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The Journal of Immunology, 1999, 163: 4213-4223.
Copyright © 1999 by The American Association of Immunologists

VIP and PACAP Differentially Regulate the Costimulatory Activity of Resting and Activated Macrophages Through the Modulation of B7.1 and B7.2 Expression1

Mario Delgado*,{dagger}, Wei Sun*, Javier Leceta{dagger} and Doina Ganea2,*

* Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and {dagger} Departamento Biologia Celular, Universidad Complutense, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), two structurally related neuropeptides produced and/or released within the lymphoid microenvironment, modulate numerous immune functions. Although primarily antiinflammatory in nature, VIP and PACAP also affect resting macrophages. In this study, we report on in vitro and in vivo dual effects of VIP/PACAP on the expression of B7.1 and B7.2 and on the costimulatory activity for T cells in unstimulated and LPS/IFN-{gamma}-activated macrophages. VIP and PACAP up-regulate B7.2, but not B7.1, expression and induce the capacity to stimulate the proliferation of naive T cells in response to soluble anti-CD3 or allogeneic stimulation. In contrast, both neuropeptides down-regulate B7.1/B7.2 expression on LPS/IFN-{gamma}-activated macrophages and inhibit the endotoxin-induced costimulatory activity for T cells. Interestingly, both the stimulatory and the inhibitory effects of VIP/PACAP are mediated through the specific receptor VPAC1 and involve the cAMP/protein kinase A transduction pathway. The dual effect on B7.1 and B7.2 expression occurs at both mRNA and protein level and correlates with the VIP/PACAP regulation of the macrophage costimulatory activity. Through their regulatory role for resting and activated macrophages, VIP and PACAP act as endogenous participants in the control of immune homeostasis. Their effects depend not only on the timing of their release, but also on the activation and differentiation state of the neighboring immune cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of an immune response involves the activation of effector cells such as macrophages, neutrophils, and T cells, and the subsequent production of cytokines, chemokines, and reactive oxygen and nitrogen intermediates. Activated macrophages play an essential role in the inflammatory process, and contribute to the initiation of the adaptive immune response, by acting as APCs. The activation of T lymphocytes requires two signals delivered by APCs. The first signal, which confers specificity, is provided by the interaction of the antigenic peptide/MHC complex with the T cell receptor. The second signal is provided by costimulatory molecules expressed on APCs and results in optimal cytokine production and proliferation. Among the costimulatory molecules, the B7 family plays a major role. Two molecules, B7.1 (CD80) and B7.2 (CD86) interact with their T cell-inducible counterreceptors, CD28 and CTLA-4 (reviewed in Refs. 1 and 2). B7.1 and B7.2 appear to be equivalent in the costimulation of T cell proliferation and IL-2 production (1, 2). However, B7.1 and B7.2 may differ in the signals provided for T cell differentiation, with B7.2 favoring Th2 development in a number of experimental systems (3, 4, 5, 6, 7, 8, 9). The relative expression of the B7 isoforms on APCs may be critical in determining the nature and extent of the immune response. The expression of B7.1 and B7.2 depends on the nature and on the activation state of the APC. Although differences exist among macrophages, B cells, and dendritic cells in terms of B7 expression, as a general rule, B7.2 is induced earlier during the activation process and at higher levels than B7.1 (1, 2).

Vasoactive intestinal peptide (VIP)3 and the pituitary adenylate cyclase activating polypeptide (PACAP) are two neuropeptides produced in the immune microenvironment (10, 11) that modulate both natural and acquired immunity (reviewed in Refs. 12 and 13), acting primarily as antiinflammatory agents. VIP and PACAP inhibit T cell proliferation and cytokine production (reviewed in Ref. 13) and affect several macrophage functions, including phagocytosis, respiratory burst, and chemotaxis (reviewed in 12). We reported recently that VIP and PACAP inhibit the in vitro and in vivo production of proinflammatory cytokines such as IL-6 and TNF-{alpha} (14, 15, 16), reduce the expression of the inducible NO synthase (iNOS) (17), enhance the production of the antiinflammatory cytokine IL-10 (18), and protect mice from endotoxic shock, presumably through the inhibition of endogenous proinflammatory mediators (19). Furthermore, we and others have recently demonstrated that VIP and PACAP inhibit IL-12 production in endotoxin-stimulated macrophages (20, 21, 22), which results in the inhibition of IFN-{gamma} synthesis by T cells (22).

Since the B7 molecules play an essential role in the stimulation of T cells by activated macrophages, we were interested in the possible effect of VIP/PACAP on the expression of B7. The aim of this study is to evaluate the effects of VIP/PACAP on B7.1 and B7.2 expression and on the costimulatory function of resting and activated macrophages. The data presented in this report demonstrate a dual effect of VIP and PACAP on the costimulatory activity of resting and activated macrophages, mediated through the differential regulation of B7.1 and B7.2 expression. To our knowledge, this is the first report of an immunomodulatory role of VIP and PACAP on the B7 costimulatory activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c mice obtained from The Jackson Laboratory (Bar Harbor, ME) were kept in pathogen-free conditions. All mice used were between 7 and 12 wk of age.

Reagents and Abs

Synthetic VIP and PACAP38 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1-antagonist [Ac-His1, D-Phe2, K15, R16, L27] VIP (3-7)-GRF (8-27) and the VPAC1-agonist [K15, R16, L27] VIP (1-7)-GRF (8-27) were kindly donated by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Belgium). The VPAC2-agonist Ro 25-1553 Ac-[Glu8, Lys12, Nle17, Ala19, Asp25, Leu26, Lys27,28, Gly29,30, Thr31-VIP cyclo (21-25) was a generous gift from Drs. Ann Welton and David R. Bolin (Hoffmann-La Roche, Nutley, NJ). The synthetic PAC1 agonist maxadilan was a generous gift from Dr. Ethan A. Lerner (Massachusetts General Hospital, Charlestown, MA). The PAC1-antagonist PACAP6-38, was obtained from Peninsula Laboratories (Belmont, CA). Monoclonal Abs to Mac-1, CD28, CD24, Thy-1, CD40, IgM µ-chain (6B2), CD11b, ICAM-1, I-Ad, B7.1 (IG10, rat IgG2a) and B7.2 (GL1, rat IgG2a), murine recombinant mrIFN-{gamma} and mrIL-10 were purchased from PharMingen (San Diego, CA). LPS (from Escherichia coli 055:B5), calphostin C, dibutyryl cAMP (dbcAMP), forskolin (FK), and PGE2 were purchased from Sigma (St. Louis, MO), and N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide (H89 from ICN Pharmaceuticals, Costa Mesa, CA).

