The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation

Pergamon Advances in Neuroimmunology Vo1.6, pp. 5-27, 1996

Copyright 0 1996. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain.

0960-5428/96 $32.00 PII:SO960-5428(96)00008-3

The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation

Denise L. Bellinger*$, Dianne Lortont, Sabine Brouxhon *, Suzanne Felten* and David L. Felten*

*Department of Neurobiology & Anatomy, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA

tSun Health Research Institute, P.O. Box 1278, Sun City, AZ 85372, USA

Summary

Evidence for VIP influences on immune function comes from studies demonstrating VIP-ir nerves in lymphoid organs in intimate anatomical association with elements of the immune system,

the presence of high-affinity receptors for VIP, and functional studies where VIP influences a variety of immune responses. Anatomical studies that examine the relationship between VIP- containing nerves and subpopulations of immune effector cells provide evidence for potential target cells. Additionally, the presence of VIP in cells of the immune system that also possess VIP receptors implies an autocrine function for VIP The functional significance of VIP effects on the immune system lies in its ability to help coordinate a complex array of cellular and subcellular events, including events that occur in lymphoid compartments, and in musculature and intramural blood circulation. Clearly, from the work described in

this chapter, the modulatory role of VIP in immune regulation is not well understood. The pathways through which VIP can exert an immunoregu-

latory role are complex and highly sensitive to physiological conditions, emphasizing the importance of in viva studies. Intracellular events following activation of VIP receptors also are not

$Corresponding author.

well elucidated. There is additional evidence to

suggest that some of the effects of VIP on cells of the immune system are not mediated through

binding of VIP to its receptor. Despite our lack of knowledge regarding VIP

immune regulation, the evidence is overwhelm- ing that VIP can interact directly with lym-

phocytes and accessory cells, resulting in most

cases, but not always in CAMP generation within these cells, and a subsequent cascade of intracellular events that alter effector cell function. VIP

appears to modulate maturation of specific populations of effector cells, T cell recognition, antibody production, and homing capabilities.

These effects of VIP are tissue-specific and are

probably dependent on the resident cell populations within the lymphoid tissue and the sur-

rounding microenvironment. Different microenvironments within the same lymphoid

tissue may influence the modulatory role of VIP

also. Effects of VIP on immune function may result from indirect effects on secretory cells, en-

dothelial cells, and smooth muscle cells in blood

vessels, ducts, and respiratory airways. Influ- ences of VIP on immune function also may vary depending on the presence of other signal molecules, such that VIP alone will have no effect

on a target cell by itself, but may greatly poten-

tiate or inhibit the effects of other hormones,

5

6 Advances in Neuroimmunology

transmitters, or cytokines. The activational state of target cells may influence VIP receptor expression in these cells, and therefore, may determine whether VIP can influence target cell activity.

Several reports described in this chapter also indicate that VIP contained in neural compartments is involved in the pathophysiology of several disease states in the gut and lung. Release of inflammatory mediators by cells of the immune system may destroy VIP-containing nerves in inflammatory bowel disease and in asthma. Loss of VIPergic nerves in these disease states appears to further exacerbate the inflammatory response. These studies indicate that altered VIP concentration can have significant consequences in terms of health and disease. In addition, the protective effects of VIP from tissue damage associated with inflammatory processes described in the lung also may be applicable to other pathologi- cal conditions such as rheumatoid arthritis, ana- phylaxis, and the swelling and edema seen in the brain following head trauma. While VIP degrades rapidly, synthetic VIP-like drugs may be developed that interact with VIP receptors and have similar protective effects. Synthetic VIP-like agents also may be useful in treating neuroendocrine disorders associated with dysregulation of the hypothalamic-pituitary-adrenal axis, and pituitary release of prolactin. Copyright 0 1996. Published by Elsevier Science Ltd

Introduction

For over a decade, evidence has accumulated to indicate that VIP can modulate immune function. High- and low-affinity receptors for VIP are present on cells of the immune system, and interaction of VIP with these receptors can alter a variety of immune responses both in vitro and in vivo. The question of whether VIP is available in vivo for interaction with immune effector cells that possess VIP receptors also has been examined; such evidence must include the demonstration that VIP is present in vivo, at suf- ficient concentrations to stimulate a predictable response. Several investigators have reported VIP- containing nerves in primary and secondary

lymphoid organs. Other studies also have demonstrated VIP in a variety of cells of the immune system. Whether VIP-containing neurons

in the central nervous system (CNS) can alter immune function by modulating neuroendocrine and/or autonomic outflow is not known; however, data showing that VIP modulates the hypothalamic-pituitary axis suggests that central VIPergic neurons also can influence immune function by interacting with CNS circuitry that is well documented to play a modulatory role in neural-immune interactions. In this chapter, we will (1) describe the distribution and availability of VIP for interactions with lymphoid cells in lymphoid tissue; (2) examine the evidence for VIP receptors on cells of the immune system; (3) review functional data demonstrating that VIP can modulate immune function; and (4) discuss

the clinical significance of VIP immunomodulation.

Availability and distribution of VIP in sites that modulate the immune system

VIP in regions of the CNS

VIP-containing neurons and nerve fibers are found in CNS regions that have been shown to influence the immune system. These sites include all regions of the cerebral cortex, limbic forebrain structures (stria terminalis, hippocampus, amygdala, and septum), hypothalamic areas (particularly, anterior and preoptic areas, arcuate nucleus, paraventricular nucleus (PVN), and pe- riventricular nucleus), pineal and pituitary glands (particularly anterior lobe), and brain stem autonomic and reticular regions (midbrain peri- aqueductal gray, dorsal raphe, locus coeruleus, area postrema) (reviewed by Fuxe et al., 1977; Loren et al., 1979). Neuroendocrine regulation of VIP content in corticotropin-releasing factor (CRF)-containing neurons (Mezey and Kiss, 1985) in the PVN is suggested by an increase in VIP- immunoreactive (ir) neurons in the PVN follow-

ing adrenalectomy (Mezey and Kiss, 1985), and by corticosterone-induced increases in hypothalamic VIP-mRNA levels (Gozes, 1988). Micro- injection of VIP into the PVN elevates plasma

VIP in immunomodulation 7

ACTH and corticosterone in fasted, freely moving rats (Alexander and Sanders, 1994), an effect that

is mediated through the release of CRF (Alexander

and Sanders, 1995). Hypothyroidism also increases VIP immunoreactivity in the PVN (Toni et al.,

1992). VIP in the PVN modulates prolactin secretion (Arey and Freeman, 1990; Kato et al., 1978), a neuroendocrine hormone that modulates immunological activity. VIP in the arcuate nucleus inhibits somatostatin (SOM) release (Epelbaum et al., 1979; Shimatsu et al., 1982). an important regulator of anterior pituitary function. In the pineal gland, VIP stimulates the activity of serotonin-N-acetyltransferase, an enzyme involved in the synthesis of melatonin (Kaneko et al., 1980; Yuwiler, 1983). Given the well documented role of the glucocorticoids, thyroid hormone, melatonin, and prolactin in the modulation of immune function, it is tempting to speculate that VIP in hypothalamic neurons (as well as other CNS regions) also has an immunomodulatory role; however, we are unaware of studies examining this possibility directly. Intracerebroventricular injection, but not peripheral injection, of li- popolysaccharide has been shown to result in a significant increase in VIP content in spleen from rats 3 hours after administration (Wan et al., 1993) indicating that altered neuroendocrine and/or autonomic outflow affects peripheral VIP content in lymphoid organs.

