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Article

The Significance of Vasoactive Intestinal Peptide in Immunomodulation

Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas, Granada, Spain (M.D.); Departamento de Bioquimica Médica y Biologia Molecular, Universidad de Sevilla, Sevilla, Spain (D.P.); and Department of Biological Sciences, Rutgers University, Newark, New Jersey (D.G.)

Abstract

I. Introduction

II. Presence of Vasoactive Intestinal Peptide in the Immune System

A. Historical Background: Discovery of Vasoactive Intestinal Peptide

B. Vasoactive Intestinal Peptide Gene and Protein Structure

C. Neuronal Vasoactive Intestinal Peptide Sources within Lymphoid Organs

D. Vasoactive Intestinal Peptide Production by Immune Cells

1. Vasoactive Intestinal Peptide Production by Mast Cells and Granulocytes.

2. Vasoactive Intestinal Peptide Production by Lymphocytes.

E. Mechanisms for Vasoactive Intestinal Peptide Release in the Immune System

III. Vasoactive Intestinal Peptide Receptor Expression in the Immune System

A. Introduction and Nomenclature

B. Biochemical, Pharmacological, and Signaling Key Features of Vasoactive Intestinal Peptide Receptors

C. Basis of Vasoactive Intestinal Peptide Signaling in the Immune System

1. Functional and Molecular Expression of Vasoactive Intestinal Peptide Receptors in Immune Cells.

a. Vasoactive Intestinal Peptide Binding Sites in Immune Cells.

b. Vasoactive Intestinal Peptide Receptor mRNA Expression in Immune Cells.

2. Vasoactive Intestinal Peptide Signaling Pathways in Immune Cells.

IV. Effects of Vasoactive Intestinal Peptide on Innate Immunity

A. Adaptive/Innate Immunity

B. Vasoactive Intestinal Peptide Effects on Macrophages as Participants in Innate Immunity

1. Vasoactive Intestinal Peptide Effects on Macrophage Phagocytosis, Adherence, Migration, and Superoxide Ion Production.

2. Vasoactive Intestinal Peptide Effects on Macrophage-Derived Inflammatory Mediators.

3. Molecular Mechanisms Involved in the Anti-Inflammatory Action of Vasoactive Intestinal Peptide.

4. Vasoactive Intestinal Peptide Effects on Macrophage-Derived Chemokines.

C. Vasoactive Intestinal Peptide Effects on Macrophages as a Link to Adaptive Immunity

D. Vasoactive Intestinal Peptide Effects on Hematopoiesis

V. Effects of Vasoactive Intestinal Peptide on Adaptive Immunity

A. Vasoactive Intestinal Peptide Effects on T Cell Activation

B. Vasoactive Intestinal Peptide Effects on CD4+ T Cell Differentiation

1. Molecular Mechanisms by which Vasoactive Intestinal Peptide Promotes Th2-Type Immune Response.

C. Effects of Vasoactive Intestinal Peptide on CD4+ and CD8+ T Cell Function

1. T Cell Traffic and Adhesion.

2. CD4+ T Cell Function: Production of Cytokines.

a. Th1 Cytokines: Interleukin-2 and Interferon {gamma}.

b. Th2 Cytokines: Interleukin-4 and Interleukin-5.

c. In Vivo Consequences of the Vasoactive Intestinal Peptide Alteration of the T Helper 1/T Helper 2 Balance.

3. Function of Cytotoxic T Cells.

D. Effects of Vasoactive Intestinal Peptide on the Survival of CD4+ T Effectors

E. Vasoactive Intestinal Peptide Favors the Directional Migration of T Helper 2 Cells through Effects on Chemokines

VI. Effects of Vasoactive Intestinal Peptide in Brain Inflammation and Neurodegeneration

VII. Clinical Implications

A. Septic Shock

B. Rheumatoid Arthritis

C. Crohn's Disease

D. Parkinson's Disease

E. Brain Trauma

VIII. Conclusions and Perspectives

First identified by Said and Mutt some 30 years ago, the vasoactive intestinal peptide (VIP) was originally isolated as a vasodilator peptide. Subsequently, its biochemistry was elucidated, and within the 1st decade, their signature features as a neuropeptide became consolidated. It did not take long for these insights to permeate the field of immunology, out of which surprising new attributes for VIP were found in the last years. VIP is rapidly transforming into something more than a mere hormone. In evolving scientifically from a hormone to a novel agent for modifying immune function and possibly a cytokine-like molecule, VIP research has engaged many physiologists, molecular biologists, biochemists, endocrinologists, and pharmacologists and it is a paradigm to explore mutual interactions between neural and neuroendocrine links in health and disease. The aim of this review is firstly to update our knowledge of the cellular and molecular events relevant to VIP function on the immune system and secondly to gather together recent data that support its role as a type 2 cytokine. Recognition of the central functions VIP plays in cellular processes is focusing our attention on this "very important peptide" as exciting new candidates for therapeutic intervention and drug development.