Culture medium

Cells were cultured in DMEM (HyClone Laboratories, Logan, UT) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 10 µg/ml streptomycin, and 10% FCS (Life Technologies Laboratories, Grand Island, NY) (complete DMEM).

Cell purification

T cells were purified by sequential passage of a single cell suspension of mesenteric lymph node cells over two nylon wool columns, followed by treatment with anti-Ia and anti-B220 mAbs and complement-mediated lysis (rabbit complement; Pel Freeze, Rogers, AR). The purity of the T cell preparations was >95% (Thy-1+ by FACS analysis). The purified T cell preparations uniformly failed to respond to stimulation with Con A or soluble anti-CD3 mAb.

Purified macrophages were prepared following i.p. injection of 2 ml of 3% thioglycollate broth (Difco, Detroit, MI). After 4 days, the mice were killed, injected i.p. with 5 ml of cold DMEM medium, followed by the harvesting of peritoneal fluid. The peritoneal exudate cells were washed, and macrophages were obtained after the elimination of T and B cells through complement-mediated lysis following treatment with anti-Thy-1 and anti-B220 mAbs. The purified macrophage preparations were >96% Mac-1+ by FACS analysis.

Activation and fixation of macrophages

Purified peritoneal macrophages (1 x 106 cells/ml) were cultured with DMEM medium, LPS (10 µg/ml), and/or mrIFN-{gamma} (100 U/ml), in the presence or absence of different concentrations of VIP or PACAP for 22 h at 37°C in a humidified incubator with 5% CO2. The cells were extensively washed and subjected to either FACS analysis or fixed with 0.5% paraformaldehyde (Sigma) in PBS, pH 7.0, at 22°C for 30 min. The reaction was quenched by the addition of 10% FCS, the fixed cells were washed three times with DMEM, and incubated in complete medium at 37°C for 4 h before their use in culture as APC.

FACS analysis

Purified peritoneal macrophages (1 x 106 cells/ml) incubated in 25-cm2 flasks (Corning Plastic, Corning, NY) were scraped gently after exposure to ice-cold DMEM medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS. Cells were incubated with various mAbs at 4°C for 1 h. Isotype-matched Abs were used as controls, and IgG block (purchased from Sigma) was used to block the nonspecific binding to Fc receptors. The cells were washed and further stained with FITC-conjugated goat F(ab')2 anti-rat IgG (Sigma) for 30 min at 4°C. After extensive washing, the cells were fixed in 1% buffered paraformadehyde. Stained macrophages, gated according to scatter characteristics, were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Fluorescence data are expressed as mean channel fluorescence (MCF) and as percentage of positive cells after subtraction of background isotype-matched values.

Assay of macrophage costimulatory activity

Allostimulation assays were performed as previously described (15). BALB/c lymph node T cells (4 x 105) were stimulated in flat-bottom 96-well plates with 1 x 105 cells C57BL/6 peritoneal macrophages pretreated with various reagents and fixed with 0.5% paraformaldehyde. Proliferation was evaluated by pulsing with 0.5 µCi [3H]TdR (spec. act. 97 Ci/mmol; Amersham Life Science, Arlington Heights, IL) for the last 16 to 18 h of a 4-day culture period. [3H]TdR incorporation was measured by using a beta scintillation counter (Beckman, Palo Alto, CA). Results are expressed as the mean cpm ± SD of triplicate assays.

The syngeneic stimulation assay using anti-CD3 treatment was performed as described previously (16). C57BL/6 T cells (4 x 105) were cultured in complete DMEM medium in flat-bottom 96-well plates with various numbers of macrophages fixed with 0.5% paraformaldehyde, in the presence of soluble anti-CD3 mAb (2C11, 100 ng/ml) for 4 days. Proliferation was detected as described above.

Semiquantitative RT-PCR analysis for B7.1 and B7.2 mRNA

Macrophage monolayers (2 x 106 cells/ml) were stimulated with LPS (10 µg/ml), in the absence or presence of VIP and PACAP (10-8 M) for different time periods at 37°C. Total RNA was extracted by the acid guanidinium-phenol-chloroform method. cDNA was prepared from 1 µg of total RNA using random hexamer primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s instructions. A volume of 5 µl of the cDNA was amplified in a PCR reaction with specific primers in the presence of 1 µCi [{alpha}-32P]dATP (spec. act. 3000 Ci/mmol; NEN/DuPont Research Products, Boston, MA). The designated primers sequences are as follows: murine B7.1 sense, 5'-GCTGTCACTAAAAGGAGAGGTGCC-3', and antisense, 5'-CCCAACCATAGTTTTCCCCACCCC-3'; murine B7.2 sense, 5'-CCTGCACGTCTAAGCA AGGTCACC-3', and antisense, 5'-TGAGCAGCATCACAAGGAGGAGGG-3'; GAPDH sense, 5'-TCCTGCACCACCAACTGCTTAGCC-3', and antisense, 5'-GTTCAGCTCTTGGATGACCTTGCC-3'. The PCR conditions were: denaturation 94°C, 30 s; annealing 60°C, 30 s; and primer extension 72°C, 60 s. At sequential cycle numbers (12–15 cycles for GAPDH, 24–27 cycles for B7.1, and 22–25 cycles for B7.2), 8 µl of the reaction mixture was sampled and electrophoresed on 6% nondenaturing polyacrylamide gels. Gels were dried and transferred, and signal quantitation was performed in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). Results are expressed as ratios of B7.1 or B7.2 to GAPDH signals determined in parallel reactions. Each assay was judged to be valid if the correlation coefficient was greater than 0.9.

