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Originally published online as doi:10.1189/jlb.0507327 on January 16, 2008

Published online before print January 16, 2008
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(Journal of Leukocyte Biology. 2008;83:972-981.)
© 2008 by Society for Leukocyte Biology

VIP differentially activates β2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1

Nabil El Zein*,1, Bassam Badran{dagger},1 and Eric Sariban*,2

* Hemato-Oncology Unit and Laboratory of Pediatric Oncology, Hôpital Universitaire des Enfants, Brussels, Belgium; and
{dagger} Laboratory of Experimental Hematology, Bordet Institute, Brussels, Belgium

2 Correspondence: Hopital des Enfants Cancer, Av. J. J. Crocq 15, Brussels, Belgium 1020, Belgium. E-mail: esariban{at}ulb.ac.be


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ABSTRACT
 
The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the β2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.

Key Words: neuropeptides • cell activation • signal transduction • adhesion molecules • exocytosis


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INTRODUCTION
 
The vasoactive intestinal polypeptide (VIP) is a pleiotropic peptide produced by neurons in different areas of the CNS as well as by peripheral neurons and endocrine cells. As a peptidergic neurotransmitter, VIP regulates sympathetic transmitter release [1 ]. VIP as a neuro-hormone stimulates prolactine and luteinizing hormone and growth hormone (GH) release from the pituitary [2 ] and as a gastrointestinal hormone, stimulates enzyme secretion from parotides [3 ], pancreatic acinar cells [4 ], and insulin from pancreatic β-cell [5 ]. VIP also stimulates exocytosis of secretory granules contained in duodenal [6 ] and tracheal [7 ] cells and regulates protein secretion in testicular germ cells [8 ]. In addition, VIP is one of the signal molecules of the neuroimmune network affecting innate and adaptive immunity [9 , 10 ]. VIP interacts with the pituitary adenylate cyclase-activating protein (VPAC1) receptor, a class II G protein-coupled receptor (GPCR) that also includes the receptor for the pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, glucagon, calcitonin, parathyroid hormone, GH-releasing factor, and glucagon-like peptide-1 [11 ]. In hematopoietic cells, the VPAC1 receptor is constitutively expressed in human resting T cells [12 ], neutrophils [13 , 14 ], monocytes [12 , 15 , 16 ], macrophages [17 ], dendritic cells [18 ], as well as bone marrow stromal cells [10 ]. In cells of hematopoietic and nonhematopoietic origin, VIP has anti-inflammatory [9 , 10 ] and proinflammatory properties [19 20 21 22 23 24 25 ].

VPAC1 is preferentially coupled to G{alpha}s protein, and many studies have identified the VPAC1 receptor and the cAMP/protein kinase A (PKA) pathway as being major mediators of VIP effects on hematopoïetic cells. However, VIP-induced, cAMP-independent pathways have also been reported. This was based on the fact that H89, a specific PKA inhibitor, was not effective in blocking some VIP-induced effects [11 , 17 , 26 ]. These reports were not taking into account that cAMP-dependent/PKA-independent pathways could also play a role in VIP effects.

Recently, exchange protein directly activated by cAMP (EPAC), a guanine nucleotide-exchange factor activated by cAMP, has been identified as an alternative cAMP target [27 ]. EPAC regulates the GTPase activity of Rap1, a member of the Ras superfamily of monomeric GTPase. Rap1 signaling regulates several important cellular process [28 , 29 ]; one of the most consistent finding is the involvement of Rap1 in the stimulation of exocytosis by cAMP in a number of cells of different origins as well as the implication of Rap1 in the activation of a variety of integrins [30 31 32 33 34 35 ]. Although at first, it was reported that human peripheral blood monocytes and neutrophils were not expressing EPAC transcripts [36 ], further studies clearly indicated the presence of EPAC mRNA or protein in primary phagocytic cells [31 , 32 , 37 ], as well as the role of EPAC in myeloid cell function [38 ].