I. Introduction

Optimal host defense is the result of interactions between the two systems implicated in homeostasis: the neuroendocrine and immune systems. For many years, the neuroendocrine system and the immune system have been viewed as being two autonomous networks functioning to maintain a balance between host and environment. The neuroendocrine system responds to external stimuli such as temperature, pain, and stress, whereas the immune system responds to exposure to bacteria, viruses, and trauma. Within the last 30 years, a direct link between the functions of these two systems has been pointed out by evidence presented in four distinct areas: 1) psychoneuroimmunology, the interaction between stress, behavior, and immune responses to diseases; 2) immunopsychiatry, abnormalities of the immune system that may be associated with mental illness; 3) immunoneurology, the actions of secreted immune factors and immunologically competent cells on the central nervous system (CNS¹); and 4) neuroimmunomodulation, the effects of the CNS on immune function (for review, see Weigent and Blalock, 1987). Neuroimmunomodulation has experienced an explosive growth not only in basic research but also expanding to the point that prospective clinical research could become reality. A crucial factor for the functioning of this intimate bidirectional network was the demonstration that the immune and neuroendocrine systems speak a common biochemical language. Indeed, it is becoming increasingly difficult, in the light of recent research, to see clear boundaries between such systems. This implies that: 1) the production of neuroendocrine hormones and neuropeptides by immune cells and of cytokines by neuroendocrine cells, 2) shared receptors on cells of the immune and neuroendocrine systems, 3) the effect of neuroendocrine mediators on immune functions, and 4) the effect of cytokines on the neuroendocrine system (Fig. 1). This raises the question of what can be actually be considered as immune or neuroendocrine. The fact that neurons and endocrine cells possessed similar substances was soon established. The "Rosetta stone" for the deciphering of the mutual chemical language for the neuroendocrine and immune systems was provided by the surprising discovery that lymphocytes are able to produce neuropeptides and hormones that were previously thought to reside exclusively in the nervous and endocrine systems. In 1980, it was shown that macrophages and lymphocytes were able to produce adrenocorticotropin and endorphins (Blalock and Smith, 1980; Smith and Blalock, 1981). To date, immune cells produce around 30 neuroendocrine mediators, including growth hormone (Weigent et al., 1988), prolactin (Montgomery et al., 1987), proenkephalin A (Rosen et al., 1989), somatostatin (Fuller and Verity, 1989; Weinstock et al., 1990), oxytocin-vasopressin (Geenen et al., 1987), atrial natriuretic peptide (Vollmar et al., 1992), substance P (Ho et al., 1997; Lai et al., 1998; Lambrecht et al., 1999), gonadotropin-releasing hormone (Chen et al., 1999), glucocorticoids (Lechner et al., 2000), procalcitonin (Oberhoffer et al., 1999), corticotropin-releasing factor (CRF) (Brouxhon et al., 1998), vasoactive intestinal peptide (VIP; see Section II.), pituitary adenylate cyclase-activating polypeptide (PACAP) (Abad et al., 2002), neuropeptide Y (Schwarz et al., 1994), calcitonin gene-related peptide (CGRP) (Singaram et al., 1991), -melanocyte-stimulating hormone (MSH) (Lolait et al., 1986; Rajora et al., 1996), and opioid peptides (Przewlocki et al., 1992). Moreover, an additional important fact demonstrated that lymphocytes competent to produce peptidic hormones and neuropeptides possess the biochemical machinery for a regulated secretory pathway as well as the necessary proteases as furin and other convertases to process neuropeptides (Taplits et al., 1988; Decroly et al., 1996). In addition to immune cells, other sources for neuropeptides such as neuropeptide Y, VIP, somatostatin, and galanin are the autonomic noradrenergic and cholinergic innervation, whereas substance P, neurokinin A, and CGRP are released by the sensory innervation present in the lymphoid organs (Felten et al., 1987, 1992; Fink et al., 1988; Nohr and Weihe, 1995; Bellinger et al., 1997). In addition, some of the neuropeptides can be released from the hypothalamic-pituitary axis as hormones or prohormones and arrive in the lymphoid organs via the circulation.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Bidirectional communication between the nervous and immune systems established through the production of neuroimmune mediators (hormones, cytokines, neuropeptides, and chemokines) and acting on specific receptors expressed by cells of both systems.

The immunological activity of neuropeptides is mediated through specific receptors. The presence of specific receptors for substance P (Cook et al., 1994; McCormack et al., 1996), somatostatin (Sreedharan et al., 1989; Hiruma et al., 1990), CGRP (McGillis et al., 1991), CRF (Agnello et al., 1998), melanocortin peptides (Catania et al., 1996; Getting et al., 1999), and VIP and related peptides (see Section III.) has been reported in immune cells.

The presence and release of neuropeptides within the lymphoid microenvironment and the existence of specific neuropeptide receptors on immune cells represent the framework for neuropeptides functioning as mediators of neuroimmune interactions. Although the list of neuropeptides/neurotransmitters affecting immune functions grows at a steady pace (for review, see McCann et al., 1998; Conti et al., 2000; Sternberg et al., 2003), here we review only the effects of VIP, one of the best studied immunoregulatory neuropeptides.

II. Presence of Vasoactive Intestinal Peptide in the Immune System

A. Historical Background: Discovery of Vasoactive Intestinal Peptide

In 1968, based on the observation that patients with severe lung injury or massive pulmonary embolism often developed systemic hypotension and shock, Sami I. Said tried to isolate a vasodilator peptide from lung extracts. Although the lung extract had vasodilator activity (Piper et al., 1970), the isolation of a pure peptide with this property was slow because of tissue paucity. Later, in collaboration with Viktor Mutt, Dr. Said decided to look for the vasodilator peptide in the intestine instead of lung based on the fact that both upper intestine and lung originate from the same embryonic bud, the foregut. The idea proved fruitful, and work on duodenal extracts quickly led to the isolation of a vasodilator, hypotensive peptide (Said and Mutt, 1970), named vasoactive intestinal peptide. Several years later, VIP was identified in the central and peripheral nervous system (Said and Rosenberg, 1976) and has since been recognized as a widely distributed neuropeptide, acting as a neurotransmitter or neuromodulator in many organs and tissues, including heart, lung, thyroid gland, kidney, immune system, urinary tract, and genital organs (Henning and Sawmiller, 2001). In light of this finding, the choice of intestine for the isolation of VIP had clearly been a fortunate one because of the dense enteric plexuses of nerves. The widespread distribution of VIP is correlated with its involvement in a wide variety of biological activities including systemic vasodilation, increased cardiac output, bronchodilation, hyperglycemia, smooth muscle relaxation, promotion of growth, hormonal regulation, analgesia, hyperthermia, neurotrophic effects, learning and behavior, bone metabolism, and some differential effects on secretory processes in the gastrointestinal tract and gastric motility.

B. Vasoactive Intestinal Peptide Gene and Protein Structure

VIP is a 28-amino acid peptide with structural similarities with other gastrointestinal hormones, such as secretin, glucagon, gastric inhibitory peptide, peptide histidine methionine (PHM; in human tissues) or peptide histidine isoleucine (PHI; its counterpart in other mammalian species), growth hormone releasing hormone, helodermin, PACAP (which exists in two amidated forms, PACAP27 and PACAP38, and shows 68% identity with VIP), and CRF (Said and Mutt, 1970, 1988; Said, 1986). The amino acid sequences of the various members of the VIP family peptide are depicted in Fig. 2 (adapted from Said, 1986).

View larger version (52K): [in this window] [in a new window]   FIG. 2. VIP structure: protein and gene. A, sequence of VIP family peptides. B, VIP gene structure. C, proteolytic generation of VIP fragments.

Conformational analysis of VIP by two-dimensional NMR and circular dichroism spectroscopy has show an initial disordered N terminus sequence of eight amino acid residues, probably with two -turns, followed by two helical segments at residues 7 to 15 and 19 to 27 connected by a region of undefined structure that confers mobility to the peptide molecule (Fry et al., 1989; Theriault et al., 1991; Filizola et al., 1997).

The three-dimensional structure of VIP exhibits substantial similarities with those of other members of the VIP/glucagon family (Braun et al., 1983; Gronenborn et al., 1987; Wray et al., 1993). In particular, both VIP and PACAP27 possess two helices separated by a disordered region, but two residues toward the C terminus shift the position of the first -helix of PACAP27, and the conformation of the second helix of PACAP27 is closer to a -helix than that of VIP. These minor conformational differences between PACAP27 and VIP may contribute to the selectivity of the peptides for their receptors (Inooka et al., 1992).