In vivo treatment of mice with VIP or PACAP

Mice were injected i.p. with medium alone, or with LPS (100 µg/mouse), in the presence or absence of VIP or PACAP (5 nmol/mouse). After 8 h, macrophages were purified from the peritoneal exudate as indicated above. Purified macrophages were analyzed by flow cytometry and, alternatively, fixed and assayed for macrophage costimulatory activity as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VIP and PACAP differentially regulate B7.1 and B7.2 expression in unstimulated and activated macrophages

To investigate the effect of VIP and PACAP on the expression of surface B7.1 and B7.2, peritoneal macrophages were cultured with medium alone, VIP, or PACAP for 24 h, and B7.1 and B7.2 expression was analyzed by flow cytometry. Only a minor fraction of the macrophages cultured with medium alone expressed B7.1 (5.1 ± 0.2% B7.1+ cells; Table IGo). In contrast, a significant number of macrophages express B7.2 (38 ± 3% B7.2+ cells; Table IGo). VIP and PACAP significantly increase B7.2 expression, whereas B7.1 expression is not affected (Table IGo, Fig. 1GoA). The stimulatory effect of VIP and PACAP on B7.2 expression is dose dependent, already apparent at a concentration of 0.1 nM and maximal at 10 nM (Fig. 1GoB, left panels). To determine whether B7.1 expression was induced at later time points, we analyzed B7.1 expression at 12, 24, 48, and 72 h; no increase in B7.1 expression was observed at any time point, whereas B7.2 expression progressively increased up to 48 h, when it reached a plateau (Fig. 1GoC, left panels).


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  		Table I. Modulation of B7.1 and B7.2 expression by VIP and PACAP1

 


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  		FIGURE 1. VIP and PACAP differentially regulate B7.1 and B7.2 expression in unstimulated and LPS-stimulated macrophages. A, Effects of VIP and PACAP on the expression of surface markers on peritoneal macrophages. Purified macrophages from C57BL/6 mice (1 x 106 cells/ml) were incubated with medium alone, VIP (10-8 M), PACAP (10-8 M), LPS (10 µg/ml), LPS plus VIP (10-8 M), or LPS plus PACAP (10-8 M) for 22 h. Expression of IAb, ICAM-1, CD11b, B7.1, and B7.2 was analyzed by flow cytometry. Thin lines represent the staining profile with isotype-matched control Abs. Data are representative of four independent experiments. B, Dose-dependent effect of VIP and PACAP on B7.1 and B7.2 expression on peritoneal macrophages. Purified macrophages (1 x 106 cells/ml) were incubated with medium alone or with LPS (10 µg/ml), in the presence or absence of different concentrations of VIP or PACAP. After 24 h, B7.1 and B7.2 expression was analyzed by flow cytometry. Results are the mean ± SD of three independent experiments performed in duplicate. C, Time course for the effects of VIP/PACAP on B7.1 and B7.2 expression. Purified macrophages (1 x 106 cells/ml) were incubated with medium alone, VIP (10-8 M), PACAP (10-8 M), LPS (10 µg/ml), LPS plus VIP (10-8 M), or LPS plus PACAP (10-8 M). At different time points, B7.1 and B7.2 expression was analyzed by flow cytometry. Results are the mean ± SD of three independent experiments performed in duplicate.

 
Next, we investigated the effect of VIP and PACAP on B7.1 and B7.2 expression on peritoneal macrophages activated with different stimuli. Expression of both B7.1 and B7.2 was significantly enhanced by stimulation with LPS, IFN-{gamma}, LPS plus IFN-{gamma}, or anti-CD40 mAbs (Table IGo). Contrary to their effect on unstimulated macrophages, VIP and PACAP inhibited the expression of B7.1 and B7.2 on macrophages stimulated with LPS and/or IFN-{gamma} (Table IGo and Fig. 1GoA). In contrast, CD40-induced B7.1/B7.2 expression was not affected by the treatment with VIP or PACAP (Table IGo). The inhibitory effect of VIP and PACAP on B7.1 and B7.2 expression was dose dependent (Fig. 1GoB, right panels), and was observed at all time points tested (Fig. 1GoC, right panels).

The effect of VIP and PACAP on B7.1 and B7.2 expression appears to be selective, because the expression of other molecules involved in Ag presentation, such as ICAM-1 and MHC class II, or of other macrophage surface markers, such as CD11b, was not affected (Fig. 1GoA).

To evaluate whether the inhibitory effect of VIP and PACAP on B7.1/B7.2 was reversible, macrophages were stimulated with LPS or IFN-{gamma} for 24 h in the presence or absence of VIP or PACAP, washed, and then recultured for an additional 24 h with LPS or IFN-{gamma}. The inhibitory effect of VIP/PACAP was reversed upon LPS/IFN-{gamma} restimulation (data not shown).

The VIP/PACAP modulation of B7.1/B7.2 expression is mediated through VPAC1

Next we investigated whether the regulatory effect of VIP/PACAP on B7.1 and B7.2 expression could be related to occupancy of specific receptors. The immunological actions of VIP and PACAP are exerted through a family of VIP/PACAP receptors that were recently reclassified (23): VPAC1 and VPAC2 exhibit similar affinities for the two neuropeptides, and activate primarily the adenylate cyclase system, whereas PAC1 exhibits a 300- to 1000-fold higher affinity for PACAP and activates both the adenylate cyclase and phospholipase C systems (reviewed in Ref. 24). To determine which of the VIP/PACAP receptors are involved in the regulation of B7.1/B7.2 expression, we used specific receptor agonists and antagonists. We investigated the effect of a VPAC1-agonist (25), a VPAC2 agonist (Ro 25-1553) (26), and of maxadilan, a specific PAC1 agonist (27) on B7.1/B7.2 expression in unstimulated and LPS-stimulated macrophages. The VPAC1-agonist, but not the VPAC2 and PAC1 agonists, stimulate B7.2 expression in unstimulated macrophages and inhibit B7.1/B7.2 expression in LPS-stimulated macrophages, with a potency similar to that of VIP/PACAP (Fig. 2Go, A and B, upper panels). In addition, we investigated the ability of PACAP6-38, an antagonist specific for PAC1 and to a lesser degree for VPAC2 (28), and of a specific VPAC1-antagonist (29), to reverse the effects of VIP and PACAP. The regulatory effects of VIP and PACAP were reversed by the VPAC1-antagonist, but not by PACAP6-38 (Fig. 2Go, A and B, lower panels). These results indicate that both neuropeptides exert their action primarily through VPAC1.