Human neutrophils express the formyl peptide receptor (FPR), the high-affinity receptor for the bacterial peptide fMLP, as well as the structurally related FPR-like 1 receptor (FPRL1), a promiscuous receptor having low affinity for fMLP as well as for numerous and chemically unrelated ligands [39 , 40 ]. FPR and FPRL1 are involved through neutrophil degranulation in exocytosis of inflammatory mediators and CD11b membrane integrin up-regulation. Recently, it has been reported that PACAP but not VIP in human neutrophils is also a functional ligand for FPRL1. Of interest, in neuronal cells, some effects of PACAP are mediated through a cAMP/EPAC-dependent, PKA-independent signaling cascade [41 , 42 ]. As we found previously that VPAC1 is differentially regulated in human neutrophils and monocytes [13 14 15 16 ], we evaluate in this study whether in monocytes, VIP can modulate signaling pathways and functions. In sharp contrast to what has been reported in neutrophils [43 ], we did find that human monocytes are sensitive to VIP.


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MATERIALS AND METHODS
 
Reagents
Synthetic VIP and PACAP 27 were purchased from Neosystem (Strasbourg, France), the FPRL1-selective antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) [44 ] was obtained from Calbiochem (La Jolla, CA, USA), and 8CPT-2Me-cAMP from Biolog Life Science Institute (Bremen, Germany). All the other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Cell preparation and treatment
PBMC were isolated by centrifugation on a Ficoll-Metrizoate density gradient (Lymphoprep, Lucron Bioproducts, Axis-Shield, Norway). PBMC were suspended in RPMI-1640 medium (BioWhittaker, Europe) containing 10% heat-inactivated FBS (NV Life Technologie, Rockville, MD, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin. Monocytes were purified further by adherence for 90 min to gelatin-coated plastic flasks, previously incubated with FBS. Differential cell counts revealed that these preparations were >95% monocytes and >99% viable. Thereafter, purified monocytes were cultured overnight and used for experiments the day after. Thus, the time for venipuncture to performance of the experiment was 24 h. Chinese hamster ovary (CHO)-K1 cells and FPRL1-transfected CHO-K1 cells [45 ] were kindly provided by Marc Parmentier (Free University, Brussels). In studies using inhibitors of biochemical pathways, cells were treated for 1 min with WRW4 (10 µM), 10 min with U73122 (10 µM), 30 min with PD98059 (10 µM), 30 min with SB203580 (10 µM), 30 min with H89 (10 µM), 60 min with LY294002 (10 µM), and 120 min with pertussis toxin (PTX; 200 ng/ml). Thereafter, cells were exposed to 10 µM VIP for 10 min and analyzed. Monocyte exposure to these different inhibitors did not alter their viability, as measured by the propidium iodide exclusion test. Dose and time exposure was similar to the ones reported in the literature for LY294002 [46 ], U73122 [47 ], PTX [48 ], PD98059 and SB203580 [49 ], WRW4 [50 ], and H89 [51 ]. Activated monocytes used as positive controls consist in monocytes exposed to fMLP (0.1 µM, 2 min) or LPS (1 µg/ml, 10 min). When indicated, forskolin (FK) was used at 10 µM for 20 min and 8CPT-2Me-cAMP at 10 µM for 30 min.

RT-PCR
RNA isolation and RT-PCR were performed as reported previously using specific primers set for VPAC1 [14 , 15 ] and for glycerol-3-phosphate dehydrogenase (G3PDH). Monocytes and neutrophils were used as positive controls for VPAC1 expression, and the T-lymphoblastoid cell line SupT-1 was used as negative control.

Measurement of calcium signals
Free intracellular calcium was determined as described previously [15 , 16 ] with a LS50B fluorescence Photometer (Perkin Elmer, Norwalk, CT, USA) in purified monocyte loaded with the calcium-sensitive dye Fluo-3/AM, and calcium concentration was calculated from fluorescence using the equation [52 ]: [Ca2+] = (dF – Fmin/Fmax–dF)Kd. In this formula, dF is the observed fluorescence, Fmin is the fluorescence at low Ca2+, 5 mM EGTA, Fmax is the fluorescence at high Ca2+, 8 µM ionomycin, and Kd= 390 nM for Fluo-3/AM. Values Fmin and Fmax were obtained at the end of each experiment. Peak values and plateau phase are recorded, this last data being calculated as the area under the curve, starting at the peak, up to 200 s after, and expressed as arbitrary units.