VIP is synthesized from a precursor molecule (prepro-VIP), which also contains PHM/PHI. The 170-amino acid prepro-VIP is metabolized by a signal peptidase in the endoplasmic reticulum to yield the 148-amino acid pro-VIP. Pro-VIP is cleaved by prohormone convertases to VIP-GKR (prepro-VIP_125-155) and PHM-GKR (prepro-VIP_81-110) (Bloom et al., 1983). VIP-GKR and PHM-GKR are then cleaved by carboxypeptidase-B-like enzymes to VIP-G and PHM-G (Itoh et al., 1983). The VIP-G and PHM-G can then be metabolized by PAM enzymes to VIP and PHM, which have an amidated C terminus.

The human VIP gene, located on chromosomal region 6q24, contains seven exons, each encoding a distinct functional domain on the protein precursor or its mRNA (Fig. 2). The gene spans 8837 bp. Exon 1 of 165 bp consists of the 5'-untranslated region of the mRNA, exon 2 of 117 bp encodes the signal peptide, exon 3 of 123 bp encodes an N-terminal peptide, exon 4 of 105 bp encodes PHM, exon 5 of 132 bp encodes VIP, exon 6 of 89 bp encodes the C-terminal peptide, and exon 7 of 723 bp consists of the 3'-untranslated region of the mRNA (Bodner et al., 1985; Tsukada et al., 1985; Gozes et al., 1986). The discovery of VIP and PHM or PHI sequences on the same gene, and mRNA suggests that both peptides are cosynthesized in the same tissue. However, one does not always find VIP and PHI in the same cell (Beinfeld et al., 1984); hence, alternative processing of the nuclear precursor RNA could take place. Alternatively, differential protein processing may occur, resulting in cells expressing either VIP or PHI. The fact that all brain cells synthesizing VIP mRNA also contain PHM mRNA (Linder et al., 1987; Card et al., 1988) points out that the differential regulation could be at the protein processing level.

C. Neuronal Vasoactive Intestinal Peptide Sources within Lymphoid Organs

Several investigators have shown by immunohistochemistry the presence of VIPergic nerve fibers in both central (thymus) and peripheral (spleen, lymph nodes, and mucosal-associated lymphoid tissue) lymphoid organs. These VIP-containing nerve terminals establish the anatomical link between the CNS and the immune system.

In the thymus, VIP⁺ nerve endings are most abundant in the capsule and extend into the interlobular septa, forming linear arrays. Fine VIPergic nerves exit this plexus to enter in thymic cortex and occasionally get into the medulla (Felten et al., 1985; Bellinger et al., 1996). The origin of these nerve terminals is not well defined. They do not derive from the sympathetic ganglia that provide the noradrenergic innervation to the thymus, because ganglionectomy does not deplete VIP from the thymus.

In spleen, VIP⁺ nerve endings are prominent in the red pulp along the venous/trabecular system and course as free fibers in the parenchyma and in the white pulp along the central arterioles and in the periarteriolar lymphatic sheath among T lymphocytes (Bellinger et al., 1996). Although the origin of VIP-containing nerve fibers in spleen is not clear, they may come from the dorsal root ganglion, from vagal input, or from the intrinsic neurons of the gut.

In lymph nodes, the VIP innervation is relatively sparse and depends on their location in the body, being present beneath the capsule particularly near the hilus, and along the vasculature in the internodal regions of the cortex (Fink and Weihe, 1988; Bellinger et al., 1996). VIP⁺ fibers occasionally leave the vascular plexus to extend among T cells in the adjacent cortex and along the medullary cords (Bellinger et al., 1990).

Regarding the mucosal-associated lymphoid tissue, an extensive nerve plexus containing various neuropeptides, including VIP, substance P, CGRP, cholecystokinin, and somatostatin, is present in the intestinal lamina propria (Bellinger et al., 1996). VIP⁺ fibers course throughout the lamina propria, distributing to the crypts, into the muscularis mucosae, into the core of the villi, and around the margins of the Peyer's patches (Ottaway et al., 1987; Ichikawa et al., 1994). Some VIP⁺ enteric fibers traverse the lamina propria to enter into the epithelium. VIPergic nerves in the Peyer's patches course predominantly along lymphatics and high endothelial venules and infrequently in the lymphoid follicle of the patches. The source of the VIP innervation of gut derives largely from intrinsic enteric VIP-containing neurons in the myenteric and submucous ganglionic plexuses and from extrinsic parasympathetic autonomic nerves and sensory fibers (Furness and Costa, 1980). In the bronchus-associated lymphoid tissue, VIP⁺ nerves are present in the walls of extra- and intrapulmonary bronchi and bronchioles (Uddman and Sundler, 1979; Dey et al., 1981), with abundant distribution to the smooth muscle layer and around submucosal mucous and serous glands of the airways. Like the gastrointestinal tract, an extensive network of VIPergic fibers courses in the lamina propria, although the anatomical association of VIP-containing nerve fibers with immune effector cells has not been investigated. VIP⁺ nerves in the lung probably arise from the vagus nerve and from microganglia present in the walls of the bronchi (Lundberg et al., 1979; Dey et al., 1981).

D. Vasoactive Intestinal Peptide Production by Immune Cells

1. Vasoactive Intestinal Peptide Production by Mast Cells and Granulocytes. The first evidence for the production of VIP by cells of the immune system was the identification of VIP-immunoreactivity (VIP-ir) in rat peritoneal, intestinal, and lung mast cells using radioimmunoassay (RIA) and immunohistochemical studies (Cutz et al., 1978). VIP-related peptides have also been detected in several mast cell lines, mouse bone marrow-derived mast cells, rat basophilic leukemia cells, and mouse peritoneal cell extracts (Wershill et al., 1993). Reversed-phase HPLC molecular characterization showed that the VIP content of mast cell lines differs structurally from naive VIP, mostly corresponding to the truncated form of VIP_10-28 with the carboxyl-terminal asparagine as a free acid and in a variable percentage to a mixture of amino-terminally extended VIP (Goetzl et al., 1988; Wershill et al., 1993), apparently derived from a novel prepro-VIP encoded by an alternatively spliced mRNA. Furthermore, mast cell lines appear to be incapable of synthesizing VIP_1-28. It is not clear whether multiple forms of VIP-ir molecules are present in normal immune effector cells. If this is the case, differential expression of VIP in specific immune effector cells may add to the complexity of VIP-immune effector cell interaction. The isolation of distinctive fragments of VIP_1-28 from suspensions of lymphocytes, mast cells, and other leukocytes after incubation in vitro confirmed the involvement of post-translational peptidolysis (Goetzl et al., 1989a) in the structural diversity of some VIPs. Therefore, subpopulations of immune cells may contain different VIP forms, which could preferentially bind to VIP receptor subtypes and have different functions.