VIPergic nerves in lymphoid organs

Immunohistochemical methods have revealed VIP-containing nerves that distribute to lymphoid organs. These VIPergic nerves establish an anatomical link between the brain and the immune system for receiving signals from the immune system, and for sending neural signals that influence lymphoid cells. VIP-positive (‘) nerves have been demonstrated in both primary (thymus, bursa fabricii of birds) and secondary (spleen, lymph nodes, mucosal-associated lymphoid tissue) lymphoid organs. VIP-ir fibers in bone marrow have not been reported thus far. In most cases, the origin of these nerves is not well known; they may come from several sources, including

sympathetic ganglia, postganglionic parasympathetic neurons, dorsal root ganglia, and intrinsic

neurons of the gut.

Bursa fabricius. In the bursa fabricii of the

chicken, a primary lymphoid organ for B lymphocyte differentiation, VIP-ir nerves distribute throughout all bursal compartments, except for the medulla of the follicles (Zentel and Weihe, 1991). VIP fibers course in the muscle layers of the bursal wall, the connective tissue of the bursal plicae, the interfollicular septae, the subepithe- lial regions, and the follicular cortex which is a site for the later stages of B lymphocyte maturation. In the follicular cortex, VIPergic nerves reside adjacent to B lymphocytes. VIP-ir fibers also closely associate with macrophages under the bursal epithelium and around the bursal follicle. In these bursal nerves, VIP coexists with galanin and tachykinin. The origin of these fibers is not clear, however, VIP+ neuronal perikarya present in intrabursal microganglia are the most likely source of VIP+ nerves in the chicken bursa fabricius. Zentel and Weihe (1991) also have suggested that VIP fibers may be sensory, since capsaicin, a neurotoxin specific for small diameter sensory neurons, can release VIP from the gut (the bursa fabricius is the most distal part of the gut in birds). However, this could result secondar- ily to the effect of capsaicin on sensory neurons that innervate the gut. VIP+ nerves often are associated intimately with arteries forming perivas- cular plexuses, and frequently reach into the media of large vessels, closely resembling the pattern typically found for sympathetic innervation. However, the lack of tyrosine hydroxylase-ir nerves in the bursa suggests only a meager input from sympathetic outflow, and also suggests that VIP+ fibers probably do not originate from sympathetic ganglia.

Thymus. In the thymus, VIP-ir nerves are most abundant in the capsule and extend into the interlobular septa (Bellinger et al., 1990, 1992). Varicose VIP-ir nerves in the capsular/interlobular septal system form linear arrays that course in close association with mast cells. Fine VIP” fibers


exit this plexus to enter the thymic cortex (Felten et al., 1985; Bellinger et al., 1992). Occasion- ally, VIP-ir fibers are found in the medulla. VIP

nerves in the thymus do not derive from sympathetic ganglia that provide noradrenergic innervation of the thymus, because surgical gang- lionectomy fails to deplete VIP content in the thymus (Bellinger et al., unpublished observation).

Spleen. In the spleen at the hilus, VIP-ir nerves enter the parenchyma of the organ along with large blood vessels (Lundberg et al., 1985). In the splenic red pulp, VIP+ fibers are prominent along the venous/trabecular system and course as free fibers in the parenchyma. In the splenic white pulp, VIPergic nerves are present along the central arterioles and in the periarteriolar lymphatic sheath (PALS) among T lymphocytes. The density of VIPergic nerves in this compartment of the spleen appears to be much greater in Long- Evans hooded rats than in Fischer 344 rats, suggesting that strain differences may occur (Bellinger et al., unpublished observation). The origin of VIP-containing nerves in spleen is not clear. Surgical removal of the superior mesenteric ganglion, which results in greater than 95% deple- tion of norepinephrine content and virtual loss of noradrenergic sympathetic nerves in the spleen, does not alter splenic VIP content (Bellinger et al., unpublished observation). These data suggest that VIPergic neurons in the ganglion contribute little, if any, VIP+ nerve fibers to the spleen. Consistent with these findings, Chevendra and Weaver (1992) have found that less than 1% of the mesenteric neurons that innervate the spleen were immunoreactive for VIP. VIP nerves in the spleen may come from dorsal root ganglion, from vagal input, or from intrinsic neurons of the gut.

Lymph nodes. VIP innervation of mesenteric lymph nodes is relatively sparse, even though VIP concentration, as measured with radioimmu- noassay, was higher in these lymphoid organs than in thymus and spleen (Bellinger et al., 1992). The high VIP content is mesenteric lymph nodes is probably from cells of the immune system that

synthesize VIP In mesenteric lymph nodes, VIP+ nerves are present beneath the capsule (Bell- inger et al., 1992) particularly near the hilus, and

along the vasculature in internodal regions of the cortex (Fink and Weihe, 1988; Bellinger et al.,

1992). Occasionally, VIP fibers exit the vascular

plexus to extend among T lymphocytes in the adjacent cortex. VIPergic nerves also are sporadi- cally seen along the medullary cords in the medulla (Bellinger et al., 1992). No VIP+ nerves have been found thus far in lymph nodules. No VIP immunoreactivity in nerves has been detected

in popliteal lymph nodes of the Fischer 344 rat, indicating that VIP innervation varies in lymph nodes depending on their location in the body.

Mucosal-associated lymphoid tissue. The mucosal immune system (mucosa-associated lymphoid tissue or MALT) includes both gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) in the lung. MALT can be divided into three discrete compartments. There are well-defined lymphoid aggregates that form the appendix, Peyer’s patches, and tonsils. These lymphoid compartments have T and B lymphocyte zones, contain antigen-presenting cells (dendritic cells), and prominent germinal centers after immunization (Craig and Cebra, 1971; Mayrhoffer, 1984). Secondly, a diffuse population of immune effector cells is present in the lamina propria, including plasma cells, cyto- toxic T cells, macrophages, eosinophils and mast cells. The majority of immune effector cells are located within this compartment of mucosa. And lastly, a population of leukocytes reside in the epithelium that line the gastrointestinal and respiratory tracts.