Western blot analysis
Cell lysates were subjected to SDS-PAGE using 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Uppsala, Sweden) using a semidry electroblot chamber. Membranes were blocked with TBST containing 5% BSA overnight at 4°C. The blots were incubated with primary antibodies diluted in TBST for 1 h at 25°C. Following 1 h of incubation with goat-antirabbit peroxidase-conjugated antibody (Sigma Chemical Co.) at room temperature, proteins were detected by the electogenerated chemiluminescence method (Amersham Biosciences), according to the manufacturer’s instructions. Blots were stripped by incubation in 62.5 mM Tris-HCl, 2% SDS, 100 mM 2-ME (pH 6.7), for 30 min at 50°C. Stripped blots were then rinsed extensively with TBST and reprobed as described above. Detection of ERK, p38, phospho-ERK (Tyr204), and phospho-p38 (Tyr182) was done using specific mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [15 , 16 ].

Assessment of H2O2 and matrix metalloproteinase-9 (MMP-9) production
Intracellular production of H2O2 was analyzed on a Perkin Elmer LS50B luminescence spectrometer (Fluometer) as reported [15 , 16 ]. Monocytes (106/ml) were incubated at 37°C for 20 min with 20 mM 2',7'-dichlorofluorescein (DCF) diacetate (Molecular Probes, Eugene, OR, USA). After labeling, cells were treated, and production of H2O2 was then monitored every 10 min by measuring DCF emission at 525 nm; results were expressed as relative fluorescence intensity ± SEM. MMP-9 production was determined by a gelatin assay as described [15 , 16 ].

Flow cytometry
Expression of CD11b (membrane-activated complex 1) and of the complement receptor 1 CD35 was analyzed on a flow cytometer (FACSort, Becton Dickinson, San Jose, CA, USA), as described previously [13 ], and results were reported as mean fluorescence ± SEM. Antibodies were from Becton Dickinson. Control of isotype-matched antibody was assayed in parallel.

Rap1 activation assays
Rap1 activation assays were performed as described [53 ]. Briefly, an equal amount of cell lysates was incubated with the Ras-binding domain of Ras-related GTPase (Ral)-GDP dissociation stimulator (GDS) fused to GST provided by Johannes Bos (Utrecht, the Netherlands). This fusion protein was precoupled to glutathione beads to specifically pull down the activated GTP-bound form of Rap1, and activation of Rap1 was analyzed by immunoblotting using a Rap1 antibody. As negative control, glutathione beads incubated with serum before tumbling with cell lysate were used.

Statistical analysis
Statistical differences were determined with Student’s t-test or Wilcoxon matched pairs test using Prism 3.0 statistical software (Graphpad, San Diego, CA, USA). In experiments where cells were exposed only to VIP, differences were regarded as significant when *, P < 0.05, **, P < 0.01, or ***, P < 0.001, versus untreated cells. In experiments where cells were pretreated with inhibitors before exposure to VIP, differences were regarded as significant when •, P < 0.05, or ••, P < 0.01, versus VIP-treated cells.


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RESULTS
 
The activation of PI-3K by VIP is cAMP-dependent but phospholipase C (PLC)-independent
As illustrated (Fig. 1A ), stimulation of monocytes with VIP results in a rapid phosphorylation of the downstream target Akt, which becomes maximal at 10 min and undetectable at 30 min. The effect of VIP on Akt phosphorylation is dose-dependent with significant phosphorylation observed at 1 µM (Fig. 1B) . To evaluate the specificity of Akt phosphorylation, we tested the ability of the specific PI-3K inhibitor LY294002 to interfere with VIP-induced phosphorylation. As shown (Fig. 1C) , LY294002 induces a concentration-dependent inhibition of Akt phosphorylation, which is also sensitive to the PKA inhibitor H89 (10 µM) but not to the PLC inhibitor U73122 (10 µM; Fig. 1D ).


Figure 1
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  		Figure 1. Activation of the PI-3K pathway by VIP. Kinetics (A) and dose response (B) of VIP-induced Akt phosphorylation (pAkt) are presented as well as the dose-response effect of the PI-3K inhibitor LY294002 (LY), the PKA inhibitor H89, and the PLC inhibitor U73122 in VIP-treated cells (10 min; C and D). Preincubation times with the different inhibitors are 10 min for PLC, 20 min for H89, and 60 min for LY294002. Cells lysates were subjected to SDS-PAGE analysis, and phosphorylation of Akt was analyzed by immunoblotting using antibodies against the phosphorylated form of the protein. Equal protein loading was confirmed by stripping the blots and reprobing them with an antibody against total p38. All of the results shown are representative of at least three separated experiments. c, Control.