In addition to mast cells, VIP-ir has been demonstrated in several types of leukocytes. O'Dorisio et al. (1980) have found VIP (1.1 ng/10⁸ cells) in human peripheral blood polymorphonuclear cells, mainly in neutrophils, but not in mononuclear cells, and suggested that VIP production may be regulated under immunopathological conditions, providing the means for the differential diagnosis of certain leukemias. However, others did not confirm these results. For example, several reports have shown much higher amounts of VIP in mononuclear cells than in polymorphonuclear cells (Madden et al., 1981; Lygren et al., 1984). Evidence for the production of VIP in rat basophilic leukemia cells has also been shown by the identification of a specific prepro-VIP/PHI mRNA fragment (Wershill et al., 1993). In addition, Murphy et al. (1981) described substantially lower VIP levels in different types of leukemic cells, and no correlation with leukemic disease states was found. Differences in experimental techniques used for cell isolation or in the specificity of the antibody used in these studies, as well as contamination with other cells or possible degranulation of leukocytes, could explain these discrepancies. Collectively, these studies suggest that VIP is present in both human mononuclear and polymorphonuclear cells. In addition, VIP (72 fmol/10⁷ cells) has been found in human eosinophils (Aliakbari et al., 1987). Similarly, Weinstock and Blum (1990) have demonstrated VIP-ir in eosinophils in granulomatous lesions induced by infection with Schistosomiasis mansoni.

2. Vasoactive Intestinal Peptide Production by Lymphocytes. After the initial reports describing the presence of VIP (0.2-0.3 pmol/10⁸ cells) in mononuclear leukocytes (Madden et al., 1981; Lygren et al., 1984), several studies, using different experimental approach, demonstrated that VIP is synthesized by lymphocytes and secreted in response to different immunological stimuli (Fig. 3). The first evidence was the presence of VIP-ir in thymus, spleen, and lymph node cells using immunohistological methods (Gomariz et al., 1990, 1992), where most VIP-ir was strongly identified in cells with lymphocyte appearance. VIP⁺ lymphocytes are more numerous in the thymic deep cortex and medulla, in the splenic periarteriolar lymphoid sheaths, and in the lymph node T cell-dependent interfollicular areas and deep cortex. The number of VIP⁺ lymphocytes is higher in peripheral organs than in thymus (Leceta et al., 1994). RIAs demonstrated that the VIP amounts in murine lymphoid organs are in the range of those found in mast cells (0.5-2.5 pmol/10⁸ lymphocytes) (Gomariz et al., 1992). To exclude the possibilities that the VIP-ir described in these studies really corresponded to nerve terminals in close proximity to lymphocytes or that lymphocytes merely took up VIP from the local microenvironment, biochemical characterization of VIP by HPLCRIA and light and electron microscopy were performed in isolated lymphoid cell suspensions (Gomariz et al., 1992; Leceta et al., 1994). HPLC-RIA showed that, although VIP_1-28 is the predominant molecular form of VIP-ir in isolated lymphocyte extracts, two additional molecular forms with longer retention times, corresponding to higher molecular weight precursors, are also detected (Gomariz et al., 1992), suggesting that VIP could be in fact synthesized by lymphoid cells. VIP occurrence in lymphocytes was definitively confirmed when VIP gene expression was demonstrated by in situ hybridization and reverse transcription (RT) followed by polymerase chain reaction (PCR) in different lymphocyte populations in both central and peripheral lymphoid organs (Gomariz et al., 1993, 1994b). In situ hybridization localized VIP mRNA in lymphocytes of the thymic corticomedullar and medullar regions and in the splenic white pulp (Gomariz et al., 1993). RT-PCR assays demonstrated the presence of mRNA for the precursor of VIP in both CD4 and CD8 T cells as well as B lymphocytes of primary and secondary lymphoid organs (Gomariz et al., 1994b). These findings were confirmed by flow cytometry analysis on double-labeled lymphocytes of thymus, spleen, and lymph nodes (Gomariz et al., 1993; Leceta et al., 1994). Regarding the thymus, VIP gene expression was shown in double positive (CD4⁺CD8⁺) and single positive (CD4⁺CD8^- and CD4^-CD8⁺) but not double negative (CD4^-CD8^-) thymocytes subsets (Delgado et al., 1996c). This suggests the possible autocrine involvement of VIP in intrathymic T cell maturation/differentiation. Northern blot analysis of mRNA from spleen lymphocytes showed two transcripts for VIP: a predominant form of 1.0 kb and a less abundant form of approximately 1.7 kb (Leceta et al., 1996). Although the 1.7-kb mRNA is preferentially expressed in most tissues, the 1.0-kb transcript predominates in the anterior pituitary gland (Lara et al., 1994), a product resulting from the utilization of the proximal polyadenylation signal of the prepro-VIP/PHI gene (Chew et al., 1994). This could also be the case in lymphoid cells (Gomariz et al., 1994b). Finally, the development of a specific enzyme-linked immunosorbent assay for VIP demonstrated that lymphocytes not only express mRNA for VIP and accumulate VIP protein in the cytoplasm but also secrete VIP in response to various inflammatory and mitogenic stimuli (Martinez et al., 1999). A recent study indicated that antigen-stimulated Th2, but not Th1, cells synthesize and secrete VIP (Delgado et al., 2001a; see below).

View larger version (45K): [in this window] [in a new window]   FIG. 3. VIP is synthesized by lymphocytes. A, immunohistochemical analysis of VIP+ thymocytes. B, HPLC analysis of VIP-ir from murine spleen lymphocytes. The major VIP-ir peak corresponds to naive VIP, and the two minor peaks with higher retention times correspond to VIP precursors (prepro-VIP and proVIP). C, RT-PCR analysis of VIP mRNA in different lymphocyte populations. Brain, positive control. DP, double positive thymocytes. DN, double negative thymocytes. D, VIP is secreted by T cells following TCR stimulation. Determination by a specific enzyme-linked immunosorbent assay. E, VIP is preferentially produced by Th2 CD4+ cells after antigenic stimulation.

Whether macrophages express VIP is still controversial. We did not find VIP-ir or VIP gene expression in macrophages from lymphoid organs and peritoneal suspensions (Leceta et al., 1994; Delgado et al., 1996d), whereas Metwali et al. (2002) described production of VIP by macrophages. Differences in state of cell activation or macrophage source could explain the discrepancy.

E. Mechanisms for Vasoactive Intestinal Peptide Release in the Immune System

Similar to other neuropeptides, VIP appears to be released in the lymphoid organs from two separate sources, the innervation and immune cells. The challenge remains to identify the exact factors that control neuronal or immune VIP release during an inflammatory reaction and to characterize the molecular mechanisms involved. The nature of the signals leading to neuronal VIP release during an inflammatory reaction is not known. However, a strong candidate is nitric oxide (NO), produced at high levels in inflammatory responses and shown to release VIP from enteric ganglia (Grider and Jin, 1993; Matsuyama et al., 2002). In addition, in a murine model of pulmonary delayed-type hypersensitivity characterized by a massive parenchymal infiltration of T lymphocytes, intratracheal antigenic challenge resulted in an increased secretion of VIP, recruitment of T cells bearing VIP receptors, and up-regulation of VIP receptors on the responding T cells, suggesting prominent antigen-evoked VIP neural immunoregulation (Kaltreider et al., 1997).

