonic lysis for 45 s and subsequent washing in Hepes buffer [75]. The final suspension was adjusted to a concentration of 6.10 6 PMNs/mL and 0.1% (Wt/vol) bovine serum albumin was added to prevent non-specific leucotoxin adherence on tube walls.
To flow cytometry measurements PMNs were suspended at a concentration of 5.10 5 cells/mL in Hepes and measurements from 3000 PMNs were carried out using a FacSortH flow cytometer (Becton-Dickinson, Le Pont de Claix, France) equipped with a 15 mW argon laser tuned to 488 nm [76].
We evaluated the intracellular calcium, using flow cytometry of cells previously loaded with 5 mM Fluo-3 (Molecular Probes, New Brunswick, USA). The increase of Fluo-3 fluorescence was measured with different CgA-derived peptides in absence or after addition of 1.1 mM extracellular CaCl 2 [77]. Fluo-3 fluorescence was measured from the fluorescence light 1 (FL1: l Em = 530 nm) using Cell Quest Pro TM software (Becton-Dickinson, Le Pont de

Time-lapse intracellular Ca 2+ imaging
PMNs were incubated for 30 min at 37uC in Hepes-buffered Krebs medium (130 mM NaCl, 4.5 mM KCl, 1.2 mM MgSO 4 , 2.5 mM CaCl 2 , 11 mM D-glucose, 10 mM Hepes, pH 7.4) with 2 mM Fura-2-AM. Cells were then washed with Hepes-buffered Krebs medium and subsequently incubated for 20 min to allow Fura-2-AM hydrolysis. After three additional washes the cells were placed on an inverted microscope (Axiovert 35M Zeiss, Germany), superfused with Krebs solution (1 mL/min), and alternatively illuminated at 350610 nm and 380610 nm. The fluorescent emission was observed using a long pass filter at 510 nm. For each excitation wavelength and every 2 s an image pair was recorded using a CCD camera (ImageEM TM , Hamamatsu Photonics, Hamamatsu, Japan) and fluorescence ratio images were calculated subsequently using Metafluor TM software (Molecular Devices, Downingtown, USA).

Spectrofluorometry determination
PMNs were loaded with Fura-2 as above and washed in EGTA buffer with 1.1 mM CaCl 2 added. Continuous variations of fluorescence intensities were recorded using a dual excitation and dual emission spectrofluorometer Deltascan (Bioritek, PTI, Chamarande, France) with slits set at 4 nm. One mL of PMNs (4.10 6 cells/mL) was added to 1 mL of the assay solution under constant stirring in a 4 ml cuve (1 cm light path) thermostated at 37uC. Wavelengths were settled at 340 nm (Ca 2+ ) and 360 nm (Mn 2+ ) for excitation, and 510 nm for emission. Data were expressed in arbitrary units.

Surface Plasmon Resonance experiments
Experiments were conducted on a Biacore 3000 TM (GE Healthcare) and peptides were immobilized on a CM5 Biacore chip using amine-coupling [78]. The surface of the chip was activated for 7 min at a flow rate of 5 mL/min with a mixture of 50 mM NHS and 0.2 mM EDC. Peptides were covalently linked to the surface giving up to 2000 resonance units (RU). Ethanolamine (1M, pH8.5) was injected for 7 min to block the remaining activated groups. 5 mM CaM (Sigma, St. Louis, MO, USA) addition was performed at an injection flow of 20 mL/min in 50 mM NaCl, 2 mM CaCl 2 , 20 mM Tris pH6.5 until stabilization was reached (10 min). Dissociation was registered for 10 min after the end of injections. All measurements were performed at 20uC. Sensorgrams were analyzed by using BIAevaluation Software.