VIP in nerve fibers occurs throughout the gastrointestinal tract in a wide variety of mammals (see Fumess and Costa, 1980 for a review). Only recently has attention focused on the relationship of these nerves to cells of the immune system in the gut. VIP-ir nerves have been found in Peyer’s patches of the mouse (Ottaway et al.,

1987) and cat (Ichikawa et al., 1994). These fibers course predominantly along lymphatics and small caliber blood vessels called postcapillary venules


or high endothelial venules, and infrequently in

the lymphoid follicle of the patches. Based on

functional studies (described below), Ottaway

(1984) has suggested that VIP nerves adjacent to postcapillary venules regulate lymphocyte trafficking in this region. An extensive nerve plexus which contains various neuropeptide transmitters, including VIP, substance P (SP), calcitonin gene-related peptide, cholecystokinin, and SOM, is present in the intestinal lamina propria (Ekblad ef al., 1987; Probert et al., 1981; Cooke, 1986). VIPergic nerves course throughout the lamina propria, distributing to the crypts, into the mus- cularis mucosae, into the core of the villi, and around the margins of Peyer’s patches (Ichikawa et al., 1994; Ottaway et al., 1987; Furness and Costa, 1980; Schultzberg et al., 1980). Some VIP+ enteric nerves traverse the lamina propria to enter into the epithelium, where they are thought to regulate epithelial and enteroendocrine function (Stead et al., 1987). The anatomical relationship of VIP-containing nerves and immune effector cells in the lamina propria and within the epithelium needs to be examined. The source of gut innervation is believed to derive largely from intrinsic enteric VIP-containing neurons in the myenteric and submucous ganglionic plexuses; however, extrinsic sources also may exist, including parasympathetic autonomic fibers and sensory fibers (Furness and Costa, 1980). Few VIP- containing fibers in the intestines derive from postganglionic sympathetic neurons (Chevendra and Weaver, 199 1). VIP-containing nerves also have been described in the rat mesentery where there is a close anatomical relationship with serotonin+ mast cells (Crivellato et al., 1991). Ad- ditionally, VIP is present in neuroendocrine cells in the epithelial lining of the gut (Polak er al., 1974), and could provide a source for VIP to interact with intraepithelial leukocytes (IEL) and immune effector cells of the lamina propria.

VIP present in nerves that distribute to the gut may be important clinically in patients with inflammatory bowel disease (Koch ef al., 1987, 1990) and inflammation of the intestines associated with infection (Palmer and Greenwood, 1993) where VIP nerve density and VIP content in the

bowel is reduced. It has been suggested that libera-

tion of neurotoxic substances from activated

granulocytes, such as eosinophils and neutrophils, is responsible for diminished VIP content during an inflammatory response.

VIP-ir nerves have been reported in the tracheo- bronchial tree from dogs, cats, rats, and humans (Dey et al., 1981; Said, 1987; Said, 1988; Dey and Said, 1985; Uddman and Sundler, 1979; Laitinen et al., 1985; Nohr and Weihe, 1991). VIP-containing nerves are present in the walls of extra- and intrapulmonary bronchi and bron- chioles (Dey et al., 1981; Uddman et al., 1978; Uddman and Sundler, 1979), with abundant distribution to the smooth muscle layer and around submucosal mucous and serous glands of airways. These nerves frequently encircle parts of the acinar units, and occasionally course within the epithelial layer of the gland. No VIP+ nerves have been found in the airway epithelium. Bronchial arteries also receive a rich supply of VIP-ir nerves. These fibers course in the walls of pulmonary and bronchial blood vessels between the media and adventitia. Like the gastrointestinal tract, an extensive network of VIP+ nerves course in the lamina propria. Many of these fibers form close associations with the epithelial basement membrane. The anatomical relationship of VIP- containing nerves in the lamina propria with immune effector cells has not been investigated. VIP in pulmonary and bronchial nerves colocal- izes with acetylcholine (Laitinen et al., 1985) and a number of neuropeptides, including peptide histidine isoleucine (PHI) (and peptide histidine- methionine (PHM) in humans) (Lundberg et al., 1984a,b), SP (Dey et al., 1988) vasopressin, and certain opioid peptides (Said, 1988). Microgan- glia, consisting of multiple neuronal cell bodies that display VIP immunoreactivity, are present in the walls of the bronchi (Dey et al., 1981), and intrinsically innervate the lung. VIP-ir nerves in the lung probably arise from the vagus nerve as well (Lundberg et al., 1978, 1979).

A loss of VIP immunoreactivity in pulmonary nerves is reported to occur in patients with asthma (Ollerenshaw et al., 1989).Based on the pattern of VIP innervation in the lung, the functions of


VIP, and the manifestation of the disease, Said (1988) has suggested that VIP also may play a role in cystic fibrosis. Functional studies where VIP is inhaled (Said et al., 1982) suggest that diminished VIP innervation of the lung may diminish VIP-mediated bronchodilation, and change pulmonary blood flow and mucus secretion in asthmatic patients. Loss of VIP nerves in the lung may result from a loss of nerves containing VIP, a reduction in the synthesis of the peptide, or increased metabolism of VIP in these nerves.

In cats, rabbits, pigs, and guinea pigs, the nasal mucosa also receives an abundant supply of VIP- containing nerves (Uddman et al., 1978; Lacroix et al., 1992). These nerves are found surrounding nasal glands, forming dense plexuses around the acini (Uddman et al., 1978), and associate with small blood vessels and venous sinusoids (Uddman et al., 1978; Lacroix et al., 1992). These nerves, which colocalize with SOM and neuropeptide Y, are presumably of parasympathetic origin (Lacroix et al., 1992).

VIP in cells of the immune system

VIP immunoreactivity in cells of the immune system was first reported in mast cells isolated from the small intestines, lung, and peritoneal cavity (Cutz et al., 1978). Additionally, Cutz et al. (1978) reported co-release of small amounts of W (1 rig/lo6 cells) and histamine (600 ng/106) in peritoneal mast cells stimulated with A23187, a calcium ionophore. Immunoreactivity for VIP in mast cells has not been further characterized neurochemically. Since this report, VIP immunoreactivity has been demonstrated in several types of leukocytes; however, reports in the literature are controversial. O’Dorisio et al. (1980) found VIP immunoreactivity in polymorphonuclear cells, but not in mononuclear cells. VIP immunoreactivity on the order of 1.1 ng/lO* cells can be extracted from human neutrophils with their antisera for VIP (O’Dorisio et al., 1980). Further, they suggest that VIP synthesis may be altered in polymot-phonuclear cells under immu- nopathological conditions, providing a means for differential diagnosis of certain kinds of leuke-

mias. Other investigators have been unable to

confirm these findings. Measurements of VIP im-

munoreactivity by Murphy et al. (198 1) in several

different types of leukemic cells are substantially

lower than in those by O’Dorisio et al. (1980), and they have not found a correlation between

VIP levels and leukemic disease states. Similarly,

Madden et al. (1981) demonstrate much higher

VIP levels in mononuclear cells than in poly-

morphonuclear cells. Lygren and colleagues (1984)

have confirmed the presence of VIP immuno-

reactivity in mononuclear, as well as polymor-

phonuclear cells obtained from humans and pigs.

