Immunology of VIP: A Review and Therapeutical Perspectives

Current Pharmaceutical Design, 2001, 7, 89-111 89

Immunology of VIP: A Review and Therapeutical Perspectives

R.P. Gomariz*, C. Martinez, C. Abad, J. Leceta and M. Delgado

Dept. Cell Biology, Faculty of Biology, Complutense University, Madrid, 28040 Spain

Abstract: Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad distribution in the body thatexerts very important pleiotropic functions in several systems. The present work reviews theimmunology of VIP. Being daring, this neuropeptide could be included in the group of cytokines since itis produced and secreted by different immunocompetent cells in response to various immune signals,plays a broad spectrum of immunological functions, and exerts them, in a paracrine and/orautocrine way,through three different specific receptors. Although VIP has been classically considered as animmunodepressant agent, and its main described role has been as an anti-inflammatory factor, severalevidences suggest that a better way to see this peptide is as a modulator of the homeostasis of theimmune system. In the last decade, the pharmacology of VIP has spectacularly grown, and VIP itself, aswell as more stable VIP-derived agents, have been used or proposed as efficient therapeutical treatments ofseveral disorders, specially inflammatory and autoimmune diseases, such as septic shock, rheumatoidarthritis, multiple sclerosis, Crohn´s disease and autoimmune diabetes. A broad field of perspectives isactually open, and further investigations will help us to definitively understand the immunology of this“very important peptide”

.

INTRODUCTION substances and posses the same receptors forthem, thus suppressing traditional differencesbetween neurotransmitters, hormones and immunemediators. The fact that nervous and endocrinesystems shared the same mediators was soonestablished, and it was later when the immunesystem was involved in this circuitry. In thissense, the first reports were performed in 1980 byBlalock and Smith [1], demonstrating that neuronsand endocrine cells were not the exclusive sourcesof hormones and regulatory peptides, anddescribing for the first time that macrophages andlymphocytes were also sources for ACTH andendorphins. Actually, there are at least 20 differentneuroendocrine peptides [2,3] that can besynthesized by cells of the immune system, andthe list of candidates is continuously growing up intwo ways, adding new members, and amplifyingthe types of cells that produced these substances.Firstly, the most recent members of the list:glucocorticoids [4] synthesized by thymicepithelial cells, procalcitonin produced by varioustypes of human leukocytes [5], and thehypothalamic decapeptide gonadotropin-releasinghormone (GnRH) whose endogenous production isreported in human peripheral lymphocytes [6].

Maintenance of health is dependent onnumerous regulatory interactions between organsystems. It is well established the communicationbetween the three main systems involved inhomeostasis: nervous, endocrine and immunesystems as well as the genetic and environmentalinfluences on this circuitry. Thus, neurological andpsychiatric components have been described inexacerbation of infections, cancer or other immuneassociated problems. Inversely, an immuneetiology has been associated with altered nervousactivities, such as Alzheimer’s disease, chronicfatigue or multiple sclerosis.

An important fact for the behavior of thiscircuitry, as the result of the interaction betweendifferent areas of research, it’s the demonstrationthat the cells of the nervous, endocrine andimmune systems synthesize and secrete similar

*Address correspondence to this author at the Dept. Biología Celular,Facultad de Biología, Universidad Complutense, Madrid, 28040 Spain.Phone: 34-91-3944971, Fax: 34-91-3944981; e-mail:[email protected]

1381-6128/01 $28.00+.00 © 2001 Bentham Science Publishers Ltd.

90 Current Pharmaceutical Design, 2001, Vol. 7, No. 2 Gomariz et al.

Secondly, Substance P was one of the firstcandidates produced in rat and humanmacrophages [7,8], one year later in humanlymphocytes [9], and recently in dendritic cells ofmouse bone marrow [10].

lymphoid cell suspensions [22,23]. Moreover, VIPsynthesis was demonstrated in T and Blymphocytes by in situ hybridization and RT-PCR [24-26]. Regarding thymus, we havedemonstrated by RT-PCR VIP gene expression indouble positive (CD4+ CD8+) and single positive(CD4+ CD8-, CD4-CD8+) thymocyte subsets,thus thymocytes express VIP mRNA at specific,later differentiation stages suggesting a possibleautocrine effect of VIP on intrathymic T-cellmaturation and /or differentiation. Finally, we haverecently demonstrated that, VIP synthesized inlymphoid organs is secreted to the lymphoidmicroenvironment. Thus, agents that mediateimportant immune functions, such as proliferationand antigenic stimulation (ConA, LPS, and anti-TCR antibody), inflammation (LPS, TNFα, IL-6and IL-1β) or apoptosis (glucocorticoids) inducethe production and release of VIP to the centraland peripheral lymphoid organ micro-environment[27].