Calmodulin-Affinity Chromatography and dotblot immunodetection
CHR and CAT were prepared in buffer A (50 mM NaCl, 2 mM CaCl2, 50 mM Tris-HCl, pH 7.5) and loaded on 0.1 ml CaM-Sepharose 4B column (GE healthcare, Buckinghamshire, United Kingdom) with molar ratio 10:1. The mixtures were incubated at room temperature for 1 h. Afterwards, the column was washed (x5) with 1 mL of buffer A, and (x3) 0.1mL buffer B (50 mM NaCl and 2 mM EGTA, 50 mM Tris-HCl, pH 7.5) were used to elute the CaM binding peptides. The elutions were analyzed by dotblot onto PVDF membrane with pore 0.2 mM (Millepore, Billerica, MA, USA) with specific antibodies anti-CHR (monoclonal anti-CGA 47-68 (dilution 1:5,000) and anti-CAT (anti-CGA 344-364 (dilution 1:20,000) [17,24]. Immunoreactivity signals 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), iPLA 2 (calcium independent phospholipase A 2 ) 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 iPLA 2 to produce LysoPL (3a) and activation of Ca 2+ 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 Ca 2+ -channels and a rise in intracellular Ca 2+ (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). doi:10.1371/journal.pone.0004501.g009 were developed using goat anti-mouse IgG conjugated to peroxidase (dilution 1:400,000, Jackson Immunoresearch laboratories, Baltimore Pike, USA).
Detection of iPLA 2 in PMNs and iPLA 2 activity assay Subcellular fractionation of PMNs. PMNs (5.10 7 /mL) were sonicated (10 W) in the absence or presence of 1% (v/v) Triton X-100, in the buffer 1 mM phenylmethysulphonyl fluoride, 1 mM EDTA, 50 mM Tris-HCl pH 7.5 for 3 times of 10 s with 15 s break between cycles, then spun down at 10,0006 g for 15 min at 4uC. The pellet containing membranes was collected and the supernatant was further centrifuged at 100,0006 g for 60 min at 4uC [44]. The amount of proteins contained in membrane and cytoplasm fractions was determined by Bradford method, and both preparations were stored at 220uC for later use.
iPLA 2 activity assay. PMNs (3.10 8 ) were collected, sonicated 3 times for 10 s with a 15 s break between cycles, and centrifuged at 15,0006 g for 20 min at 4uC. The supernatant was removed and kept on ice. After determination of the protein concentration (Bradford assay), iPLA 2 activity measurement was obtained in presence of different peptide concentrations using the cPLA 2 assay kit, according to the modified method previously reported (cPLA 2 assay kit, Caymen chemical), [36]. Results were from two similar experiments, each performed in triplicates.