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  		FIGURE 2. VIP/PACAP regulation of B7.1/B7.2 expression is mediated through VPAC1. Upper panels, Comparative effects of VIP and PACAP agonists on B7.1/B7.2 expression in unstimulated (A), and LPS-stimulated (B) macrophages. Purified macrophages (1 x 106 cells/ml) were incubated for 24 h with medium alone (A) or LPS (10 µg/ml) (B), and different concentrations of maxadilan (a PAC1 agonist), Ro 25-1553 (a VPAC2 agonist), or [K15, R16, L27]VIP (1-7)-GRF(8-27) (a VPAC1-agonist). B7.1 and B7.2 expression was analyzed by flow cytometry. Results are the mean ± SD of four experiments performed in duplicate. Lower panels, Effect of PAC1- and VPAC-antagonists on the modulation of B7.1/B7.2 expression by VIP and PACAP. Purified macrophages (1 x 106 cells/ml) were incubated for 24 h with medium alone (A) or LPS (10 µg/ml) (B), and treated simultaneously with VIP or PACAP (10-8M) and a VPAC1 antagonist, Ac-His1, D-Phe2, K15, R16, L27]VIP(3-7)-GRF(8-27) (10-6 M), or a PAC1/VPAC2-antagonist (PACAP6-38) (10-6 M). B7.1 and B7.2 expression was determined by flow cytometry. VPAC1- and VPAC2/PAC1-antagonists alone did not affect B7.1/B7.2 expression in LPS-stimulated macrophages: [VPAC1 antagonist: 115 ± 12 B7.1, 221 ± 16 B7.2; PACAP6-38 : 124 ± 15 B7.1, 209 ± 12 B7.2]. Results are the mean ± SD of four experiments performed in duplicate.

 
cAMP as a second messenger for the effects of VIP and PACAP on B7.1/B7.2 expression

Since VPAC1 activates primarily the adenylate cyclase, we compared VIP/PACAP with FK and PGE2 (two strict cAMP-inducing agents), and dbcAMP (a cAMP analogue). Similar to VIP and PACAP, FK, PGE2, and dbcAMP stimulated B7.2 expression in unstimulated macrophages (Fig. 3GoA, upper panel) and inhibited B7.1/B7.2 expression in LPS-stimulated macrophages (Fig. 3GoB, upper panel), suggesting the involvement of cAMP in both the stimulatory and the inhibitory action. The role of cAMP as a second messenger is supported by the fact that the PKA inhibitor H89 reversed in a dose-dependent manner the effects of VIP and PACAP (Fig. 3Go, lower panels). In contrast, calphostin C, a PKC inhibitor, did not reverse the effect of the two neuropeptides. These results suggest that both the stimulatory effect of VIP/PACAP on B7.2 expression in unstimulated macrophages, and the inhibitory effect on B7.1/B7.2 expression in activated macrophages, are mediated through increases in intracellular cAMP.



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  		FIGURE 3. cAMP as a second messenger for the regulatory activity of VIP and PACAP on B7.1/B7.2 expression. Upper panels, Effects of various cAMP-inducing agents. Peritoneal macrophages (1 x 106 cells/ml) were incubated for 24 h with medium (A), or with LPS (10 µg/ml) (B), in the presence or absence of VIP (10-8 M), PACAP (10-8 M), FK (10-7 M), PGE2 (10-7 M), or dbcAMP (10-7 M). Control cultures were incubated with medium (A), or LPS (B). B7.1 and B7.2 expression was determined by flow cytometry. Each result is the mean ± SD of four experiments performed in duplicate. Lower panels, Comparative effects of calphostin C (a PKC-inhibitor) and H89 (a PKA-inhibitor) on the regulatory activity of VIP and PACAP. Peritoneal macrophages (1 x 106 cells/ml) were incubated for 24 h with medium (A), or LPS (10 µg/ml) (B), with or without VIP or PACAP (10-8 M). Cultures were treated with or without different concentrations of calphostin C or H89. B7.1 and B7.2 expression was determined by flow cytometry. The dashed lines represent control values from cultures incubated with medium (A) or LPS alone (B). Each result is the mean ± SD of four experiments performed in duplicate.

 
VIP and PACAP differentially regulate B7.1 and B7.2 mRNA levels

Having demonstrated that VIP and PACAP affect the expression of surface B7.1 and B7.2, we sought to determine whether this action occurred at a transcriptional level. We incubated peritoneal macrophages with medium (unstimulated) or with LPS (activated), in the presence or absence of 10-8 M VIP or PACAP for 24 h, followed by total RNA preparation and semiquantitative RT-PCR. Although little B7.1 and B7.2 mRNA is detectable in unstimulated cells (Fig. 4Go), significantly higher B7.2 mRNA levels were present in the VIP or PACAP-treated cells (Fig. 4Go). In addition, increased mRNA levels for both costimulatory molecules were observed in LPS-stimulated cells (Fig. 4Go), and treatment with VIP or PACAP significantly decreased the levels of both B7.1 and B7.2 mRNA (Fig. 4Go).



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  		FIGURE 4. VIP and PACAP differentially regulate B7.1 and B7.2 expression in macrophages at the mRNA level. Peritoneal macrophages (2 x 106 cells/ml) were incubated for 24 h with medium or with LPS (10 µg/ml), in the absence or presence of VIP and PACAP (10-8 M). Expression of B7.1, B7.2, and GAPDH mRNA was analyzed by semiquantitative RT-PCR as described in Materials and Methods. Results are expressed in arbitrary densitometric units normalized for the expression of GAPDH in each sample. One representative experiment of three is shown.

 
Differential regulation of the macrophage costimulatory activity

Since the levels of B7.1 and B7.2 expression correlate with the costimulatory activity of macrophages, we investigated whether VIP and PACAP regulate the costimulatory capacity of unstimulated and activated macrophages. We used a system in which macrophages could develop into effective costimulatory cells in the absence of T lymphocytes by treating macrophages with VIP or PACAP, followed by fixation with paraformaldehyde. Fixed LPS- or IFN-{gamma}-treated macrophages were previously shown to function as potent accessory cells for the activation of purified T lymphocytes with soluble anti-CD3. Fixed VIP- or PACAP-treated macrophages were added to purified T cells treated with soluble anti-CD3 Abs. Macrophages cultured with medium alone had a low costimulatory activity for anti-CD3-induced T cell proliferation; in contrast, VIP and PACAP-treated macrophages function as good costimulators (Fig. 5GoA). The rate of T cell proliferation depends on the number of added macrophages. The stimulatory effect of VIP and PACAP was, however, lower than that of LPS, a potent inducer of the macrophage costimulatory activity (Fig. 5GoA). The ability of VIP and PACAP to induce the costimulatory activity in unstimulated macrophages was dose dependent (Fig. 5GoB). In addition, we determined the effects of VIP/PACAP on allostimulation. BALB/c (H-2d) macrophages were pretreated with VIP or PACAP for 24 h, fixed, and added to purified allogeneic C57BL/6 (H-2b) T cells. Although C57BL/6 T cells stimulated with allogeneic macrophages cultured with medium alone did not proliferate, those stimulated with VIP- or PACAP-treated macrophages proliferated (Fig. 5GoC). However, the stimulatory effect of the VIP/PACAP-treated macrophages was lower than that of IFN-{gamma}-treated macrophages (Fig. 5GoC). These data indicate that VIP/PACAP enhance the costimulatory function of macrophages, and this increase correlates with the effect of VIP/PACAP on B7.2 expression.