In 1969, Said and Mutt [11] published a paperentitled “Long acting vasodilator peptide from lungtissue” describing for the first time to the scientificcommunity the existence of vasoactive intestinalpeptide (VIP). VIP is today considered as amultifunctional and pleiotrophic peptide, with awell conserved sequence in vertebrates, a fact thatis consistent with its important biological role[12]. VIP is also an important signal molecule thatparticipates in the neuroendocrine-immunecircuitry, and it is well established that it isproduced by neurons and endocrine cells.Regarding the immune system, in 1985 in aninteresting supplement of Journal of Immunologyabout the proceedings of the conference on“Neuroimmunomodulation of immunity andhypersensitivity” two indicative papers werepublished about the origin of VIP in lymphoidorgans. Felten et al. [13] described VIP-likeimmunoreactivity in varicose profiles in thymuscortex suggesting for the first time a neural originof VIP. Moreover, O´ Dorisio´s group [14]pointed out that some circulating immune cellsappeared to synthesize several neuropeptidesincluding VIP. In this sense, the first identificationof VIP-like material was in rat mast cells byimmuno-cytochemistry [15] and in humanneutrophils by radioimmunoassay [16].

Taken together these data, we can summarizethat VIP, in the immune system, is locatedanatomically in nerve fibers and in lymphocytes[20,28]. Suzanne and David Felten and theircolleagues described recently these two locationsfor VIP, demonstrating by using radioimmuno-assay that surgical sympathectomy did not alterthymic or splenic VIP content, suggesting that thegreater amount of VIP is originated in lymphoidcells [20].

In view of these data we can postulate that VIPpresent in nerve terminals of lymphoid organsfrom postganglionic neurons of the autonomicnervous system could play a role in general orsystemic aspects of blood circulation as alteringblood flow and vascular permeability throughlymphoid organs. However, our in vitro and invivo data support the hypothesis that VIP, whichis synthesized and released by thymocytes and Tand B lymphocytes of peripheral lymphoidorgans, is biologically involved in the directinteraction between immune cells in aparacrine/autocrine way, independently of sensorynerves.

A neural origin for VIP has been reported bydifferent research groups. VIP immunoreactivenerves have been demonstrated in the respiratorytract [17], Peyer´s patches [18], spleen [19] andthymus [20].

Since 1990, our group has proposed a clearcellular origin for VIP in lymphoid organs. Usingdifferent technique approaches we havedemonstrated that VIP is synthesized and secretedby lymphocytes. The first evidence was thepresence of VIP immunoreactivity in thymus,spleen and lymph node cells usingimmunohistochemical methods [21]. In order toexclude the possibility that we are including nerveterminals, these results were later confirmed bybiochemical characterization of VIP by HPLC andRIA, and light and electron microscopy in

VIP RECEPTORS AND SIGNALING TRANS-DUCTION IN THE IMMUNE SYSTEM

VIP exerts its diverse biological actions througha pathway initiated by specific binding to cell