VIP regulates pathways, which are cAMP/p38- and PI-3K/ERK-dependent
In human polymorphonuclear neutrophils, VIP is reported to have no effect on ERK [43 ]. In contrast, we show that monocytes exposed to VIP present a rapid phosphorylation of ERK as well as p38 MAPK; this is dose-dependent, with maximal phosphorylation observed at 10 min for ERK and 5 min for p38 (Fig. 2A 2B 2C 2D ). ERK phosphorylation is sensitive to the specific inhibitor PD98059 (10 µM; Fig. 2E ), and p38 phosphorylation is inhibited by the specific inhibitor SB203580 (10 µM; Fig. 2F ). Lower doses of these two inhibitors (1 µM) were without any effect on ERK or p38 phosphorylation. As VIP increases cAMP in human monocytes [54 ], we evaluated whether MAPK activation was a downstream target of PKA and/or PI-3K. As shown (Fig. 2G and 2H) , the PKA inhibitor H89 (10 µM) decreases p38 phosphorylation without having any effect on ERK phosphorylation, and the reverse was found in cells pre-exposed to the PI-3K inhibitor LY294002 (10 µM; Fig. 2 I and J). Lower concentration of these two inhibitors (1 µM) was without any effect on p38 or ERK phosphorylation.


Figure 2
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  		Figure 2. Activation and regulation of ERK and p38 MAPK by VIP. VIP-treated monocytes were analyzed for phosphorylation of ERK (pERK; A, C, E, G, I) and p38 (pp38; B, D, F, H, J). Kinetics in cells exposed to 10 µM VIP (A and B) and dose response of VIP-treated cells for 10 min (C and D) are presented as well as the effect of preincubation with inhibitors of ERK (E), p38 (F), PKA (G and H), and PI-3K pathways (I and J) in VIP-treated cells (10 µM). Unstimulated cells were run as a negative control (C). Preincubation times with the different inhibitors are 20 min for H89, 30 min for PD098059 (PD), 30 min for SB203580 (SB), and 60 min for LY294002. Cells were analyzed as described in Figure 1 , and results are representative of at least three separated experiments.

VIP regulates a cAMP/EPAC/PI-3K/ERK pathway and a cAMP/p38 pathway, which is EPAC-independent
To determine whether EPAC was activated in VIP-exposed monocytes, cells were analyzed by Western blotting using Rap1 activity as an indicator of EPAC activation. The specific EPAC activator 8CPT-2Me-cAMP (10 µM) and VIP activate Rap1 with maximal effect of VIP at 10 µM drug concentration after 10–20 min cell exposure (Fig. 3A and 3B ). As expected, the PKA inhibitor H89 (10 µM) was without any effect on Rap1 activation (Fig. 3A) . We further investigated downstream signaling pathways activated by EPAC and found that similar to VIP, monocyte exposure to 8CPT-2Me-cAMP (10 µM) results in an increase in Akt (Fig. 3C) and ERK phosphorylation (Fig. 3D) . The level of Akt and ERK phosphorylation in cells treated with 10 µM 8CPT-2Me-cAMP is similar to the one obtained with 10 µM VIP or with the positive controls (LPS- or fMLP-treated cells). However, in sharp contrast to VIP, 8CPT-2Me-cAMP does not modulate p38 phosphorylation (Fig. 3D) .


Figure 3
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  		Figure 3. Role of EPAC in VIP-treated monocytes, which were exposed to VIP for the indicated time (A) or the indicated concentration (B) or to 10 µM 8CPT-2Me-cAMP (A, B), with or without pretreatment with H89 (A). Cells were then lysed and analyzed for GTP-bound Rap1 (upper blot) as well as total Rap1 (lower blot). Cells exposed to VIP or to the specific EPAC activator 8-CPT-2Me-cAMP were analyzed by Western blotting for Akt (C) and ERK and p38 (D) phosphorylation using the appropriate antibody.