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  		FIGURE 5. VIP and PACAP increase the costimulatory activity of macrophages through the up-regulation of B7.2. A-C, VIP and PACAP increase the costimulatory activity of macrophages. A, C57BL/6 peritoneal macrophages (1 x 106 cells/ml) were incubated with medium alone, VIP (10-8 M), or PACAP (10-8 M) for 24 h. After washing with medium and PBS, macrophages were fixed with 0.5% paraformaldehyde and added in various numbers to purified C57BL/6 T cells (4 x 105 cells) in the presence of soluble anti-CD3 (100 ng/ml). [3H]TdR was added to the wells for the last 18 h of a 4-day culture period. Each result is the mean ± SD of four experiments performed in duplicate. B, Purified lymph node T cells (4 x 105 cells) were cultured for 96 h in the presence of soluble anti-CD3 (100 ng/ml) and of fixed macrophages (1 x 105 cells) pretreated with different concentrations of VIP or PACAP for 24 h before fixation. [3H]TdR was added during the final 18 h of culture. C, Fixed C57BL/6 macrophages (1 x 105 cells) pretreated with medium, VIP (10-8 M), or PACAP (10-8 M), were added to purified BALB/c lymph node T cells (4 x 105 cells) and cultured for 96 h. [3H]TdR was added during the final 18 h of culture. Each result is the mean ± SD of four experiments performed in duplicate. D, VIP and PACAP increase the costimulatory activity of macrophages through the up-regulation of B7.2. Peritoneal macrophages (1 x 105 cells) were incubated for 24 h with medium or VIP (10-8 M). After washing with medium and PBS, macrophages were fixed with 0.5% paraformaldehyde and added to purified lymph node T cells (4 x 105 cells) in the presence of soluble anti-CD3 (100 ng/ml), and of anti-B7.1, anti-B7.2, and control (IgG) Abs (10 ng/ml). [3H]TdR was added during the final 18 h of culture of a 4-day culture period.

 
We investigated next the effect of VIP and PACAP on the costimulatory function of LPS- or IFN-{gamma}-activated macrophages. Peritoneal macrophages were stimulated with LPS or IFN-{gamma} in the presence or absence of VIP or PACAP, followed by fixation and addition to responder T cells. LPS- and IFN-{gamma}-treated macrophages expressed significant costimulatory activity, and VIP and PACAP inhibited the costimulatory activity in a dose-dependent manner (Fig. 6Go, A and B).



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  		FIGURE 6. VIP and PACAP inhibit the costimulatory activity of LPS/IFN-{gamma}-activated macrophages through the down-regulation of B7.1 and B7.2 expression. A and B, VIP and PACAP inhibit the costimulatory activity of LPS-activated macrophages. A, C57BL/6 peritoneal macrophages (1 x 106 cells/ml) were incubated for 24 h with medium or LPS (10 µg/ml), in the presence or absence of VIP (10-8 M), or PACAP (10-8 M). Increasing numbers of fixed macrophages were added to purified C57BL/6 lymph node T cells (4 x 105 cells) in the presence of soluble anti-CD3 (100 ng/ml). [3H]TdR was added to the wells for the last 18 h of a 4-day culture period. Each result is the mean ± SD of four experiments performed in duplicate. B, Purified peritoneal macrophages (1 x 106 cells/ml) were incubated for 24 h with medium or LPS (10 µg/ml) or IFN-{gamma} (100 U/ml) in the presence or absence of various concentrations of VIP or PACAP. After fixation, the macrophages (1 x 105 cells) were added to purified lymph node T cells (4 x 105 cells), and the cells were cultured for 96 h in the presence of soluble anti-CD3 (100 ng/ml). [3H]TdR was added during the final 18 h of culture. C and D, The inhibitory effect of VIP/PACAP on the costimulatory function of LPS-stimulated macrophages is mediated through the down-regulation of B7.1 and B7.2 expression. C, Blocking of B7.1 and B7.2 decreased the costimulatory activity of LPS-stimulated macrophages. Peritoneal macrophages (1 x 106 cells/ml) were stimulated with LPS (10 µg/ml), fixed, and added to purified lymph node T cells (4 x 105 cells), in the presence or absence of anti-B7.1, anti-B7.2, or control Abs (20 ng/ml). The macrophage costimulatory activity was determined as described above. Each result is the mean ± SD of three experiments performed in duplicate. D, Reconstitution of the costimulatory activity by anti-CD28 Abs. Peritoneal macrophages (1 x 106 cells/ml) were stimulated with LPS (10 µg/ml) with or without VIP or PACAP (10-8 M) for 24 h, followed by fixing. The fixed macrophages (1 x 105 cells) were added to purified lymph node T cells (4 x 105 cells) stimulated with soluble anti-CD3 mAbs (100 ng/ml). The cultures were treated with or without anti-CD28 (2 µg/ml) or control Abs. The macrophage costimulatory activity was determined as described in Materials and Methods. Results are the mean ± SD of four independent experiments performed in duplicate.

 
Regulation of B7.1 and B7.2 expression by VIP/PACAP is responsible for the effect on the macrophage costimulatory activity

To determine whether the increased costimulatory activity of unstimulated macrophages treated with VIP/PACAP is due to the increase in B7.2 expression, we treated the cultures with anti-B7.2 mAbs. Addition of anti-B7.2 mAbs, but not of anti-B7.1 mAbs, significantly reversed the stimulatory effect of VIP and PACAP on the costimulatory function of macrophages (Fig. 5GoC). Control isotype-matched Igs had no significant effect. Similar effects were observed in allogeneic stimulation experiments (data not shown).