Immunology of VIP Current Pharmaceutical Design, 2001, Vol. 7, No. 2 91

surface receptors. Via binding techniques and using125I-VIP as a ligand, specific binding sites for VIPhave been described on a variety of immune cellsand lymphocytic cell lines [reviewed in Ref. 29].Thus, VIP receptors have been identified in humanperipheral blood lymphocytes and monocytes,murine lymphocytes and alveolar and peritonealmacrophages, and several human cell lines (i.e.Molt 4B lymphocytes, Jurkat T cells, U266myeloma cells, SKW 6.4 B cells, Sup-T1 lympho-blasts, Raji B cells, Nalm 6 pre-B cells, Dakikiplasma cells). Biochemical and pharmacologicalstudies of VIP receptors suggest the existence ofheterogeneous VIP receptor populations in varioustissues including immune system [30-32]. Sincethe first recombinant receptor for VIP from ratlung was identified by Ishihara et al. in 1992 [33],a major breakdown in our understanding of themolecular structure of VIP receptors has emergedas a result of the cloning and functional expressionof cDNAs corresponding to different VIP receptormolecules. Three types of VIP receptors have beencloned, which according to the International Unionof Pharmacology (IUPHAR) nomenclature [34]have been classified as follow: the VPAC1receptor (also known as VIP1, VIP/PACAP typeII, or PVR2), and the VPAC2 receptor (alsotermed VIP2, VIP/PACAP type III, or PVR3)bind both VIP and the structurally relatedneuropeptide PACAP with equal affinity, andactivate primarily the adenylate cyclase pathway;and the PAC1 receptor (also known asVIP/PACAP type I receptor, or PVR1), thePACAP preferring receptor, binds PACAP with a300-1000 fold higher affinity than VIP, andactivates both adenylate cyclase andphospholypase C [35].

have demonstrated that in most organs VPAC1and VPAC2 do not overlap anatomically, and insome cases, particularly in brain, the distribution iscomplementary and they point out that theexpression of VPAC2 appears to bedevelopmentally regulated in several systems [43].However, in the immune system we have shownthat, at least in the stimulatory state, bothreceptors coexist in the same cell. Involvement ofdifferent transduction signals could explain thedifferential distribution and regulation of bothreceptors. The best characterized effect of VIP invarious tissues, including immune cells, is theaccumulation of intracellular cAMP and thesubsequent activation of protein kinase A(reviewed in Ref. 29). Although both receptorsinduce intracellular cAMP accumulation, moresubtle differences may exist between transductionpathways affected specifically by VPAC1 andVPAC2.

Regarding PAC1, macrophages are the onlyimmunocompetent cells that has been shown toconstitutively express PAC1 [41,44]. Although ithas been extensively reported (at least in centralnervous system) a highly preference of thisreceptor for PACAP in comparison to thanshowed for VIP, we have described that PAC1expressed in peritoneal macrophages shows asimilar affinity for both PACAP and VIP [44];moreover, both neuropeptides activate in a similarway the protein kinase C/phospholypase Csystem coupled to PAC1 [44,45], suggesting thatPAC1 expressed in macrophages is essentiallydifferent to that expressed in nervous system,since it has been described at least five differentsubtypes of this receptor [46].

IMMUNOMODULATORY EFFECTS OF VIPThe expression and distribution of the different

receptors in various immune cell populations havebeen recently studied by our group. VPAC1 wasshown to be expressed in rat thymocytes,peripheral T and B lymphocytes, murinethymocytes, and peripheral CD4 and CD8 Tlymphocytes [25,36-39], and murine macrophages[40,41]. In contrast to VPAC1, which seems to beconstitulated and stimulated lymphocytes andmacrophages, VPAC2 is expressed only followingstimulation through the TCR-associated CD3molecule in lymphocytes [37] or endotoxin inmacrophages [41], and represents the only VIPreceptor expressed in T cell lines such as EL4.IL-2,D10-G4.1, 2B4.11, and A1.1 [37,42]. Usdin et al.

VIP, synthesized and secreted in the micro-environment of the lymphoid organs, can regulate,in an autocrine/paracrine manner, natural andacquired immunity affecting lymphocyte adhesionand traffic, T and B cell proliferation,immunoglobulin production, NK and macrophageactivity, and more recently described Th1/Th2differentiation.

When we try to review the effects of VIP onimmune and inflammatory functions, it is frequentto find opposite results, thus, VIP can either


enhance or inhibit a particular function. Thisduality could partially be explained by dose-dependency, different activation and developmentstate of the cellular target, and by the fact that in avariety of systems, heterogeneous cell populationsare commonly used. For example, modulation ofphagocytic and lymphocyte functions, cellproliferation, immunoglobulin synthesis and NKactivity could be enhanced, inhibited or notaffected at all by this neuropeptide. Moreover, theVIP receptor heterogeneity and the different actionmechanisms of VIP-mediated immunoregulationcould explain, at least in part, the different VIPeffects observed on lymphoid and phagocyticcells. In the next paragraphs, we will examine theeffects of VIP on these functions.