They also report significantly higher levels of immunoreactivity in mononuclear fractions of

blood leukocytes, compared with that measured

in polymorphonuclear cells. Several explana- tions have been put forth by O’Dorisio and cow-

orkers (198 1 b) to account for these discrepancies,

including differences in antisera recognition of immunological determinants on the VIP molecule (and VIP-like peptide), differences in experi-

mental techniques used for the isolation of leu-

kocyte fractions, possible contamination of fractions by platelets, degranulation of leuko-

cytes, and differences in recovery of VIP during extraction. Collectively, these studies suggest that VIP is present in both mononuclear and poly-

morphonuclear cells in human peripheral blood. Future studies that examine the presence of VIP mRNA in subpopulations of human blood leu-

kocytes may help to clarify which immune

effector cells contain VIP Aliakbari and coworkers (1987) report VIP im-

munoreactivity (72 fmol/lO’ cells) in human eosinophil fractions. These investigators have

chromatographically demonstrated that the extracted immunoreactive VIP is identical to VIP from neuroendocrine tissues. In support of their findings, VIP-ir eosinophils have been demonstrated in granulomatous lesions induced by infection with schistosomiasis mansoni in CBA mice (Weinstock and Blum, 1990). Immunore-

active VIP is released from rat basophilic leukemia (RBL) cells, a mast cell-like cultured line (Goetzl et al., 1988). In this cell line, they have found a


mixture of VIP-like peptides that differ structur-

ally from VIP (Goetzl et al., 1988), the

predominant form being VIP,,_,, with the carboxyl-terminal asparagine as a free acid. Further, RBL cells appear to be incapable of synthesizing VIP,_,,. It is not clear whether

multiple forms of VIP immunoreactive molecules are present in normal immune effector cells. If this turns out to be the case, differential expression of VIP in specific immune effector cells may add to the complexity of VIP-immune effector

cell interaction. Subpopulations of immune effector cells may contain different forms of VIP. Dif- ferent forms of VIP molecules may preferentially bind to VIP receptor subtypes, or upon binding with its VIP receptor class may have different functional outcomes.

Immunoreactive VIP also is present in cell suspensions from primary and secondary lymphoid organs. Extracts from normal human bone marrow cells, consisting of immature erythroid, myeloid and lymphoid cells, contain approximately 0.67 ng/108 cells (Fisher et al., 1982). Examination of bone marrow extracts from patients with acute lymphocytic leukemia or chronic and acute my- elogenous leukemia indicate that the majority of VIP in bone marrow extracts is present in myeloid cells. Expressed as pmol immunoreactivity/lO’ cells, VIP content in cell suspensions from murine spleen (2.3 + 0.3) and thymus (1.8 * 0.7) is about two-fold higher than that seen in the same organs from rats (0.9 f 0.1 and 0.7 + 0.2, respectively) (Gomariz et al., 1992). VIP content in axillary lymph nodes from these two species is about the same (1.5 f 0.6 and 1.3 & 0.2, respectively) (Gomariz et al., 1992). In this study, immunoreactive VIP eluted in three peaks, one corresponding to the native peptide and two larger molecules, presumably precursors of VIP.

Using in situ hybridization for VIP mRNA and immunocytochemistry for VIP, Gomariz and colleagues (1992) also have described the distribution of VIP immunoreactivity in lymphoid cells in thymus, spleen and axillary lymph nodes from mice and rats. In thymus, VIP-ir, lymphoid cells are found predominantly in the deep cortex, and scattered in medulla. In spleen, VIP+ lymphoid

cells are scattered in the outer regions of the periarteriolar lymphatic sheath of the white pulp. In

axillary lymph nodes (Gomariz et al., 1992), interfollicular areas, deep cortex, and germinal centers of the lymphoid follicles contain lymphoid

cells immunoreactive for VIP. In our search for VIP-containing nerves in primary and secondary lymphoid organs, we also have observed cytoplasmic immunoreactive staining for VIP in cells that have morphological features of eosinophils, masts, dendritic cells, and macrophages

(Bellinger et al., 1992) in the same sites as described by Gomariz and colleagues (1992). The presence of VIP mRNA in thymocytes, and in T and B lymphocytes from spleen and lymph nodes, has been conlirmed using reverse transcrip- tion and polymerase chain reaction (rtPCR) (Gomariz et al., 1994). Demonstration of VIP mRNA in lymphoid organs with in situ hybridization reveals overlapping distribution of mRNA with the VIP peptide (Gomariz et d., 1993); however, fewer VIP mRNA+ cells are detected with this method. It is possible that some of the VIP-ir cells result from uptake of the neuropeptide into lymphoid cells following release by VIPergic nerves and lymphoid cells. Activation of the VIP gene also may be regulated such that the gene is only ‘turned on’ during certain cellular functions (i.e. cell differentiation and activation). The precise distribution of VIP mRNA in deep thymic cortex suggests that thymocytes express VIP during the later stages of differentiation. In the spleen, the distribution of VIP mRNA- containing lymphoid cells in the outer PALS near the marginal zone, a site of antigen processing, suggests that VIP released from this compartment may modulate the immune response.

VIP receptors

General characteristics of VIP receptors

VIP-containing nerves can release at least four peptides that can cross-react with VIP receptors: VIP, PHI 1_27-NH2, PHI-Gly, and peptide histidine valine (PHV),,, (Fahrenkrug et al., 1985,


1989; Cauvin et al., 1989; Yiangou et al., 1985). Additionally, studies by Goetzl and colleagues

(1988) suggest that cells of the immune system may be capable of releasing multiple VIP-related peptides that can interact with VIP receptors. The

physiological response subsequent to binding of these peptide molecules may differ, depending on VIP receptor heterogeneity. Thus far, it has been difficult to clearly demonstrate functional heterogeneity using several VIP analogues, or when comparing VIP, PHI, PHI-Gly and PHV,,, binding to VIP receptors from a variety of rat tissues (Inoue et al., 1985; Turner and Bylund, 1987; Robberecht et al., 1982; Dehaye et al., 1986; Laburthe et al., 1983, 1986). Additionally, the media conditions in which binding assays are performed, and the presence of GTP in the medium can alter both VIP receptor affinity and selectivity (Robberecht et al., 1990; Paul, 1989). Robberecht and coworkers (Robberecht et al.,

1989~) have found that pretreatment of SUP-T1 lymphoblasts with cholera toxin alters VIP receptor selectivity. The plasticity of these receptors has been attributed to a change in receptor conformation after interaction with altered guanine nucleotide-binding stimulatory proteins (Gs). Although, VIP receptor subtypes have not yet been classified, a variety of evidence suggests that multiple forms exist. As more tools are developed to evaluate VIP receptors in a variety of mammalian tissues, such as methods for receptor purification, oligonucleotide probes to screen genomic libraries, and development of highly selective VIP antagonists and agonists, it is likely that we will discover multiple VIP receptor subclasses that are structurally, and pharmaco- logically distinct. Currently, only a few VIP antagonists are available, and these molecules show low affinity and may act as partial agonists in some systems (Thompson et al., 1988; Ottaway, 1988; Robberecht et al., 1986b).

Both high- and low-affmity VIP receptors have been described in a variety of tissues from rats, guinea pigs and humans (Provow and Velicelebi, 1987; Robichon and Marie, 1987; Robberecht et al., 1988a, 1990). Both receptor types display similar selectivity, differing only by their affinity

for VIP. They can be readily distinguished in binding studies using radiolabeled-helodermin and

(His’,D-Ala’) growth hormone releasing factor

(GRF),_,,NH2 which bind selectively to high- affinity VIP receptors (Robberecht et al., 1984).

On the other hand, VIP does not distinguish between the two receptor types (Robberecht et

al., 1986a, 1988b). Most VIP receptors described thus far have a greater specificity for VIP than for the homologous peptides including helodermin, GHRF, PHI and secretin, with an order of potency VIP>helodermin>GHRF>PHI>secretin.