VIP increased in membrane expression of the integrin CD11b is ERK-, p38 MAPK-, cAMP/PKA-, cAMP/EPAC-, and PI-3K-dependent and PLC-independent
It has been reported that VIP does not increase the expression of CD11b in human neutrophils [55 ]. In contrast, kinetic studies in monocytes show that expression of CD11b increases within 10 min of VIP exposure with a plateau achieved by 30 min after neuropeptide exposure (Fig. 4A ). The minimum concentration necessary for CD11b up-regulation is in the micromolar range (Fig. 4B) . ERK and p38 MAPK are controlling CD11b up-regulation with dose-dependent inhibition of CD11b expression in cells pretreated with PD98059 or SB203580 (Fig. 4C) . Compared with untreated cells, levels of CD11b up-regulation in cells treated with the MAPK inhibitors were higher, indicating an incomplete inhibition by each of these inhibitors. However, cell exposure to both MAPK inhibitors results in a complete inhibition of CD11b up-regulation (data not shown). Inhibition of PKA by H89 (10 µM), which controls p38 phosphorylation, and inhibition of PI-3K by LY294002 (10 µM), which regulates ERK activation, result in a decrease in VIP-induced CD11b expression, and inhibition of PLC by U73122 (10 µM) does not have any effect (Fig. 4D) . To evaluate, in addition to the presence of a cAMP/PKA pathway, the role of the cAMP/EPAC pathway in VIP-activated monocytes, cells were exposed to 8CPT-2Me-cAMP and analyzed for CD11b expression. As shown (Fig. 4E) , 10 µM VIP or 10 µM 8CPT-2Me-cAMP produces a similar increase in CD11b expression. The PI-3K inhibitor LY294002 (10 µM) prevents 8CPT-2Me-cAMP induction of CD11b up-regulation, and the PLC inhibitor U73122 (10 µM) is without any effect (Fig. 4F) .


Figure 4
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  		Figure 4. VIP increased in CD11b integrin membrane expression is ERK-, p38-, PI-3K-, cAMP/PKA-, and cAMP/EPAC-dependent. Kinetics (A) and dose-response (B) of VIP increased in CD11b integrin expression are presented as well as the effect of inhibitors of p38 and ERK (C) and PKA and PI-3K pathways (D) in monocytes exposed to 10 µM VIP. CD11b expression was evaluated in cells exposed to VIP and 8CPT-2Me-cAMP (E), and the effect of PI-3K and PLC inhibitors on 8CPT-2Me-cAMP-mediated CD11b up-regulation is shown in F. CD11b expression was measured by means of PE-labeled anti-CD11b and was analyzed by flow cytometry. Mean fluorescence intensity for control samples is 100 ± 6.9. The results are expressed as mean fluorescence ± SEM from three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005, versus control; •, P < 0.05, versus VIP or 8CPT-2Me-cAMP-treated cells.

VIP-associated CD35 activation and MMP-9 secretion are cAMP/PKA/p38-dependent and EPAC/ERK-independent
As additional markers of monocytes activation, we investigated whether VIP could regulate CD35 expression, a receptor having a large, intracytoplasmic storage pool in the membrane of secretory vesicles and MMP-9 secretion, a protein contained in the tertiary (gelatinase) granules. VIP increases CD35 expression (Fig. 5A 5B 5C ) and MMP-9 secretion (Fig. 5D 5E 5F) in a time- and dose-dependent manner. This is also observed when monocytes are exposed to the cAMP-elevating agent FK but not with 8CPT-2Me-cAMP (10 µM; Fig. 5C and 5F ). The PKA inhibitor H89 (10 µM) or the p38 MAPK inhibitor SB203580 (10 µM) prevents such activation, and the ERK inhibitor PD98059 (10 µM) is without any effect (Fig. 5C and 5F) .


Figure 5
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  		Figure 5. VIP increased in CD35 and MMP9 expression is cAMP/PKA/p38-dependent but EPAC/ERK-independent. Cells exposed to VIP were analyzed for CD35 up-regulation by flow cytometry. Kinetics (A) and dose-response (B) are shown as well as the effects of the cAMP-elevating agents 8CPT-2Me-cAMP and FK on CD35 up-regulation (C) and the effects of the ERK (PD), p38 (SB), and MAPK and PKA (H89) inhibitors on CD35 expression in VIP-stimulated cells (C). Statistical evaluation is as in Figure 3 . Exocytosis of MMP-9 is evaluated by a gelatinase assay (D–F). Time course of MMP-9 release in response to VIP stimulation (D), dose-response effects of VIP on MMP-9 secretion (E), and effects of the cAMP-elevating agents 8CPT-2Me-cAMP and FK on MMP-9 secretion (F) and of the inhibitors of p38 (SB), ERK (PD), and MAPK and PKA (H89) pathways on VIP-induced stimulation of MMP-9 release is presented (F). Mean fluorescence intensity for control samples in CD35 experiments is 27 ± 4.1. Data from one experiment are representative of three others. *, P < 0.08; **, P < 0.01; ***, P < 0.005, vs. control; ••, P < 0.01, vs. VIP-treated cell 5.