Next, we assessed whether the VIP/PACAP down-regulation of B7.1 and B7.2 expression on LPS-stimulated macrophages led to their defective costimulatory function. First, the costimulatory activity of LPS-stimulated macrophages was determined in the presence or absence of Abs to B7.1 and B7.2. Indeed, the addition of neutralizing anti-B7.1 or anti-B7.2 mAbs blocked the costimulatory activity of LPS-treated macrophages (Fig. 6GoC). If the reduction in the macrophage costimulatory activity by VIP/PACAP is due to the decrease in B7.1/B7.2 expression, the anti-CD28 Abs should restore the costimulatory signal. In a second set of experiments, the macrophages were stimulated with LPS with or without VIP/PACAP, fixed, and added to purified T cells in the presence of anti-CD28 Abs. As expected, the anti-CD28 Abs restored the proliferation of T cells to the levels observed with LPS-treated macrophages in the absence of VIP/PACAP (Fig. 6GoD). These experiments indicate that VIP/PACAP inhibit the costimulatory function of activated macrophages by targeting the expression of B7.1 and B7.2.

The effect of VIP/PACAP on the costimulatory activity of macrophages is mediated through VPAC1 and cAMP. Similar to VIP/PACAP, the VPAC1 agonist, but not the PAC1 and VPAC2 agonists, inhibited the costimulatory activity of the LPS-activated macrophages (Fig. 7GoA), and the VPAC1 antagonist, but not the PAC1/VPAC2 antagonist, reversed the effect of VIP/PACAP (Fig. 7GoB). Similar to VIP/PACAP, FK, PGE2, and dbcAMP inhibited the costimulatory function of LPS-stimulated macrophages (Fig. 7GoC), and H89, but not calphostin C, reversed the VIP/PACAP-mediated inhibition of the costimulatory activity (Fig. 7GoD).



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  		FIGURE 7. Involvement of VPAC1 and cAMP in the VIP/PACAP inhibition of the costimulatory activity of LPS-stimulated macrophages. A and B, Inhibition of the costimulatory activity of macrophages by VIP and PACAP is mediated through VPAC1. A, Comparative effects of VIP and PACAP agonists on the functional costimulatory activity of LPS-stimulated macrophages. Purified macrophages (1 x 106 cells/ml) were incubated for 24 h with LPS (10 µg/ml) and different concentrations of maxadilan (a PAC1 agonist), Ro 25-1553 (a VPAC2 agonist), or [K15, R16, L27]VIP (1-7)-GRF(8-27) (a VPAC1-agonist). The macrophage costimulatory activity was determined as described in Fig. 6Go. Results are the mean ± SD of four experiments performed in duplicate. B, Effect of PAC1 and VPAC antagonists on the inhibitory effect of VIP and PACAP. Purified macrophages (1 x 106 cells/ml) were stimulated for 24 h with LPS (10 µg/ml) and treated simultaneously with VIP or PACAP (10-8M) and a VPAC1 antagonist, Ac-His1, D-Phe2, K15, R16, L27]VIP (3-7)-GRF(8-27) (10-6M), or a PAC1/VPAC2-antagonist (PACAP6-38) (10-6M). The macrophage costimulatory activity was determined as described in Fig. 6Go. VPAC1 and VPAC2/PAC1 antagonists alone did not affect the costimulatory activity of LPS-stimulated macrophages (VPAC1 antagonist, 63456 ± 2363 cpm; PACAP6-38, 61596 ± 4677 cpm). Results are the mean ± SD of four experiments performed in duplicate. C and D, cAMP as a second messenger for the VIP/PACAP inhibition of the costimulatory activity of LPS-stimulated macrophages. C, Effects of various cAMP-inducing agents. Peritoneal macrophages (1 x 106 cells/ml) were stimulated for 24 h with LPS (10 µg/ml) in the presence or absence of VIP (10-8 M), PACAP (10-8 M), FK (10-7 M), PGE2 (10-7 M) or dbcAMP (10-7 M). Control cultures were incubated with LPS alone. The macrophage costimulatory activity was determined as described in Fig. 6Go. Each result is the mean ± SD of four experiments performed in duplicate. D, Comparative effects of calphostin C (a PKC inhibitor) and H89 (a PKA inhibitor) on the inhibitory activity of VIP and PACAP. Peritoneal macrophages (1 x 106 cells/ml) were stimulated for 24 h with LPS (10 µg/ml) and incubated with or without VIP or PACAP (10-8 M), in the absence or presence of different concentrations of calphostin C or H89. The macrophage costimulatory activity was determined as described in Fig. 6Go. Each result is the mean ± SD of four experiments performed in duplicate.

 
VIP and PACAP regulate macrophage B7.1/B7.2 expression and costimulatory function in vivo

An attempt was made to reproduce the in vitro observations in vivo. First, we injected BALB/c mice i.p. with medium, VIP, or PACAP (5 nmol/mouse). After 8 h, peritoneal macrophages were isolated, and the B7.1/B7.2 expression was analyzed by flow cytometry. The in vivo administration of VIP and PACAP resulted in increased B7.2 expression, with no effect on B7.1 (Fig. 8GoA, upper panels, and Fig. 8GoB). Peritoneal macrophages harvested from VIP/PACAP-injected mice also exhibited increased costimulatory activity for anti-CD3-stimulated T cells (Fig. 8GoC, left panel). We conclude that, similar to the in vitro experiments, the in vivo administration of VIP or PACAP stimulates B7.2 expression and induces the costimulatory activity of peritoneal macrophages.



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  		FIGURE 8. In vivo effects of VIP and PACAP on B7.1/B7.2 expression and macrophage costimulatory activity. Mice (groups of four) were injected i.p. with medium or LPS (100 µg/mouse), in the presence or absence of VIP or PACAP (5 nmol/mouse). After 8 h, macrophages were purified from the peritoneal exudate. Purified macrophages were analyzed by flow cytometry for B7.1 and B7.2 expression (A and B). The gates (dashed lines) were set based on staining with unrelated isotype control Abs (A). Data are representative of four similar experiments. Results in B are the mean ± SD of four independent experiments performed in duplicate. C, In vivo treated macrophages were fixed with 0.5% paraformaldehyde and added in various numbers to purified lymph node T cells (4 x 105 cells) in the presence of anti-CD3 (100 ng/ml). [3H]TdR was added to the wells for the last 18 h of a 4-day culture. Results are expressed as cpm [3H]TdR incorporation; each value is the mean of three determinations.