Using SDS-PAGE after covalent cross-linking with radiolabeled VIP, two VIP receptor molecules with molecular weights of 65-75 kDa (pancreas and intestines) and 45-50 kDa (liver, lung, and brain) have been isolated in a variety of rat tissues (Inoue et al., 1986; Turner and Bylund, 1987; Robberecht et al., 1982; Dehaye et al., 1986; Laburthe et al., 1983, 1986); however, crosslink- ing studies must be interpreted with caution as the structural heterogeneity may actually reflect a variable degree of glycosylation and/or partial proteolysis (reviewed by Robberecht et al., 1990).

Several laboratories have begun to sequence VIP receptors. cDNA clones of the VIP receptor have been isolated from a rat lung cDNA library (Ishihara et al., 1992), and from human HT29 intestinal epithelial cells, lung and kidney (Sreed- haran et al., 1993). The cloned rat VIP receptor consists of 459 amino acids, has a molecular weight of 49 kDa, and contains seven transmem- brane segments, whereas the cloned human VIP receptor is 457 amino acids in length, and has a molecular weight of 52 kDa. The VIP receptor is expressed as a prominent 5.5 kb transcript in rat lung, with moderate expression in liver and intestines, and weak expression in thymus and brain (Ishihara et al., 1992). A 2.8 kb human VIP receptor transcript is expressed in human lung, HT29 cells and Raji cells, with weaker expression in the brain, heart, liver, kidney and placenta (Sreedharan et al., 1993). Transfection of the cDNA clones into COS-7 cells (Sreedharan et al., 1993) or murine COP cells (Ishihara et al., 1992) results in specific binding of VIP (K. of 0.8 nM, and KD of 0.17 and 2 1 r&I, respectively).


Interaction of VIP with VIP receptors on trans- fected cells results in an increase in intracellular CAMP.

VIP receptors are closely associated with Gs- adenylate cyclase complex (Couvineau et al., 1986), and interaction with VIP stimulates adenylate cyclase activity in lymphoid cells (Beed et al., 1983; O’Dorisio et al., 1981a). VIP synergizes the effects of guanine nucleotide,

5guanylyl imidodiphosphate (GppNHp) and forskolin. These agents promote the interaction of Gs with the catalytic subunit of adenylate cyclase (Gilman, 1984; Seaman et al., 1981) and subsequent cascade effects mediated via a CAMP- dependent protein kinase network (Guerrero et

al., 1984; O’Dorisio and Campolito, 1989).

VIP receptors on immune effector cells

A variety of human and rodent lymphoid cell lines have been used to demonstrate specific binding sites for VIP. Most of the VIP receptors on these cell lines have a selectivity and/or a molecular weight (based on cross-linking studies) comparable to that encountered in normal tissue preparations. However, new affinity profiles have been encountered that suggest either a mutation of the receptor or expression of a receptor subtype that is, as yet, undetectable or barely detectable in normal tissues (reviewed by Robberecht et al.,

1990). VIP binding sites have been characterized in the cell membrane of Molt-4b (Beed et

al., 1983; O’Dorisio et al., 198.5), SUPT-1 (Rob- berecht et al., 1989a), and Jurkat (Finch et al.,

1989) T cell lines, and Raji (Robichon et al.,

1993) Dakiki (O’Dorisio et al., 1989), Nalm6 (O’Dorisio et al., 1989), U266 (Finch et al., 1989), and SKW 6.4 (Cheng et al., 1993) B cell lines. These binding studies suggest that lymphocytes of both B and T cell lineage can express VIP receptors. The high density of receptors on the Molt-4b cell line (15,000 sites/cell) has made this a useful line for characterizing VIP receptors (O’Dorisio et al., 1985). VIP receptors on Molt4b bind VIP with similar characteristics to that seen in normal tissue. In contrast, the human lymphoblastic SUPT-1 T cell line (Robberecht et al.,

1988c, 1989a,b) and the THP-1 monocyte/

macrophage cell line (Gespach et al., 1989) bear

unusual VIP receptors in that the high-affinity re-

ceptors recognize helodetmin with higher affinity

than VIP and PHI. This receptor phenotype also

is expressed on a murine T lymphoblastic cell

line induced by thymic irradiation (Abello et al.,

1989).

Several studies have examined VIP interac-

tion with VIP receptors on lymphoid cells in human blood (Guerrero et al., 1981; Ottaway et

al., 1983; Calvo et al., 1986a; Danek et al., 1983; Roberts et al., 1991; Wiik et al., 1985). Wiik et

al. (1985) report binding on monocytes and in

T cell-depleted populations, but not in T cell-

enriched fractions. This is in contrast with other

studies showing that the predominant VIP binding occurs in lymphocyte-enriched fractions (Danek

et al., 1983; Calvo et al., 1986a; Roberts et al.,

1991). Further fractionation of mononuclear cell suspensions suggests that VIP binding sites are

present on B lymphocytes and/or cells of K/natural killer (NK) system from mononuclear cell suspensions (Calvo et al., 1986a). O’Dorisio and col-

leagues (198 1 a) have been unable to demonstrate VIP receptors or VIP-induced stimulation of adenylate cyclase in suspensions of human blood

monocytes, neutrophils and erythrocytes. Guerrero et al. (1981) report both high-(Kr,s of 0.24 nM) and low-affinity (Kos of 80 nM) VIP binding sites

in human circulating mononuclear leukocytes.

Danek et al. (1983) and Ottaway et ul. (1983) demonstrate only a single class of VIP recep-

tors (K,=O.47 nM and 0.24 nM, respectively) in

human monocyte-depleted, lymphocyte-enriched cell suspensions. VIP binds to human blood lym-

phocytes with CD8+ (32%), CD4+ (23%) and/or CD2+ phenotypes (Ottaway et al., 1990; Roberts et al., 1991). Discrepancies in VIP receptor

distribution on blood leukocytes and their binding characteristics may result from differences in the methods used to isolate cell suspensions, and indicate that further studies are required to characterize VIP receptors on subpopulations of

lymphoid and accessory cells of the immune system.


Rodent lymphocytes from primary and secondary lymphoid tissue (Ottaway and Greenberg,

1984; Calvo et al., 1986b; Weinstock et al., 1991) possess a single class of high-affinity receptor sites for VIP (Ko of 0.10-0.24 nM). Binding of VIP on murine lymphocytes is rapid, saturable, and reversible. Regional differences occur in binding capabilities of specific subsets of T and B lymphocytes. T cells generally have a much higher binding capacity for VIP than do non-T cells. The density of VIP binding sites on T cells

from mesenteric and superior cervical lymph nodes, spleen, and Peyer’s patches appears to be similar (about 2600 sites per cell). Thymocytes have few VIP binding sites (150 sites per cell), which suggests that expression of these receptors occurs as T cells differentiate and mature. Radioligand binding studies and autoradiographic studies by Lacey et al. (1991) demonstrate high- (1.12 nM and 0.459 nM, respectively) and low- (88.5 nM and 70.8 nM, respectively) affinity- specific binding sites in both the avian thymus and bursa. VIP receptors are expressed in the medulla and interlobular septa of the thymus, and in the bursa they are present in interfollicular regions, the epithelial border of the plicae, and the muscular layer surrounding the organ.