VIP biological effects are mediated in part by FPRL1 through PI-3K/ERK activation
WRW4 is a FPRL-1-selective antagonist, which does not activate but does bind to this receptor. Having defined the intracellular signaling pathways and functions modulated by VIP, we assessed the effect of WRW4 to evaluate the eventual role of FPRL1 in VIP-treated monocytes. WRW4 (10 µM) does not affect VIP-induced cAMP activation (Fig. 6A ); however, WRW4 dose-dependently decreases VIP-associated phosphorylation of Akt and ERK (Fig. 6 6B 6C 6E ), with inhibition observed at 10 µM and no effect at 1 µM. The level of CD11b in cells exposed to VIP and pretreated by WRW4 is higher than in control cells but not statistically different from the level of CD11b observed in VIP-treated cells. Phosphorylation of p38, which is found in Figure 2 to be sensitive to PKA (10 µM) and resistant to PI-3K (10 µM) inhibition, is not affected by WRW4 (10 µM; Fig. 6D ).


Figure 6
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  		Figure 6. VIP biological effects are mediated in part by FPRL1. Monocytes pretreated or not with WRW4 (10 µM, 30 min) were exposed to VIP and analyzed for cAMP production (A), Akt (B), ERK (C), p38 (D), and phosphorylation and CD11b up-regulation (E). (A) cAMP elevation was measured as described in Materials and Methods; VIP pretreatment time was 10 min. **, P< 0.01, versus control. (B–D) Cells were evaluated by Western blotting for Akt, ERK, and p38 phosphorylation using the appropriate antibody. Cells exposed to LPS (Akt and p38) or fMLP (ERK) were used as positive control. (E) WRW4-treated cells exposed to VIP for 30 min were evaluated for CD11b expression as described in Figure 3D . *, P < 0.05; **, P < 0.01, versus control.

VIP signaling in FPRL-1-transfected cells
CHO-K1 cells do not express VPAC1 (Fig. 7A ). Thus, FPRL1-transfected CHO-K1 cells were used to confirm that VIP also signals through FPRL1. Cell exposure to VIP results in PI-3K/ERK activation (Fig. 7B) without p38 phosphorylation (data not shown), and no effects were seen in nontransfected CHO-K1 cells on ERK (Fig. 7B) or PI-3K (Fig. 7C) phosphorylation. Maximal phosphorylation was observed at 5 mn for ERK and 10–20 mn for PI-3K. This effect of VIP on ERK and Akt phosphorylation was dose-dependent and observed at 1 µM with further increase in phosphorylation at 10 µM VIP (Fig. 7D) .


Figure 7
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  		Figure 7. VIP signals through FPRL-1. Wild-type (CHO Wt) and FPRL-1-transfected CHO-K1 cells (CHO t) were analyzed for VPAC1 mRNA expression by RT-PCR using primers specific for VPAC1 (A). Human neutrophils and monocytes were used as positive control (+), and the human T lymphoid SupT-1 cells were used as negative control (–). G3PDH was used as control to test the integrity and the quantity of the mRNAs. CHO cells were exposed to VIP for the indicated times (B, C) or the indicated concentration (D, E) and analyzed as in Figures 1 and 2 for ERK (B, D), Akt (C, D), phosphorylation, as well as for the inhibitory effect of WRW4 on VIP-induced ERK and Akt phosphorylation (E).

G{alpha}s and G{alpha}i proteins participate in VIP activation of monocytes
Phosphorylation of GPCR by the G{alpha}s/cAMP/PKA pathway diverts its attention from G{alpha}s to G{alpha}i, resulting in the activation of β{gamma} subunits with downstream activation of the PI-3K and ERK pathways. Interrupting this line of communication by pretreating cells with PTX allowed us to evaluate the role of G{alpha}i in VIP-mediated signaling; such treatment dose-dependently prevents Akt (Fig. 8A ) and ERK phosphorylation (Fig. 8B) and inhibits CD11b up-regulation (Fig. 8D) without having any effect on p38 phosphorylation (Fig. 8C) .