lymphatics of popliteal lymph nodes in sheep,reduces egress of lymphocytes from the nodes byincreasing intracellular cAMP in lymphocytes.VIP alters the composition of the lymphocytepool in lymph flow through the popliteal lymphnodes causing a marked depression in the outputof both small and blast lymphocytes into poplitealefferent lymph, with a selective effect on CD4 Tlymphocytes. In accordance with these results,continuous infusion of VIP into the superior arteryof the rat significantly reduces lymphocytemigration through intestinal and mesenteric lymph[52]. Similarly, in vivo intra-arterial infusion or invitro incubation with VIP significantly inhibitedtransendothelial migration in rat Peyer’s patchesand also significantly blocked the interstitialmigration of T cells and inhibited their subsequentappearance in the interfollicular lymphatics [53].

Lymphocyte Adhesion and TrafficIn vitro, incubation of T lymphocytes with VIP

results in downregulation of VIP receptors ontreated cells and a decrease in localization of thesecells in mesenteric lymph nodes and Peyer’spatches [54,55]. Bondesson et al. [56] havedemonstrated that VIP at concentrations rangingfrom 10-7 to 10-9 M inhibits, and from 10-12 to 10-

14 M stimulates mononuclear leukocyte migration.We have recently shown that VIP decreasesmobility of rat peritoneal lymphocytes,thymocytes and splenocytes showing the maximaleffect at concentrations around 1 nM [reviewed inRef. 57]. Our results do not show any effect ofVIP on the chemoattractant capacity of ratlymphocytes from peritoneum, thymus and spleen[reviewed in Ref. 57]. In contrast, Johnston et al.[48], using matrix-coated filters, find that VIPstimulates in vitro chemotaxis of T lymphocytesfrom both CD4+ and CD8+ subsets. However, inagreement with our results, in their studies, usinguncoated filters, T cells were unable to migrate inresponse to VIP. Recently, Schratzberger andcollaborators [58] have found that VIP stimulatedchemotaxis into micropore filters of both normalhuman peripheral blood T and B cells, and suggesta possible VPAC1 involvement. However, VIPsignificantly inhibited HuT 78 human lymphomaT cell chemotaxis in response to both IL-4 andTNF-α as a result of suppression of chemotacticmobility assessed by migration through microporefilters without matrigel, and in situ matrixmetalloproteinase activity [59].

Adherence of lymphocytes to the microvascularendothelium represents the initial step in theelicitation of these cells to sites of inflammationand/or immune response. We have demonstratedthat VIP stimulates the adherence of rat peritoneallymphocytes to inert substrates such as plasticsurfaces in a PKA-dependent way [47], suggestingan activation of these immune cells by VIP.According to these results, Johnston andcolleagues [48] have reported that pre-incubationof human resting and anti-CD3-stimulated T cellswith VIP induces increases in adhesion to ICAMand VCAM integrins and to the extracellular matrixprotein fibronectin. In human B lymphoblastic celllines, Robichon et al. [49] have shown that VIPinduces homotypic aggregation, a responsedependent on cAMP generation and LFA-1 andICAM-1. This could suggest that a single exposureto nanomolar concentrations of VIP may increasethe capacity of resident B cells to bind to migratingB cells, and thus to increase the local accumulationof B cells, thereby enhancing the production ofanti-bodies at that site.

Although adherence leads to a firm attachmentof leukocytes which allows the migration of thesecells, mobility is necessary for carrying out theimmune response. The evidence in experimentalmodels suggest that VIP may influence the tissuedistribution of lymphocytes. In vivo studiescarried out by Moore and collaborators [50,51]showed that direct infusion of VIP into afferent


T and B Cell Proliferation Differentiation of Th1 and Th2 Cells

The first evidence showing that VIP affectslymphocyte proliferative response wasdemonstrated in 1984 by Ottaway and Greenberg[60] in mouse lymphocytes. In this study, the VIPaddition resulted in an inhibition of theproliferative response of T cells to mitogenicstimulation. Later, numerous authors haveinvestigated VIP effects on mitogen and antigen-induced T and B cell proliferation in a variety oflymphoid tissues as well as on different cell lines(reviewed in Ref. 57). The effects of VIP onlymphocyte proliferation are summarized in Table1. Although VIP does not affect B cellproliferation in response to LPS, it has a profoundeffect on the proliferation of T cells stimulated bymitogens or through the T cell receptor [57]. Onthe other hand, the regulatory effects of VIP onproliferation are variable depending whether cellsare preincubated with VIP before mitogens orwhether VIP is added simultaneously withmitogens.