No VIP receptor mRNA expression in spleen, and weak VIP receptor mRNA expression in thymus have been demonstrated using Northern hyb-idization analysis (Ishihara et al., 1992). However, using a recently cloned cDNA probe that codes for VIP receptor protein isolated in rat lung, Gomariz and colleagues (1994) have demonstrated VIP receptor mRNA in rat thymocytes, splenocytes, and lymph node cells with rtPCR. From these reports it appears that expression of VIP receptor mRNA and VIP receptor protein expression of the surface of lymphoid cells in primary and secondary lymphoid organs does not correlate well, and suggests that transcrip- tion of the VIP receptor mRNA is regulated in lymphoid cells. In mesenteric lymph nodes from dogs, VIP receptors distribute to the internodu- lar and T cell regions, to the medullary cords, and germinal centers (Popper et al., 1988).

T lymphocytes from the lamina propria of the

small intestines from mice also bear VIP receptors of a single class that binds VIP with intermediate affinity (K,=9.08 nM) (Blum et al.,

1992). These receptors are distinct from VIP receptors expressed on murine intestinal epithelial cells (K,=41.7 nM). VIP also specifically binds to VIP receptors on lymphocyte membranes from the lamina propria of human colon (Pallone et al., 1990), but not human IEL, which are mainly CD8+ T cells (sensitivity of their assay was lO-15%) (Roberts et al., 1991). Roberts and coworkers (1991) have suggested that high concentrations of VIP in the gut may downregulate VIP receptor expression in IEL, or alternatively, that the lack of VIP receptor expression in IEL may result from continual internalization in situ in IEL, whole cell preparations. Collectively, these findings suggest that VIP receptor expression on subsets of lymphocytes (i.e., CD4+, CD8+, etc) differ depending on their microenvironment, and that VIP may interact selectively with epithelial cells and T cell subsets.

Weinstock et al. (1991) have demonstrated VIP receptor bearing T lymphocytes (Ko=O. 1 nM) in granulomas in liver and intestines in mice infected with Schistosorniasis munsoni. Stimulation of granuloma T cells with VIP increases CAMP production in vitro, indicating that VIP alters CAMP metabolism in these cells through a receptor-coupled mechanism similar to that observed in other tissues. They have proposed that interaction of VIP with receptors on T lymphocytes dampens lymphocyte responsiveness within the granuloma and helps to modulate the intensity of the granulomatous inflammation. Recently, VIP receptors have been reported on murine (Calvo et al., 1994) and rat (Segura et al., 1991) peritoneal macrophages.

Immunomodulatory effects of VIP

Migration and chemotuxis

Several studies indicate that VIP can influence the regional distribution and trafficking of lymphocytes, particularly in GALT. Using a sealed capillary migration test, Bondesson and colleagues (1991) have demonstrated that VIP at


concentrations ranging from 10m7 to 10m9 inhibits,

and at lo-” to lo-i4 stimulates, mononuclear leu-

kocyte migration. In mice, transfer of labeled-

syngeneic lymphocytes after incubation with VIP

in vitro results in downregulation of VIP recep-

tors on treated cells and a decrease in localiza-

tion of these treated cells in mesenteric lymph

nodes and Peyer’s patches, while migration of

VIP-treated cells to other lymphoid or nonlym-

phoid compartments is not altered (Ottaway, 1984,

1985). Moore and colleagues (1988) attribute VIP-

induced changes in lymphocyte migration to

increased CAMP production. They find that direct infusion of VIP into afferent lymphatics of pop-

liteal lymph nodes in sheep, increases intracel-

lular CAMP in lymphocytes, and reduces egress

of lymphocytes from the nodes. Conversely, agents that increase cGMP, i.e. SP, have an opposite effect on lymphocyte egress from the lymph nodes.

Further, VIP alters the composition of the lym-

phocyte pool in lymph flow through the pop-

liteal lymph nodes (Moore et al., 1988), particularly increasing the egress of CD4+ T cells,

consistent with decreased homing of CD4+ VIP- treated cells to GALT (Ottaway et al., 1990;

Ohkubo et al., 1994). Similarly, continuous infusion of VIP into the

superior mesenteric artery of the rat significantly reduces lymphocyte migration through intestinal (especially T helper cells) and mesenteric lymph

(particularly T suppressor cells) without changing

lymph flow (Ohkubo et al., 1994). Histochemi- cal staining in GALT of rats infused with VIP

reveals a decrease in pan T cells in lamina propria, especially T helper cells, and a selective decrease in IgA-containing cells in lamina propria. A selec-

tive decrease in CD4+ lymphocytes in the intestines is consistent with the findings of Ottaway (1984, 1985), Moore (1984) and Moore

et al. (1988); the increased homing of CD8+ into mesenteric lymph nodes is not, and may result from species differences or route of VIP administration. The lack of change in systemic blood pressure or intestinal lymph flow between control and VIP-treated animals suggests no drastic change in the hemodynamics of the intestine by

VIP and supports a direct effect on lymphocytes

(Ohkubo et al., 1994). VIP modulation of the regional distribution and

recirculation of lymphocyte subpopulations in particular lymphoid compartments has important implications during an immune response. VIP’ nerves associate with postcapillary venules in mesenteric lymph nodes and Peyer’s patches, through which lymphocyte trafficking proceeds, lending support for an in vivo role for VIP modulation of lymphocyte traflicking in these lymphoid organs (Ottaway et al., 1987). In popliteal lymph nodes, we have not been able to demonstrate VIP containing nerves, raising the question as to whether VIP influences lymphocyte migration through this lymphoid organ under normal physiological conditions. Of course, VIP could be released from non- neural sources in the popliteal lymph nodes, as well as in other lymphoid organs. A selective decrease in the number of CD4+ IgA+ cells in GALT may diminish T helper cell-driven inhibition of suppressor T cell activity, promoting reduced antibody response to presentation of antigen (Ottaway et al., 1987).

That VIP exerts a direct effect on lymphocytes, influencing their ability to home to particular organs, is also indicated by the work of Johnston et al. (1994) and Robichon et al. (1993). Johnston and coworkers (1994) find that VIP stimulates in

vitro chemotaxis of T lymphocytes horn both CD4+ and CD8+ subsets (as well as monocytes but not neutrophils). Chemotactic effects are more potent on unstimulated T cells compared with anti-CD3- activated cells resulting from reduced VIP receptor expression on stimulated T cells. Pre-incubation

of unstimulated T cells with VIP also increases cell adhesion to intercellular adhesion molecule (ICAM) and vascular (VCAM) integrins, and significantly increases unstimulated T cell adhesion to fibronectin, an extracellular matrix protein. Similarly, Robichon et al. (1993) recently have demonstrated that VIP enhances the aggregation of Raji cells, a response dependent on CAMP generation and lymphocyte function-associated adhesive protein (LFA)-1 and ICAM-1. Col- lectively, these studies suggest that VIP influences the regional accumulation of specific


subpopulations of lymphoid cells through upregu- lation of adhesion molecules important for tran- sepithelial migration of lymphocytes and for intercellular adhesion within lymphoid tissue. Diminished responsiveness to VIP by activated

lymphocytes is suggested by Johnston et al. (1994); during an immune response this may lead to decreased T cell infiltration into sites of VIP

production. The findings of increased binding of VIP-treated cells to fibronectin may indicate VIP modulation of lymphocyte infiltration into inflamed tissue follow extravasation (Johnston et al., 1994).