Regarding the mechanisms of VIP production by immune cells, initial studies indicated that activation of lymphocytes with mitogenic stimuli such as concanavalin A, with inflammatory stimuli such as lipopolysaccharide (LPS) or cytokines (TNF, IL-6, and IL-1), or stimulation through the T cell receptor (TCR) with an anti-TCR antibody leads to VIP expression and secretion (Leceta et al., 1996; Martinez et al., 1999). In addition, agents that increase intracellular cAMP levels or activate protein kinase C (PKC) pathway also induce the production of VIP by lymphocytes (Martinez et al., 1999). These findings are in agreement with the fact that in endocrine and neuroblastoma cells, VIP secretion is positively regulated by cAMP, calcium, PKC, and cytokines, and a cAMP response element (CRE), a phorbol ester-response element, and a cytokine response element have been located in the VIP gene promoter (Sena et al., 1994; Symes et al., 1995, 1997; Jones et al., 2000; Pitts et al., 2001). These mechanisms might also regulate the expression of VIP in immune cells, and inflammatory cytokines or signals that activate these pathways (antigen stimulation, bacterial products or mitogens) might play a role and represent potent inducers of its secretion. Of important physiological significance is the fact that Th2, but not Th1, effector cells express and secrete VIP following specific antigenic stimulation in vitro and in vivo (Delgado and Ganea, 2001a) and that the Th2-derived VIP is functional and promotes Th2 type responses in vivo (Delgado et al., 1999b, 2000b, 2002c; Delgado and Ganea, 2001b; Goetzl et al., 2001; Vassiliou et al., 2001; Voice et al., 2001, 2003). In fact, as we will discuss below, VIP is being seriously considered a type 2 cytokine.

An interesting question that should be addressed in a future is the relative role/relevance of both VIP sources (nerve terminals or immune cells) in immunomodulation. Several reports have suggested that VIP from autonomic nerve terminals in the lymphoid organs is involved in general or systemic aspects of blood circulation such as blood flow and vascular permeability, whereas VIP synthesized by lymphocytes is biologically involved in immune responses. Sympathectomy of thymus and spleen does not significantly alter their VIP contents, suggesting that lymphoid cells produce most VIPs on lymphoid organs (Bellinger et al., 1997). In addition, it has been described that, although mesenteric lymph nodes show few VIP-containing nerves, their VIP content is higher than that of thymus and spleen (Bellinger et al., 1990, 1996). This suggests again that in lymph nodes, VIP is produced mostly by the immune cells. The confirmation of the role of endogenous, T cell-derived VIP, as an important immunomodulator in T helper cell differentiation, has came recently from studies in which elimination of VIP from TCR-stimulated T cells with VIPase IgG resulted in the readjustment of the Th1/Th2 balance (Voice et al., 2003). Several reports indicate that VIP contained in the neuronal compartments is involved in the pathophysiology of several diseases in gut and lung. Loss of VIPergic nerves in these disorders appears to further exacerbate the inflammatory response. Thus, VIP present in nerve endings that distribute to the gut and lung may be important clinically in patients with inflammatory bowel disease (Koch et al., 1987, 1990), inflammation of the intestines associated with infection (Palmer and Greenwood, 1993), and asthma (Ollerenshaw et al., 1989). It has been suggested that liberation of neurotoxic substances from activated granulocytes, such as eosinophils and neutrophils, is responsible for the diminished VIP content during an inflammatory response.

Whether VIP-containing neurons in the CNS affect immune function by modulating neuroendocrine and/or autonomic outflow is not known. However, neurons and nerve fibers containing VIP have been found in CNS regions that influence the immune system, including all regions of the cerebral cortex, limbic forebrain structures (septum, amygdala, hippocampus, and stria terminalis) and hypothalamic areas (paraventricular and periventricular nuclei, arcuate nucleus, and anterior and preoptic areas), and data showing that VIP modulates the hypothalamic-pituitary axis suggest that central VIPergic neurons also influence immune function by interacting with the CNS circuitry that is well documented to play a modulatory role in neural-immune interactions (for review, see Fuxe et al., 1977; Loren et al., 1979).

III. Vasoactive Intestinal Peptide Receptor Expression in the Immune System

A. Introduction and Nomenclature

Recent advances in molecular and cell biology enabling the cloning, expression, and mutagenesis of VIP receptors has prompted an explosion of knowledge in the field, facilitating the discovery of new classes of regulatory proteins and providing a basis and means for manipulating receptor function. VIP binding to specific sites was characterized soon after VIP was first isolated from the small intestine, in liver and fat cell plasma membranes (Bataille et al., 1974), exocrine pancreas (Christophe et al., 1976), intestinal epithelium (Laburthe et al., 1979; Prieto et al., 1979), and human cancer cells (Laburthe et al., 1978). Combined functional information merged from early stages in VIP receptor research showed several common features such as a strong coupling to the activation of adenylyl cyclase and the ability to interact in a species-specific manner with related peptides such as secretin, helodermin, growth hormone-releasing factor, or peptide histidine isoleucine amide (Bataille et al., 1974; Laburthe et al., 1986). Some of these peptides were encoded within the VIP precursor polypeptide mRNA as additional biologically active peptides acting most likely through VIP receptors instead of different and specific sets of receptors. The isolation of the biologically relevant pituitary adenylate cyclase-activating polypeptides PACAP38 and PACAP27 (Miyata et al., 1989), which have a high degree of identity with VIP, led to the concept that VIP receptors interact with both VIP and PACAP as endogenous ligands. The receptors for VIP and PACAP belong to family B or group II of the G-protein-coupled receptors (GPCRs), also called the secretin receptor family (Kolakowski, 1994; Harmar, 2001). So far, three receptors that display high affinity for VIP and PACAP have been cloned and according to the established International Union of Pharmacology nomenclature (Harmar et al., 1998) are named VPAC₁ and VPAC₂ (high affinity for both VIP and PACAP) and PAC₁ (selective affinity for PACAP).