Figure 8
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  		Figure 8. G{alpha}s and G{alpha}i participate in VIP-mediated monocyte activation. Monocytes, pretreated or not with PTX (60 min), were exposed to VIP and analyzed by Western blotting for Akt (A), ERK (B), and p38 (C) phosphorylation or by flow cytometry, as described in Figure 3 for CD11b up-regulation (D). **, P < 0.01, versus control; •, P < 0.05, versus VIP-treated cells.


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DISCUSSION
 
Most of the biological action of VIP is exerted through the activation of the cAMP/PKA pathway after interaction with VPAC1. Within myeloid cells, human resting monocytes and neutrophils expressed VPAC1 exclusively [12 13 14 15 16 ], and VPAC2 expression is also found following LPS stimulation [17 ]. Pro- and anti-inflammatory effects of VIP/PACAP on myeloid cells might be related to the fact that cells at different stages of activation expressed different VIP/PACAP receptors with different transduction pathways involved in the stimulatory versus inhibitory effects of these neuropeptides [9 ]. Differential effects of VIP on cells involved in innate immunity might also depend on the ability of the VIP/PACAP receptors to crosstalk with other GPCRs [14 ] or with another class of receptors such as tyrosine kinase receptors [16 ].

Recently, it was reported that in neutrophils, the proinflammatory effects of PACAP were mediated by VPAC1 and FPRL1, the low-affinity FPR receptor [43 ]; in this study, VIP was reported to have no effect on calcium or ERK phosphorylation. In contrast, we found that in monocytes, VIP increases cAMP, phosphorylates Akt, ERK, and p38, and up-regulates CD11b, CD35 membrane expression, and MMP-9 secretion, indicating that monocytes are sensitive to VIP. Using the FPRL1-blocking hexapeptide WRW4, we found that part of the VIP-mediated signaling was WRW4-dependent. However, cAMP increase, a second messenger not reported to be linked to FPR or FPRL1 [39 , 40 ], and p38 phosphorylation were not affected by WRW4, indicating that at least both receptors, VPAC1 and FPRL1, are interacting with VIP. This was translated to functional tests: Thus, CD35 up-regulation and MMP-9 secretion are cAMP/p38-sensitive and WRW4-resistant, indicating that they are under the control of VPAC1 through a cAMP/p38 pathway. Similar results were recently reported by our group using PACAP instead of VIP to activate monocytes. In these cells, the proinflammatory effects of PACAP linked to Akt/ERK activation were inhibited to WRW4, and p38 phosphorylation was not affected by WRW4 [56 ]. CHO-K1 cells do not express VPAC1. The activation of PI-3K and ERK by VIP in FPRL-1 transfected CHO-K1 cells further confirms that VIP, in addition to VPAC1, uses FPRL1 to initiate signaling pathways. Thus, VIP and PACAP interact with myeloid cells via different receptors to fully activate the cell.

On agonist binding, heterotrimeric Gi proteins coupled to VPAC1 or FPRL1 dissociate into {alpha} and β{gamma} subunits, the latter activating the PI-3K pathway, which we found in monocytes to be involved in ERK activation and CD11b up-regulation. In agreement, PTX, which inactivates the G{alpha} subunits of the {alpha}i class, also inhibits Akt and ERK phosphorylation as well as integrin activation.