Mitogen and antigen-induced proliferation

There is general agreement that VIP (in concentrations ranging from 10.’ to lo-” M) inhibits mitogen-induced proliferative responses and decreases interleukin (IL)-2 production in murine T lymphocytes from a variety of tissues (spleen, Peyer’s patches, subcutaneous lymph nodes, and mesenteric lymph nodes) (Kroc et al., 1986a,b; Ottaway and Greenberg, 1984; Stanisz et al., 1986, 1988; Scicchitano et al., 1988; Metwall et al.,

1993; Yiangou et al., 1990; Boudard and Bastide, 1991). Likewise, VIP inhibits mitogen-induced (concanavalin A (Con A)) proliferation of splenocytes from rabbits (Peuriere et al., 1991). Peuriere and coworkers (1991) also have examined the effect of long-term preincubation of splenocytes with VIP with dexamethasone presented at the time of mitogen stimulation. An 18 hour pre- incubation with low concentrations of VIP fails to modulate the mitogenic response, but induces a ‘loss in lymphocyte sensitivity’ to dexamethasone. The presence of high concentrations of VIP added 18 hours before the mitogen, results in increased proliferation, and this effect is blocked by the addition of dexamethasone to the culture.

In contrast, the effects of VIP on mitogen- stimulated proliferation in human peripheral blood lymphocytes, and in cell lines, have not been consistent. According to Roberts et al. (1991), VIP (lo-s-lo-i2 M) has no effect on Con A-induced proliferation of either human peripheral blood lymphocytes or IEL. Nordlind and Mutt (1986) report inhibition of mercuric chloride-

induced proliferative response in peripheral blood mononuclear cells; however, this effect is realized only when micromolar concentrations of the peptide are added to the cultures. Ottaway (1991) finds highly variable effects of VIP on Con A- and phytohemagglutinin-driven proliferation of

peripheral blood monocytes, even in repetitive assays using the same donor tissue. Drew and Shearman (1985) show enhancing effects of VIP on mitogen-induced B cell proliferation in circulating human lymphocytes. Two studies have examined the effects of VIP on mitogen-induced proliferation of human lymphocytes obtained from sites other than blood. In human mononuclear cells from the lamina propria of the colon, VIP (10~13-10~‘1 M) enhances Con A-induced thy- midine uptake (Nio et al., 1993). Similarly, in purified B cells obtained by tonsillectomies for chronic tonsillitis from nonatopic children (Kimata et al., 1992), VIP increases mitogen-stimulated proliferation.

In Molt-4b lymphoblasts, VIP inhibits proliferation in a dose-dependent manner (O’Dorisio et al., 1985). Further, VIP synergizes the inhibitory effect of cholecystokinin on Molt-4b proliferation, but does not block the SP-induced stimulatory effects on Molt-4b cell proliferation (Tang et al., 1992). Conversely, a modest augmentation of Con A-stimulated IL-2 production in the A049.1 T lymphoblast cell line occurs when VIP is added to the culture (10“’ M) (Nio et al., 1993). VIP increases spontaneous proliferation by the GM-1056 B cell line in a dose- dependent fashion in the absence of CAMP generation (Ishioka et al., 1992). Other studies indicate that VIP-mediated effects on immune function can occur in the absence of CAMP generation. VIP does not alter CAMP generation in murine T cell enriched cultures (Boudard and Bastide, 1991), findings that conflict with similar studies by Ottaway (1987). Calvo et al. (1986a) also have shown that VIP does not stimulate the production of CAMP by enriched T cells from human peripheral blood lymphocytes, whereas it does stimulate CAMP production in a population containing all blood mononuclear cells, suggesting that macrophages and other


cellular elements influence T cell proliferation.

In the homogeneous T cell line, A040.1 hybrid, Nio ef al. (1993) have shown that VIP at low

concentrations ( lo-l4 M to 10m9 M) can act directly on antigen-driven T cells to cause a marked (three- fold) increase in IL-2 release without a major change in cell proliferation.

The first indication that VIP influences antigen driven responses has come from work by Kroc and colleagues (1986a,b). They have demonstrated VIP-induced inhibition of murine lymphocyte proliferation in mixed lymphocyte reaction assays. Since this report several other studies have sup- ported VIP modulation of antigen-induced lymphocyte proliferation. VIP decreases soluble egg antigen (SEA)-induced T cell proliferation in granuloma and splenic T cells from mice infected with Schistosoma mansoni (Metwall et al., 1993). Inhibition is most evident in T cells stimulated submaximally with antigen and results from a decrease in IL-2 production by CD4+ T cells. They also provide evidence that the VIP effects on T lymphocyte proliferation may in part be mediated through its interaction with nonlymphoid cells.

Cytokine production

VIP inhibits IL-2 production from mitogen- stimulated T lymphocytes presumably through the generation of CAMP (Ottaway, 1987; Ganea and Sun, 1993; Sun and Ganea, 1993). In unfractionated murine splenocytes and purified CD4” T cell cultures stimulated with antibodies directed against CD3, VIP also inhibits IL-2 and IL-4, but not interferon-y (IFNy), in a dose-dependent manner (Sun and Ganea, 1993; Ganea and Sun, 1993). VIP-induced inhibition of IL-2 and IL-4 occurs through different molecular mechanisms, since mRNA for IL-2, but not for IL-4, is affected by this peptide (Sun and Ganea, 1993). VIP also decreases IL-2 production, with no effect on IL-5 or IFNy release, in granuloma and splenic T cells from mice infected with Schistosoma mansoni stimulated with Con A- or soluble egg antigen (SEA) in vitro (Metwall et al., 1993). The differential effect of VIP on IL-2 versus IFNy production is shared by only one other naturally occurring

factor, transforming growth factor-& which selectively inhibits IFNy production, while having a minimal effect on IL-2 production (Epsevik et

al., 1987; Hardy et al., 1987). In murine schistosomiasis, VIP evokes IL-5 release from activated T cells that are not undergoing immediate T cell receptor stimulation (Mathew et al., 1992). These

investigators suggest that VIP may signal pre- activated T cells to secrete IL-5 as part of a regula-

tory mechanism for eosinophil maturation at the site of the granulomatous lesions (Mathew et al.,

1992).

Antibody production

VIP effects on antibody production are isotype- and tissue-specific (Stanisz et al., 1986, 1988). VIP (lo-* M) increases the IgA response in mesenteric lymph nodes (20%) and spleen (30%) but inhibits IgA synthesis in cells from Peyer’s patches (60%). IgM synthesis in Peyer’s patches is increased by VIP (80%), but is not affected by lo-* M VIP in spleen and mesenteric lymph nodes. IgG synthesis is not altered in these lymphoid organs by VIP The effects of VIP on immunoglobulin synthesis are thought to be T cell-mediated, and differences in organ responses may be related to differences in local T cell subpopulations in these organs. In contrast to findings by Stanisz and colleagues (1986), Neil et al. (1991) find no effect of VIP on immunoglobulin production by splenic and liver gmnuloma cell preparations from S. mansoni-infected mice incubated with VIP for 4 hours. The differences in the outcomes of these studies may result from differences in B cell subpopulations in their cultures that vary in their responses to VIP, may be related to differences in antigen used to activate B cells, or may reflect the length of time that cells are cultured in the presence of VIP.