Characterization of secretions obtained after stimulation of PMNs by CHR and CAT
Stimulation and purification. PMNs secretory products were obtained after stimulation of 1.10 8 PMNs with 20 mM CHR or 20 mM CAT in EGTA buffer for 20 min. PMNs were centrifuged at 8006 g for 10 min at 4uC and the supernatant was recovered. Desalting and elimination of low molecular weights protein were achieved by centrifugation with a molecular cut-off 3 kDa Centricon (Pall, New York, USA), then the material was stored at 220uC for further analyses.
PMNs secretions induced by CHR and CAT were purified using a DIONEX Dual Gradient system (Dionex, Sunnyvale, USA) and a Nucleosil RP300-5C18 column (46250 mm, 5 mm particle size, 300-Å porosity; Macherey-Nagel, Hoerdt, France). Absorbance was monitored at 214 nm, and the solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.1% (v/v) trifluoroacetic acid in 70% (v/v) acetonitrile-water (solvent B with a flow rate of 1 mL/min. Gradient was indicated on chromatograms and fractions of 700 mL were collected. Mass spectrometry analysis. The cysteine residues from the RP-HPLC fractions were reduced by 50 mL of 10 mM dithiothreitol at 57uC and alkylated by 50 mLof 55 mM iodoacetamide. The proteins were digested with 40 mL of 12.5 ng/mL of modified porcine trypsin (Promega, Madison, WI, USA) in 25 mM NH 4 HCO 3 at 37uC for 14 h.
The generated peptides were analyzed directly by NanoLC-MS/MS on an Agilent 1100 Series HPLC-Chip/MS system (Agilent Technologies, Palo Alto, USA) coupled to an HCT Ultra ion trap (Bruker Daltonics, Bremen, Germany). The chip contained a Zorbax 300SB-C18 (43 mm675 mm, with a 5 mm particle size) column and a Zorbax 300SB-C18 (40 nL, 5 mm) enrichment column. The solvent system consisted of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid in water (solvent A) and 2% (v/v) water, 0.1% formic acid in acetonitrile (solvent B). The sample was loaded into the enrichment column at a flow rate set to 3.75 mL/min with solvent A. Elution was performed at a flow rate of 300 nL/min with a 8-40% linear gradient (solvent B) over 12 min and followed by a 70% stage (solvent B) over 5 min before the reconditioning of the column at 92% of solvent A. The complete system was fully controlled by ChemStation Rev. B.01.03 (Agilent Technologies).
All the analyses were performed using an HCT+ ion trap (Bruker Daltonics, Bremen Germany). The peptides were ionised by positive electrospray and fragmented by CID. The trap was externally calibrated with tunning mix for LC/MSD ion trap G2431A (Agilent Technologies). The voltage applied to the capillary cap was optimized to 21750 V. The MS scanning was performed in the standard enhanced resolution mode at a scan rate of 8.100 m/z per second. The mass range was 250 to 2500 m/z. The Ion Charge Control was 100000 and the maximum accumulation time was 200 ms. A total of 4 scans were averaged to obtain a MS spectrum and the rolling average was 2.
For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The 3 most abundant peptides with an isolation width of 4 m/z, preferring doubly charged ions (absolute threshold of 2000 and a relative of 5%) were selected on each MS spectrum for further isolation and fragmentation. Ions were excluded after 2 spectra and released after 1 min.
Smart Parameters Setting was used for selected precursor ions. The MS/MS scanning was performed in the ultrascan resolution mode at a scan rate of 26.000 m/z per second. The mass range was 50 to 2800 m/z. The Ion Charge Control was 300000. A total of 6 scans were averaged to obtain a MS/MS spectrum. The values 30% and 200% give a start and end for fragmentation relative to the fragmentation amplitude selected (1.5 V).
The complete system was fully controlled by EsquireControl 6.1 Build 90 (Bruker Daltonics) software. Mass data collected during analysis were processed, converted into .mgf files using DataAnalysis 3.4 Build 179 (Bruker Daltonics) software. The standard parameters were used.
The smoothing algorithm was Savitzky Golay with a smoothing width of 0.2 m/z in 1 cycle. 250 compounds were selected with a threshold of 60000. The algorithm ''Accu Mass'' was used.
The MS and the MS/MS data were searched using a local Mascot server (version MASCOT 2. 2.0, MatrixScience, UK). Searches were performed with a mass tolerance of 200 ppm in MS mode and 0.3 Da in MS/MS mode and with the following parameters: trypsin specificity, one missed cleavage, cysteine carbamidomethylation and methionine oxidation (and protein amino-terminal acetylation) as variable modifications, trap fragmentation, without constraining protein molecular weight or isoelectric point and without any taxonomic restriction.
To minimize false positive identifications, the results were subjected to very stringent filtering criteria. For the identification of a protein with two peptides or more, at least two unique peptides had to a Mascot ion score above 20. In the case of single peptide hits, the score of the unique peptide must be greater than the 52. For the estimation of the false positive rate, a target-decoy database search was performed [80]. In this approach, peptides are matched against a database consisting of the native protein sequences found in the database (target) and of the sequencereversed entries (decoy). The evaluations were performed using the peptide validation software Scaffold (Proteome Software, Portland, USA). This strategy was used to obtain a final catalogue of proteins with an estimated false positive rate below 1%.

Statistical analysis
Group data are presented as mean6SD (as specified in the text and figure legends). Student's t test was used to determine the significance of data (*), p,0.05.

Supporting Information
Figure S1