In FK-induced murine macrophage proliferation, PI-3K phosphorylation is dependent on cAMP and EPAC activation [32 ]. Over the past years, EPAC, the cAMP-dependent guanine exchange factor for the small GTPase Rap1 and Rap2, has emerged as an important, alternative target of cAMP in a number of processes, including exocytosis and cell adhesion through integrin regulation [27 , 34 , 57 ]. PACAP in neuronal cells promotes cell survival, neurite outgrowth, and excitability through a cAMP/EPAC-dependent but PKA-independent manner [41 , 42 ], and in lymphocytes, VIP effects on Th2 cell survival are mediated via cAMP through EPAC [58 ]. Specific roles for PKA versus EPAC in the regulation of phagocyte functions have been reported [31 , 38 , 59 ]. VIP in our study was shown to activate Rap1. In addition, using the cAMP analog 8CPT-2Me-cAMP, which specifically activates EPAC but not PKA [31 ], we found that VIP and 8CPT-2Me-cAMP are activating similar biochemical pathways and functions. Thus, as reported for VIP, 8CPT-2Me-cAMP phosphorylates Akt and ERK and up-regulates CD11b. On the other hand, as opposed to VIP, 8-CPT-cAMP is not acting on p38, on CD35 up-regulation, or MMP-9 secretion. Altogether these results indicate the presence in VIP-activated monocytes of three pathways: Two are VPAC1-dependent, including a cAMP/PKA/p38 pathway, which regulates CD35 and MMP-9, and a cAMP/EPAC/PI3K/ERK pathway, which regulates integrin activation; the third pathway is mediated by VIP/FPRL1 interaction and involved the PI-3K/ERK pathway already activated by VPAC1 through EPAC (Fig. 9 ). Thus, myeloid cells should be added into the growing list of cells where EPAC plays an important role through exocytosis regulation and integrin modulation.


Figure 9
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  		Figure 9. Conceptual model for the signal transduction cascades initiated in human monocytes exposed to VIP. Occupation of the VPAC1 receptor results initially in the activation of a heterotrimeric G{alpha}s protein triggering a cAMP/PKA transduction pathway as well as a cAMP/Rap 1 pathway. Activation of the cAMP/PKA pathway leads to p38 MAPK phosphorylation and functional cell activation with CD11b, CD35 membrane up-regulation, and MMP-9 secretion. PKA activation is also responsible for a switch from {alpha}s/β{gamma} to {alpha}i/β{gamma} containing VPAC1, with subsequent activation of PI-3K. This last pathway plays a key role, as it is activated by the cAMP/Rap 1 pathway under the control of VPAC1 and by the β,{gamma} subunit of {alpha}i/β{gamma} containing FPRL1. PI-3K downstream regulates ERK and CD11b expression. Tertiary granules (dotted lines) are reservoirs of membrane proteins such as CD11b, fMLP receptor, and FPRL1, which become incorporated into the surface membrane when upon activation by proinflammatory agents, these granules fuse with the plasma membrane. We hypothesize that VIP through mobilization of tertiary granules up-regulates not only CD11b expression but also FPRL1 and primes monocytes to further activation by proinflammatory agents. The participation of other GPCR or non-GPCR receptors is not excluded.

In monocytes, Rac1, a component of the Rho family GTPase, has recently been found to activate a CD11b-proadhesive pathway in a PI-3K-dependent manner [60 ]. In neuronal cells, cAMP-signaling effects by PACAP or VIP involved the Rho family of small GTPase [61 , 62 ]. Preliminary works in our laboratory show that PACAP in monocytes also activates Rac1 in a PI-3K-dependent manner. Thus, experiments are in progress to evaluate the role of the Rho family proteins in VIP/PACAP-activated monocytes.

During inflammation, myeloid cells become primed (i.e., hyper-responsive) with respect to a number of proinflammatory mediators. The priming effect of bacterial LPS has been shown to lie at the level of granule mobilization [63 ]. Thus, LPS-induced priming mobilizes the tertiary granules, which store a considerable pool of FPRL1, thereby up-regulating FPRL1 expression at the cell surface [63 ]. As we show in this work that VIP mobilizes the tertiary granules, it is quite possible that the FPRL1-dependent, proinflammatory effects of VIP are partially dependent on VIP-induced priming of a FPRL1-mediated response at the level of tertiary granule mobilization (Fig. 9) . Thus, GPCR-induced exocytosis of granules containing preformed membrane receptors expands considerably the concept of crosstalk between GPCRs and between GPCR and non-GPCR receptors.


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ACKNOWLEDGEMENTS
 
This work was supported by the Fonds National de la Recherche Scientifique grants 7.4593.03, 7.4592.05, and 7.4603.06; the Lambeau-Marteaux and Wajnman-Mandelbaum Foundations; and the Fondation Aide aux Enfants Atteints de Cancer du Luxembourg. We thank Dr. J. Bos (Utrecht, The Netherlands) for the provision of the GST Ral-GDS construct and Dr. M. Parmentier (Brussels) for the gift of FPRL1-transfected CHO-K1 cells.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received May 25, 2007; revised November 19, 2007; accepted December 3, 2007.


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