Several studies by Kimata and colleagues (1992, 1993) demonstrate VIP modulation of antibody production in human tonsillar mononuclear cells, several B cell lines (Ishioka et al., 1992) and fetal B cells (Kimata and Fujimoto, 1995). In human mononuclear cells obtained by


tonsillectomies for chronic tonsillitis from nonatopic children, VIP (10 pM to 10 nM) inhibits

IL-4 stimulated IgE, IgG, and IgG, production (Kimata et al., 1992). Both T cells and monocytes are required for VIP to exert its effects on IL-4 induced antibody production. VIP elevates IgG, and IgE production in sIgG, and sIgE_ B cells, respectively, indicating IL-4 induces isotype switching. VIP effects on antibody production in their cultures are not seen at concentrations higher than 100 nM. In cultures of mononuclear cells from human atopic tonsils, VIP inhibits both

spontaneous IgE and IgG, production (Kimata et al., 1993). In unfractionated small resting B cells stimulated with anti-CD40 monoclonal antibodies, VIP induces IgA, and IgA, production (Kimata and Fujimoto, 1994). VIP can induce isotype switching in anti-CD40-stimulated tonsillar B cells, since it stimulates IgA, and IgA, production in sIgA; and sIgA; B cells (Kimata and Fujimoto, 1994). VIP induces IgA,, IgA, and IgM production in sIgM+ and sIgM_, CD 19+ fetal B cells (sIgA‘) stimulated with anti-CD40 monoclonal antibody (Kimata and Fujimoto, 1995). In IgM- B cells, the effects of VIP are enhanced by IL-7. Collectively, these studies show selective modulation of different antibody classes and subclasses by VIP that involve isotype switching. Further, effects of VIP on antibody production require the presence of both T cells and monocytes in B cell cultures.

VIP increases immunoglobulin production and proliferation in several lymphoblastoid B cell lines, in a dose-dependent manner (Ishioka et al., 1992). As little as lo-‘* M VIP is effective in stimulat- ing a five-fold increase in IgA production in GM- 1056 cells (Ishioka et al., 1992). VIP also enhances IgM in CBL cells, and IgG production in IM-9 (Ishioka et al., 1992). Stimulatory effects on these B cell lines occur in the absence of CAMP generation.

NK cell activity

The effects of VIP on NK cell activity are variable depending on the source of NK cells, activational state, the target cells, whether cells are pre-

incubated with VIP before, but not during the

assay, or whether VIP was present throughout the

assay period (co-incubation), assay incubation

time, and concentration of VIP. The continual

presence of VIP in a 4-hour cytotoxicity assay

produces a potent inhibition of NK cell activity

in human peripheral blood lymphocytes to K-562

target cells (lOme M only, Drew and Shearman,

1985; 10“’ to 10s, optimal 10m8 M, Rola-

Pleszczynski et al., 1985; 10.’ to 10e6 M, Sirianni

et al., 1992). The presence of 2’,5’-dideoxy-

adenosine (DDA), an inhibitor of adenylate

cyclase, does not block VIP-induced inhibition

of NK cell activity (Rola-Pleszczynski et al.,

1985). Alternatively, in human peripheral blood

lymphocyte cultures pre-incubated in VIP lo-” to 10m8 M) for 30 or 60 min and washed before the assay, VIP-enhanced cytoxicity of cells against

the target occurs (Rola-Pleszczynski et al., 1985). VIP 15-28 is a more potent augmenter of NK cell activity than VIP,_,,. Addition of DDA to the pre-incubation media blocks the enhancing effect

of VIP alone, indicating that CAMP generation

is necessary for VIP stimulation of NK cell activity. Assessment of effector-target cell

conjugate formation and subsequent target cell lysis in agarose has revealed that pre-incubation

with VIP increases conjugate formation but not lytic efficiency. These studies suggest that enhanced NK cell activity results from increased

interaction of target cells with effector cells, and inhibition of NK cell activity by co-incubation with VIP is mediated by another transduction

pathway. Van To1 and colleagues (1991, 1993) find that pre-incubation of VIP (10.’ M) stimulates

cytotoxicity of human peripheral blood mono-

nuclear cells, and lamina propria mononuclear cells for normal mucosa, against CaCo-2 human colon cancer cells. Contrary to the findings of Rola-Pleszczynski et al. (1985), they report no change in NK cell activity in these lymphocyte cultures against K-562 target cells. Ginsburg and coworkers (1983) report a decrease in NK cell activity without a decrease in NK cell number in patients with inflammatory bowel disease. It is possible that loss of VIP innervation in the gut


in these patients (Koch et al., 1987, 1990) may

contribute to NK cell dysfunction.

Accessory cell function and injlammatory

response

VIP has a protective effect on inflamed lung tissue

from injury caused by HCI (Foda et al., 1988), xanthine/xanthine oxide (Berisha et al., 1990), and toxic oxygen metabolites (Kurosawa and Ishizuka, 1993). Some effects of VIP on the inflammatory response are indirect, acting on vascular smooth muscle (vasodilator) and endothelial cells of the vasculature to change blood vessel diameter, plasma extravasation, and cell adhesion molecule expression (reviewed by Said, 1988). Still VIP does directly influence accessory cells of the immune system. Micromolar concentrations of VIP inhibit superoxide anion (0;) formation in N-formyl-methion-leucyl- phenyalanine (fMLP)-activated int%unmatory cells (peripheral blood neutrophils and mononuclear cells) from healthy human subjects (Kurosawa and Ishizuka, 1993). A similar effect of VIP on superoxide anion (0;) formation is seen in the human monoblast cell line, U937, peripheral blood eosinophils and alveolar macrophages obtained by bronchoalveolar lavage (Kurosawa and Ishizuka, 1993). Wiik (1989) has shown that na- nomolar concentrations of VIP inhibit the respiratory burst of human monocytes in response to zymogen by a CAMP-mediated mechanism. Conversely, pre-incubation with VIP ( 10m7 M) for 2 min is shown by Pedrera and colleagues (1994) to prime respiratory bursts in human neutrophils induced by either phorbol myristate acetate or fMLP; this effect does not appear to be mediated through binding of VIP with VIP receptors.

VIP has a moderate inhibitory effect on antigen- induced release of histamine from guinea pig lung (Undem et al., 1983), and on peptidoleukot- riene release from platelet-activating factor- stimulated rat lung tissue (Di Marzo et al., 1986). The influence of VIP on antigen-induced histamine release from guinea pig lung, and from other mucosal sites (adenoids, tonsils, and intestines) appears to differ from that seen in rat peritoneal

mast cells and in human skin where VIP evokes histamine release in a noncytolytic manner

(Fjellner and Hagermark, 1981; Church et al.,

1991; Shanahan et al., 1985). In a single case

study, Westley and coworkers (1982) report subcutaneous mast cell tumors that primarily produce VIP, and suggest that increased VIP production inhibited histamine production through the generation of CAMP in these tumor cells.

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The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation

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