B. Biochemical, Pharmacological, and Signaling Key Features of Vasoactive Intestinal Peptide Receptors

VPAC receptors belong to the B1 subfamily (Harmar, 2001) together with receptors that respond to structurally related ligands [growth hormone releasing hormone, glucose-dependent insulinotropic polypeptide, glucagon-like peptides (GLP-1 and GLP-2), and glucagon]. These receptors share a common molecular architecture, made of seven transmembrane domains (7TM), three extracellular loops (EC1, EC2, and EC3), three intracellular loops (IC1, IC2, and IC3), a long amino-terminal extracellular domain, and an intracellular carboxyl terminus. As previously reported for other members of the B1 family, the amino-terminal extracellular region, the transmembrane domains, and the first intracellular loop consist of a large number of amino acid residues that are well conserved among VPAC receptors. VPAC receptors have a relatively low extension of N-glycosylation sites as cross-linking experiments with ¹²⁵I-PACAP27 and ¹²⁵I-VIP to cell membrane preparations revealed (Couvineau et al., 1986; Buscail et al., 1990). PAC₁ binding sites were first characterized in the hypothalamus and the anterior pituitary (Buscail et al., 1990; Gottschall et al., 1990) and showed a high affinity for PACAP38 and PACAP27 with a K_d value around 0.5 nM, whereas the affinity for VIP was more than 500 times lower, with differences among different regions of the central nervous system (Cauvin et al., 1991). In vitro, recombinant rat and human PAC₁ stably expressed in CHO cells recognized PACAP38 and PACAP27 with higher potencies (IC₅₀, 1 nM) than VIP (IC₅₀, 1000 nM) (Ciccarelli et al., 1995). VPAC₁ receptors show important species selectivity differences between rat and human, particularly in the recognition of PHI, based in three nonadjacent amino acids within a sequence comprising part of the first extracellular loop and third transmembrane domain (Couvineau et al., 1996; Igarashi et al., 2002). This domain is responsible for the low affinity for PHI (IC₅₀, 1000 nM) of the human VPAC₁ receptor. Thus, the order of affinity for rat VPAC₁ receptors is as follows VIP (IC₅₀, 1 nM) > PACAP38 = PHI > PACAP27 >> GRF (IC₅₀, 80 nM) > secretin (IC₅₀, 300 nM) (Couvineau et al., 1996). Rat and human VPAC₂ receptor recognizes VIP with lower affinities (IC₅₀, 3-4 nM), and similar or minor differences in the affinity to PACAP38 have been reported (Lutz et al., 1993; Usdin et al., 1994).

With regard to receptor specificity, natural peptides that are structurally related to VIP have a limited utility: only secretin, which has higher affinity for VPAC₁ than for VPAC₂, can be relied upon. An important point in establishing the role of VPAC receptors was the development during the past few years of selective agonists and antagonists. The cyclic analog of VIP, Ro 25-1392, is a selective agonist for the human VPAC₂ receptor (Xia et al., 1997) and the molecular modeling designed VIP analog, [Ala^11,22,28]VIP, is a selective agonist for the human VPAC₁ receptor (Nicole et al., 2000). The chimeric VIP/GRF molecule, [K¹⁵,R¹⁶,L²⁷]VIP_(1-7)/GRF_(8-27), is a selective agonist for rat VPAC₁ receptor (Gourlet et al., 1997b). Ro 25-1553 has been used as a general high VPAC₂ preference agonist (Dewit et al., 1998; Delgado et al., 2000a), although cross interaction with the rat PAC₁ receptor has been reported (Gourlet et al., 1997c). Very recently, sequence alignment of PACAP, VIP, [K¹⁵,R¹⁶,L²⁷]VIP_(1-7)/GRF_(8-27), Ro 25-1553, and Ro 25-1392 was analyzed to identify VPAC₂ selective determinants by saturation recombinant mutagenesis, leading to the production of a high selective and potent VPAC₂ agonist as a recombinant peptide (Yung et al., 2003). The strategy followed by the above cited authors overcame important technical issues such as the absence of exogenous amino acids without losing stability and the analysis of a large number of peptides and provides a feasible method of industrial manufacture. A potent general VPAC₁ receptor antagonist is the chimeric VIP/GRF derivative, PG 97-269 (Gourlet et al., 1997c), whereas no generally used VPAC₂ receptor antagonists have been developed. Recently, the endogenous VIP fragment generated at the surface of lymphocytes by protease activity (Goetzl et al., 1989b), VIP_4-28, has been shown in vitro to be a potent agonist for VPAC₁ and a potent antagonist for VPAC₂ (Summers et al., 2003). The role of this VPAC₂ endogenous antagonist could be of physiological importance since fine modulation can be achieved in cells differentially expressing VPAC1 or VPAC2 receptors. The combined use of PACAP_6-38 as an antagonist for PAC₁ and to a lesser degree for VPAC₂ (Gourlet et al., 1995) and of the PAC₁ agonist maxadilan (Moro and Lerner, 1997) has been employed to discriminate PAC₁ receptor-mediated mechanisms (Delgado et al., 1999h; Ganea and Delgado, 2001).

VPAC receptors couple to: 1) stimulation of adenylyl cyclase triggering a protein kinase A (PKA)-cAMP transduction pathway and 2) activation of phospholipase C (PLC) and phospholipase D (PLD) (Delporte et al., 1995; Rawlings and Hezareh, 1996; Van Rampelbergh et al., 1997; Harmar et al., 1998; McCulloch et al., 2000, 2002). VPAC receptors induce responses by activating transduction systems that involve different G-proteins, with G as the best characterized in different tissues and cell lines expressing recombinant receptors. Others G proteins that have been shown to be coupled to VPAC receptors belong to Gi/Go and Gq families (Pozo et al., 1997a; Van Rampelbergh et al., 1997; McCulloch et al., 2000; Shreeve et al., 2000; MacKenzie et al., 2001). Also, other VPAC partners different of G proteins have been reported, such as the small G-protein ADP-ribosylation factor (McCulloch et al., 2001) or receptor activity-modifying proteins (Christopoulos et al., 2003), resulting in alterations of receptor phenotype or pharmacological profile (McLatchie et al., 1998).