 
Second, we assessed the in vivo effect of VIP/PACAP in LPS-injected mice. BALB/c mice were injected i.p. with LPS (100 µg/mouse), with or without VIP or PACAP (5 nmol/mouse). Macrophages harvested 8 h later were analyzed in terms of B7.1/B7.2 expression and costimulatory activity. In vivo VIP/PACAP administration reduced both B7.1 and B7.2 expression (Fig. 8GoA, lower panels, and Fig. 8GoB). Also, VIP/PACAP completely abolished the costimulatory activity (Fig. 8GoC, right panel). These effects are consistent with the in vitro effects of VIP/PACAP on LPS- or IFN-{gamma}-stimulated macrophages.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of naive T lymphocytes, including clonal expansion and differentiation, requires both a stimulatory and costimulatory signal provided by the interactions with an APC (30). Dendritic cells, activated B cells, and macrophages function as professional APCs, delivering both signals to T cells expressing the appropriate receptors (31). The B7 family (B7.1 and B7.2) on APCs and the T cell counterparts CD28/CTLA-4 represent the major system for delivering the costimulatory signal (1, 32). B7.1 (CD80) and B7.2 (CD86) are constitutively expressed on dendritic cells and up-regulated on activated macrophages, B cells, and T cells (2, 33, 34). B7.2 is induced more rapidly than B7.1 and expressed at higher levels (33, 35).

VIP and PACAP are two multifunctional neuropeptides that affect a variety of immune functions. Several reports showed that VIP and PACAP inhibit T cell proliferation (reviewed in Ref. 13) and modulate macrophage functions (reviewed in Ref. 12). Recently, VIP and PACAP were shown to act as true "macrophage deactivating factors" by significantly reducing the levels of proinflammatory cytokines such as TNF-{alpha}, IL-6, and IL-12 (14, 15, 16, 20, 21, 22), inhibiting the expression of the iNOS (17, 21) and enhancing the production of the antiinflammatory cytokine IL-10 (18). In the present study, we investigated the role of VIP and PACAP in the regulation of the B7.1/B7.2 expression and its correlation with the costimulatory function of macrophages. The data presented here demonstrate that VIP and PACAP have a dual effect depending on the macrophage activation stage. In unstimulated macrophages, VIP and PACAP increase B7.2 expression and induce the costimulatory activity. In contrast, in LPS and/or IFN-{gamma}-activated macrophages, VIP and PACAP inhibit the expression of both B7.1 and B7.2 and abolish the costimulatory activity. The effect on B7 expression was specific, since VIP/PACAP did not affect the expression of other macrophage surface markers such as ICAM-1, MHC class II, and CD11b. Also, the effect of VIP/PACAP on B7 expression was limited to macrophages. The neuropeptides failed to inhibit B7.1/B7.2 expression and the costimulatory function of activated B cells (results not shown). The unique susceptibility of macrophages to VIP/PACAP was consistent with the potent activity of these neuropeptides on the production of macrophage-derived agents (14, 15, 16, 17, 18, 19, 20, 21, 22). Of relevance is also the fact that the inhibition of B7.1/B7.2 expression by VIP/PACAP is reversible. This observation rules out the possibility that the VIP/PACAP treatment of macrophages results in a nonspecific toxic effect.

The macrophage costimulatory activity correlates closely with the expression of B7.1/B7.2. However, the contribution of each of the B7 molecule varies with the nature of the APC population and the nature of the stimulus (33, 36, 37). Some authors concluded that both B7.1 and B7.2 alone can provide costimulation in a partially redundant manner (33, 36, 37, 38, 39, 40, 41) and that blocking of B7.1 and B7.2 with specific mAbs decreases the costimulatory activity (Refs. 40 and 42, 43, 44 , and this study). However, some studies suggested that B7.2 has a more pronounced costimulatory effect for CD4+ T cells (36, 37), and that B7.1 provides a stronger proliferative signal for CD8+ T cells (45). In our experiments we used total lymph node T cell populations consisting of both CD4+ and CD8+ T cells. Therefore, it is not surprising that both B7.1 and B7.2 act as costimulatory signals, and that treatment with either anti-B7.1 or anti-B7.2 Abs reduced the proliferative signals.

The effects of VIP and PACAP on the costimulatory activity appear to be mediated through the modulation of B7 expression. In unstimulated macrophages, where VIP/PACAP induce B7.2, but not B7.1, expression, the costimulatory activity was reduced to control levels following treatment with anti-B7.2, but not anti-B7.1, Abs. In LPS-stimulated macrophages, where VIP/PACAP reduce B7.1/B7.2 expression and abolish the costimulatory activity, the VIP/PACAP inhibition was reversed by treatment with anti-CD28 Abs, which restored the CD28-signaling pathway in T cells.

Peritoneal macrophages have been previously shown to express VPAC1 and PAC1 mRNA, and both high and low affinity VIP/PACAP binding sites (46, 47). In a recent study we showed that, whereas VPAC1 and PAC1 expression is constitutive in peritoneal macrophages, VPAC2 expression is induced, relatively late, following LPS stimulation (22). A similar conclusion was reached for the Raw 264.7 macrophage cell line (16). Our agonist studies suggest that VPAC1 is the mediator for the effects of VIP/PACAP on macrophage B7 expression and costimulatory activity. The role of VPAC1 as the unique mediator is supported by the fact that a VPAC1 antagonist, but not PACAP6-38, an antagonist specific for both PAC1 and VPAC2, reverses the effects of VIP/PACAP.

VPAC1 is coupled primarily to the adenylate cyclase system (24, 48), suggesting that cAMP is the major second messenger in the modulation of B7 expression and costimulatory activity. This is supported by the fact that H89, a PKA inhibitor, reverses the effects of VIP/PACAP, and that FK, PGE2, and dbcAMP mimic the effects of VIP/PACAP. VIP and PACAP induce B7.2 expression in unstimulated macrophages and inhibit both B7.1 and B7.2 expression in LPS-stimulated macrophages. The actions of VIP and PACAP appear to be paradoxical, since both the stimulatory and the inhibitory effects are mediated by the same receptor, VPAC1, through the same secondary mediator, cAMP. However, similar effects have been previously reported for cAMP. cAMP-elevating agents and dbcAMP induce B7.2 on monocytes/macrophages and B cells (Refs. 42 and 49, 50, 51 , and this study). Also, cAMP elevating agents inhibit B7.1/B7.2 expression in LPS-stimulated peritoneal macrophages, LPS/GM-CSF-stimulated Langerhans cells, and LPS/IFN-{gamma}-stimulated microglia (Refs. 52, 53, 54 , and this study).