Genes that encode VPAC receptors have been cloned from frog, fish, chicken, rat, mouse, and human. The VPAC₁ gene (Vpr1) was originally cloned by cross-hybridization with the secretin receptor from a rat lung cDNA library (Ishihara et al., 1992). The human Vpr1 gene is located on region p22 of chromosome 3 (Sreedharan et al., 1995). VPAC₁ mRNA is particularly abundant in the cerebral cortex and hippocampus, whereas no messenger is detected in the suprachiasmatic nucleus (Usdin et al., 1994). VPAC₂ receptors showed a rather complementary distribution, although Vpr2 mRNA overlapped with Vpr1 in the hippocampus (Ishihara et al., 1992; Usdin et al., 1994). The VPAC₂ gene (Vpr2) was cloned from the rat pituitary gland (Lutz et al., 1993) and human SUP-T1 lymphoblasts (Svoboda et al., 1994). The human Vpr2 gene is located in region q36.3 of chromosome 7 (Mackay et al., 1996). There are presently no reported isoforms for the Vpr1 or Vpr2 genes. The PAC₁ gene (Adcyap1r) was first cloned from the AR4-2J rat pancreatic carcinoma cell line (Pisegna and Wank, 1993). PAC₁ mRNA is expressed mainly in the central nervous system and in endocrine glands (Basille et al., 2000; Vaudry et al., 2000). Nine variants resulting from alternative splicing have been described so far. Five alternative isoforms differing in the third intracellular loop have been characterized in rat (Spengler et al., 1993) and human (Pisegna and Wank, 1996). The PAC_1-null is the isoform without cassette insertion, three variants contain one of three 28-amino acid cassettes (PAC_1-hip, PAC_1-hop1, PAC_1-hop2), and an isoform contains a double insert, PAC_1-hip-hop1. Despite the conservation of alternative splicing of the Adcyap1r gene from rats to humans, there are differences regarding the role of hip and hop coupled to AC and PLC. Rat PAC_1-null, PAC_1-hop1, VPAC₁, and VPAC₂ receptors have been reported to stimulate PLD, although less potently than AC (McCulloch et al., 2001). Interestingly, a facilitating role for the rat hop1 cassette in receptor coupling to ADP-ribosylation factor-dependent PLD activation has been reported (McCulloch et al., 2001). The rat isoform PACAP_1-TM4 is another variant that differs from the canonical PAC₁ receptor only by discrete sequences located in transmembranes II and IV and the extracellular domain (Chatterjee et al., 1996). PACAP_1-TM4 is exclusively expressed in -cells; it is not coupled to AC, PLC, or PLD but mediates increases [Ca²⁺]_I by stimulating Ca²⁺ influx via L-type Ca⁺² channels (Chatterjee et al., 1996). Recent works pointed out that the insulinotropic effect of PACAP involves in part a cAMP but not an inositol phosphate pathway triggered by both PAC₁ and VPAC receptors (Jamen et al., 2000, 2002). There are two additional PAC₁ variants with deletions within the amino-terminal domain, the so-called PAC_1-short and PAC_{1-very short}, the former missing residues 89 to 109, and the latter missing residues 53 to 109 (Pantaloni et al., 1996; Dautzenberg et al., 1999). These variants have altered specificity; PAC_1-short receptor has no selectivity to PACAP compared with VIP, whereas PAC_{1-very short} exhibits a lower affinity for PACAP.

The mRNA-encoding PAC_1-short receptor is expressed exclusively in pituitary and adrenal glands and hypothalamus (Pantaloni et al., 1996), whereas PAC_{1-very short} is expressed in the nervous system (Dautzenberg et al., 1999). The last splice variant identified, PAC_1-3a, encodes a rat full-length receptor with an extra 24-amino acid cassette between exons III and IV, is coupled to both cAMP and inositol phosphate pathways, and is expressed during the spermatogenic cycle (Daniel et al., 2001).

C. Basis of Vasoactive Intestinal Peptide Signaling in the Immune System

VPAC receptors in the immune system share the same molecular basis of ligand-receptor interaction as in other cells and tissues. We shall focus on the present knowledge regarding various specific aspects of VPAC receptors such as functional expression and regulation, pharmacological tools, and signaling pathways in immunocompetent cells.

1. Functional and Molecular Expression of Vasoactive Intestinal Peptide Receptors in Immune Cells.
a. Vasoactive Intestinal Peptide Binding Sites in Immune Cells. The expression of fully functional VIP receptors in the immune system was first claimed in human peripheral blood lymphocytes in the early 1980s (Guerrero et al., 1981; O'Dorisio et al., 1981) by binding techniques (using ¹²⁵I-VIP as a ligand) and adenylyl cyclase enzyme measurements. Later on, VIP binding sites were identified in non adherent human peripheral mononuclear cells depleted of B lymphocytes and monocytes (Danek et al., 1983). Binding sites in human peripheral blood lymphocytes were confirmed (Ottaway et al., 1983) and extended to both mouse (Ottaway and Greenberg, 1984) and rat lymphocytes (Calvo et al., 1986b).

Depending on the source, the Scatchard analysis is consistent with a single class of high-affinity binding sites or two classes of binding sites (one of high affinity and low binding sites and the other of low affinity and high binding sites). The K_d value of the single class binding site and the high-affinity binding site is similar. For the high-affinity binding sites, the reported K_d values are 0.05 nM in rat blood mononuclear cells (Calvo et al., 1986b) and 0.24 nM in human blood mononuclear cells (Guerrero et al., 1981), whereas in the case of single class affinity binding sites, K_d values are 0.24 nM (Ottaway et al., 1983) and 0.47 nM (Danek et al., 1983) in human blood mononuclear cells and around 0.2 nM in spleen, Peyer's patches, and mesenteric and subcutaneous lymph node lymphocytes (Ottaway and Greenberg, 1984). Early experiments pointed out a lack of adenylyl cyclase stimulation by VIP in human monocytes (O'Dorisio et al., 1981), so it was reasoned that the low binding sites reported in human mononuclear cells (Guerrero et al., 1981) corresponded to the monocyte component (Danek et al., 1983). Despite that, only high-affinity VIP binding sites are considered to be functionally coupled to the effectors system (Laburthe et al., 2002), and discrepancies may be due to different methods of calculating K_d and B_max and to the concentrations of unlabeled ligand used to define specific binding, leading to different numbers of receptor classes rather than actual diverse biologically relevant entities. Later on, specific binding sites of VIP by human blood monocytes were reported, with a K_d value of 0.25 nM for the high-affinity receptor (Wiik et al., 1985).

Early attempts to identify different cells population expressing VIP receptors showed preferential expression of VIP binding sites by mouse T cells throughout the secondary lymphoid organs but not in thymus (Ottaway and Greenberg, 1984; Ottaway, 1987). In humans, although it was initially claimed that B and/or natural killer (NK) cells (Calvo et al., 1986a) could represent the only populations to specifically bind VIP, later experiments proved that CD4⁺ and CD8⁺ T cell-enriched suspensions showed high binding capacity (K_d of 0.25 nM in the CD4⁺-enriched fraction; K_d of 0.42 nM in the CD8⁺-enriched fraction) (Ottaway et al., 1990). Regarding the human thymus, it has been reported that ¹²⁵I-VIP binding sites in the cortex and medulla colocalize with anti-CD3⁺ cells (Reubi et al., 1998; Reubi, 2000). Despite the identification of VIP receptor protein, the subtype of receptors present in human thymus has not been identified. In murine thymocytes, initial studies suggest that murine thymocytes express VIP receptors, although there is no agreement on the number of binding sites due to different experimental procedures. Although thymocytes have relatively few binding sites as compared with purified T cells (Ottaway and Greenberg, 1984; Nguyen et al., 1987), functional receptors were identified on T-cell lymphomas of thymic origin (Robberecht et al., 1989), whereas VPAC₁ and VPAC₂ mRNA expression has been reported in different sets of mouse native thymocytes and in an immature T-cell line from a spontaneous thymic lymphoma (Pankhaniya et al., 1998). In rat thymocytes, flow cytometry analysis demonstrates a predominant expression of VPAC₂ and a more restricted pattern in the expression of VPAC₁ (Delgado et al., 1999c). Although it is clear that VIP affects several aspects of thymus biology such as cytokine production, apoptosis, differentiation, and mobility through direct effects on T cells (Ernstrom et al., 1995; Delgado et al., 1996a; Xin et al., 1997; Jiang et al., 1998; Pankhaniya et al., 1998), few data are available concerning the contextual role of epithelial VPAC receptors in thymus (Head et al., 1998; Reubi et al., 1998; Marie et al., 1999).

Human acute lymphocytic and myeloid blasts express functional VIP receptors as well as the transformed preB cell line (Nalm 6) and the T cell line (Molt 4b), whereas the histiocytic line (U937) and the myelocytic line (HL 60) do not appear to express VIP receptors (O'Dorisio et al., 1992). In fact, as we mentioned above, the VPAC₂ receptor was cloned from SUP-T1 lymphoblasts (Lutz et al., 1993). In most cases, human transformed cell lines have higher VIP binding affinities and capacities compared with lymphocytes from healthy donors (Beed et al., 1983; Finch et al., 1989; Cheng et al., 1993; Robichon et al., 1993).

The differential expression of specific VIP receptors by lymphocyte subpopulations is a remarkable characteristic. Human intestinal intraepithelial CD8⁺ T lymphocytes had no high-affinity VIP receptors (Roberts et al., 1991), whereas murine mucosal T cells have a single class of binding sites with a K_d of 9.08 nM (murine intestinal epithelial cell was K_d = 0.41 nM) functionally coupled to VIP-stimulated IL-5 release (Blum et al., 1992). These binding data are in close agreement with the VPAC₁ and VPAC₂ mRNA expression studies (Qian et al., 2001). There are few VPAC receptor binding studies in nontransformed and isolated B cells (Ottaway et al., 1990; Tatsuno et al., 1991) despite the biological effects mediated throughout VPAC receptors (Kimata et al., 1992, 1996; Fujieda et al., 1996; Shimozato and Kincade, 1997). Available data came from transformed B cell lines, i.e., SKW 6.4 B cells with a K_d of 59 nM (Cheng et al., 1993), Raji B cells with a K_d of 0.8 nM (Robichon et al., 1993), Nalm 6 preB cells with a K_d of 12.6 nM, and Dakiki plasma cells with a K_d of 9.1 nM (O'Dorisio et al., 1989, 1992).

NK cells account for 10% to 15% of blood lymphocytes and are found in low numbers in the peripheral lymphoid system. NK cells regulate certain aspects of T and B cell activation and hematopoiesis and defend against certain tumors and intracellular infections. In contrast to cytotoxic T cells, the NK cell-mediated cytotoxicity neither requires previous sensitization nor is MHC restricted. NK cells can be activated to produce cytokines (IL-2, IFN-, IFN-, and TNF-) that aid in immunomodulation. The first experimental evidence of VIP modulation on natural killer function indicated a decrease in the cytotoxicity (Rola-Pleszczynski et al., 1985). Although later experiments confirmed VIP inhibition (Yiangou et al., 1990; Sirianni et al., 1992), VIP restoration of NK cell activity has also been reported (Azzari et al., 1992; Peruzzi et al., 2000). There are no data available on PACAP modulation on NK cell activity, and in general, the effects of VIP are not completely understood. The binding studies suggest the presence of VPAC high affinity receptors in NK cells, although they have not been fully characterized (Ottaway et al., 1990).

Despite the immunoregulatory properties of PACAP and VIP in isolated neutrophils (Palermo et al., 1996; Kinhult et al., 2002), the presence of receptors is controversial. It has been reported that VIP increases the cAMP levels in human neutrophils (Palermo et al., 1996), although it is claimed to be mediated through nonreceptor mechanisms (Pedrera et al., 1994). In human eosinophils, VIP is able to stimulate migration apparently through VPAC₁ receptors (Dunzendorfer et al., 1998). In general, further research will be needed to evaluate the mechanisms of action of VIP in granulocytes, either eosinophils or neutrophils.

Macrophages are key components in the regulation of immune responses as well as being effectors cells of innate immunity. They are also involved in many other physiological processes, such as the development of the hematopoietic system, bone remodeling, and wound healing (Morrissette et al., 1999; Janeway and Medzhitov, 2002). The first report on VIP regulation of macrophage activation (Koff and Dunegan, 1985) clearly foresighted what became one of the main functions of VIP and PACAP as macrophage-deactivating factors (Ganea and Delgado, 2001a,b). Characterization of functional VIP and PACAP binding sites have been reported in both rat and mouse peritoneal macrophages (Segura et al., 1991; Calvo et al., 1994; Pozo et al., 1997a) and in rat alveolar macrophages (Sakakibara et al., 1994). The analysis of the binding data indicate two classes of binding sites, with the high-affinity site having a K_d between 0.2 and 1.05 nM depending on the cell type. It has been suggested that VIP binding sites on peritoneal macrophages could be considered a marker of the activation state (Segura et al., 1996). Thus, unstimulated or resident macrophages do not posses functional VIP binding sites on the cell surface. Studies regarding the expression of VPAC receptors in murine peritoneal macrophages and the human monocytic cell line THP-1 demonstrated that PAC₁ and VPAC₁ mRNA are constitutively expressed, whereas VPAC₂ is induced upon activation (Delgado et al., 1999e; Delgado and Ganea, 2001c). VPAC receptors and their immunological effects have been extensively studied, but most of the studies on peritoneal macrophages, including our own, have been performed using casein or thioglycolate-elicited macrophages and classical activation by exposure with LPS (Ganea and Delgado, 2001a,b). This represents a particular kind of activated macrophages, with a role as an effector cell in Th1 cellular immune responses. Activated macrophages are in fact a more heterogeneous group of cells than originally appreciated, with different physiologies and performing distinct immunological functions. Therefore, other types of activated macrophages (Goerdt and Orfanos, 1999; Anderson and Mosser, 2002) should be analyzed in terms of VPAC receptors.

Dendritic cells (DCs) are the best professional antigen-presenting cells with key roles in important regulatory circuits related with immunological disorders. There is no extensive characterization of VIP or PACAP binding sites in DCs, although it has been reported that VIP induces human DCs maturation (Delneste et al., 1999). Functional VPAC receptors coupled to adenylyl cyclase have been observed in murine Langerhans cells (Asahina et al., 1995).

Osteoclasts are multinucleated giant cells originated by fusion of mononuclear cells from hematopoietic precursors closely related to monocytes/macrophages (Teitelbaum, 2000; Boyle et al., 2003). Imbalance osteoclast activation affects bone remodeling which favors resorption, leading to diseases such as osteoporosis or rheumatoid arthritis (Rodan and Martin, 2000). Recent observations have demonstrated a role for VPAC receptors on both osteoclasts and osteoblasts (Lundberg and Lerner, 2002). We focus here on osteoclasts binding sites since osteoblasts are derived from mesenchymal progenitor cells (Harada and Rodan, 2003). Although ¹²⁵I-VIP and ¹²⁵I-PACAP38 studies demonstrated higher and more specific binding for PACAP than for VIP in mouse osteoblasts ^</