VIP and PACAP affect B7.1 and B7.2 expression at the mRNA level. We have recently reported that VIP and PACAP regulate the binding and composition of several transcription factors in stimulated macrophages, such as NF-{kappa}B, CREB, and IFN regulatory factor (IRF) 1 (16, 17, 18). The mechanisms by which VIP/PACAP stimulate B7.2 gene expression in resting macrophages and inhibit B7.1/B7.2 expression in activated macrophages are difficult to ascertain without knowing the structure of the B7 promoters. The B7.2 promoter has not been characterized. The induction of B7.2 by cAMP-elevating agents, including VIP/PACAP, suggests the presence of an active CRE site. The B7.1 promoter has been partially characterized as containing putative AP1, CRE, and NF-{kappa}B sites (55, 56). However, neither CREB, nor p65/cRe appear to play a role, since the CRE site is not protected in DNase footprinting experiments, and specific Abs for any of the known members of the NF-{kappa}B family fail to supershift (57). The fact that the CRE sequence appears nonfunctional is in agreement with the lack of VIP/PACAP effect on B7.1 expression in resting macrophages, as reported here. The mechanism by which cAMP-inducing agents including VIP/PACAP inhibit B7.1/B7.2 expression in activated macrophages is not clear. We speculate that, in addition to CREB, cAMP may induce transcriptional repressors in macrophages. Such a repressor, ICER, has been described in thymocytes (57), neurons (58, 59), and other cell types (45, 60, 61, 62). In T cells, ICER acts as a transcriptional repressor by replacing AP1 from complexes with NF-AT (57). VIP/PACAP-induced cAMP could affect B7 gene expression in macrophages in a similar way; e.g., an ICER-like-induced repressor could compromise the activation of the B7.1/B7.2 genes. However, prior characterization of the B7.1/B7.2 promoters is necessary to clarify the nature of such a repressor and its molecular mechanism.

Of obvious biological significance is the fact that the in vitro effect of VIP and PACAP on both the macrophage costimulatory activity and the B7.1/B7.2 expression was reproduced in vivo. The administration of VIP/PACAP in LPS-injected mice led to a significant reduction in B7.1/B7.2 expression and to the complete inhibition of the costimulatory activity of peritoneal macrophages. This is consistent with the antiinflammatory effects of the two neuropeptides in endotoxemic mice, where VIP and PACAP inhibit the production of TNF-{alpha}, IL-6, IL-12, IFN-{gamma}, and NO, and stimulate IL-10 production (14, 15, 17, 18, 19, 22). The in vivo effects of VIP/PACAP might be clinically relevant, since the proinflammatory macrophage-derived agents are involved in the detrimental effects of ischemia-reperfusion and septic shock. In fact, the in vivo administration of VIP or PACAP protected mice from lethal endotoxemia, in a high-endotoxic model for septic shock (19).

The in vivo administration of VIP/PACAP in nonimmunized mice resulted in the induction of B7.2 expression and of the costimulatory activity in peritoneal macrophages. The biological consequences of the up-regulation of B7.2, but not B7.1, in unstimulated macrophages are not clear. The functions of B7.1 and B7.2 in terms of induction of T cell proliferation and IL-2 production appear to be quantitatively, but not qualitatively, different (45). The role of B7.1 vs B7.2 in selectively activating the differentiation of T cells into Th1 or Th2 cells remains controversial. Some of the in vitro studies suggest equivalent stimulatory signals (63, 64), whereas others concluded that differentiation into Th2 cells is dependent on B7.2 (7, 65, 66), or on B7.1 (67). There is also disagreement regarding the in vivo experiments. In an EAE model, the blockage of B7.2 increased disease severity (4). In contrast, in the nonobese diabetic (NOD) mouse, which develops autoimmune diabetes, blockage of B7.1 enhances disease severity (3). Recent data suggest that B7.2, the early inducible costimulatory ligand, may play a critical role in the initiation of the Th2 response, whereas B7.1 may be more important in the maintenance of the Th1 response (9, 68). We have recently demonstrated that VIP and PACAP enhance the macrophage induction of the Th2 response (69). The enhancement of B7.2 expression by VIP and PACAP may at least partially explain why the two neuropeptides stimulate predominantly the Th2 responses in vivo.

VIP and PACAP have been described as components of the lymphoid microenvironment, including the peritoneal immune population (10, 11). Since VIP and possibly PACAP are locally released from the peptidergic innervation and/or immune cells (11, 70), and interact with specific receptors present on various immune cells, we propose that VIP and PACAP act as endogenous immune modulators. Although VIP and PACAP were described primarily as antiinflammatory agents that inhibit the functions of stimulated T cells and macrophages, some recent observations suggest a more complex regulation depending on the activation and developmental stage of the cellular targets. Resting macrophages, for example, are stimulated by VIP/PACAP to secrete IL-6 (71), and to express B7.2 (this study), whereas, in contrast, VIP/PACAP inhibit IL-6 production and B7 expression in activated macrophages (Ref. 14 , and this study). Therefore, the physiological consequences of the VIP presence in the immune microenvironment may depend on the timing of its release and the activation state of the neighboring immune cells.


   Acknowledgments
 
We thank Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium) for the VPAC1 agonist and antagonist, Drs. David Bolin and Ann Welton (Hoffmann-LaRoche, Inc., Nutley, NJ) for the VPAC2 agonist Ro 25-1553, and Dr. Ethan Lerner (Massachusetts General Hospital, Charlestown, MA) for the PAC1 agonist maxadilan.


   Footnotes
 
1 This work was supported by Grant PHS AI 41786-02 (D.G.) and a Busch Biomedical Award 98-00 (D.G.), by Grant PB98-0381 (J.L. and M.D.), and by a postdoctoral fellowship from the Spanish Department of Education and Science (M.D.). Back

2 Address correspondence and reprint requests to Dr. Doina Ganea, Department of Biological Sciences, Rutgers University, 101 Warren Street, Newark, N.J. 07102. Back

3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; CRE, cAMP-regulatory element; FK, forskolin; iNOS, inducible NO synthase; IRF, IFN-regulatory factor; PACAP, pituitary adenylate cyclase-activating polypeptide; PAC1, PACAP receptor; VPAC1, type 1 VIP receptor; VPAC2, type 2 VIP receptor; mrIFN-{gamma}, murine recombinant IFN-{gamma}; MCF, mean channel fluorescence; dbcAMP, dibutyryl cAMP; PKA, protein kinase A; PKC, protein kinase C; CREB, cAMP regulatory element binding protein; ICER, inducible cAMP repressor. Back

Received for publication March 26, 1999. Accepted for publication August 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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