It was shown that CD4 T cells produced twodistinct patterns of cytokine production, whichwere designated T helper 1 (Th1) and T helper 2(Th2). Th1 cells produce interleukine-2 (IL-2),interferon-γ (IFN-γ) and lymphotoxin (LT), whileTh2 clones produce IL-4, IL-5, IL-6 IL-9, IL-10and IL-13. Additional cytokines, such as IL-3,granulocyte-macrophage colony-stimulating factor(GM-CSF), and tumor necrosis factor-α (TNF-α)were found to be secreted by both Th1 and Th2cells. Evidence provided that Th1 and Th2 developfrom a common precursor, which may be a naive,small, resting T cell, or an activated, proliferatingT-cell precursor (Thpp). In addition, some studiesshowed the existence of polarized mouse andhuman Th1 and Th2 cells designated Th0 cells thatmay be intermediate precursors in thedifferentiation of Th1 or Th2 cells.

It is clear that the single most important factorinfluencing Th differentiation are the types andamounts of cytokines present in the culture. IL-12,

Table 1. Effects of VIP on Lymphocyte Proliferative Response

Cellular Type Mitogen incubation VIP effect cAMP levels Ref.

Mouse peripheral lymphoid T cell PHAConA - ↑ 60-62

Mouse spleen T cells Con A - ↑ 63

Murine thymus, spleen, lymph node, Peyer’spatches and peripheral blood cells

None - 64

Rabbit spleen cells

Simultaneous incubation of VIPwith Con A or PWM

Preincubation with VIP beforeCon A

-

+

65

Human peripheral blood lymphocytes Preincubation with VIP beforemercuric chloride or nickel sulfate

+ 66,67

Human intraepithelial lymphocytes Con A No effects 68

Murine splenocyte in a model of leishmaniasis None + 69

Human Molt-4 lymphoblast cell line None - ↑ 70,71

Murine lymphocytes LPS No effect 60,63,70,72

Rabbit spleen cells PWM - 65

Lymphobastoid B cell lines and tonsillar B cells None + 73

↑, elevation of cAMP levels; -, inhibition; +, stimulation


IFN-γ, IFN-α, IFN-ß and IL-18 influencedifferentiation toward Th1 phenotype, whereasIL-4, IL-2, IL-6 and IL-1 affect Th2differentiation. In addition to cytokines, anotherinfluence on Thelper cell differentiation is theinteraction of costimulatory molecules on thesurface of antigen-presenting cells (APCs) withtheir cognate receptor on T cells. Among theaccessory molecules, the B7 family appears to bethe most potent. The B7 costimulatory pathwayinvolves at least two molecules, B7.1 (CD80) andB7.2 (CD86), both of which can interact with theircounterreceptors, CD28 and CTLA-4respectively, on T cells [74]. It was suggested thatB7.1 and B7.2 costimulation enhanced theproduction of IFN-γ and IL-4, respectively [75].The expression of B7.1 and B7.2 depends on thenature and on the activation state of the APC. Interms of B7 expression, as a general rule, B7.2 isinduced earlier during the activation process and athigher levels than B7.1. Recently, VIP was shownto act modulating Th1-Th2 differentiation. Wehave demonstrated that VIP has a dual effectdepending on the macrophage activation stage [76].VIP up-regulates B7.2, but not B7.1, expressionand induces the capacity to stimulate theproliferation to naive T cells in response to solubleanti-CD3 or allogeneic stimulation. In contrast, itdown-regulates B7.1/B7.2 expression on LPS/IFN-γ-activated macrophages and inhibits theendotoxin-induced costimulatory activity for Tcells. Their effects depend not only on the timingof their release, but also on the activation anddifferentiation state of neighbouring immune cells.The in vitro effect of VIP on both the macrophagecostimulatory activity and the B7.1/B7.2expression was reproduced in vivo. Moreover,VIP-treated macrophages gain the ability to induceTh2-type cytokines such as IL-4 and IL-5 andreduce Th1-type cytokines such as IFN-γ and IL-2[77]. In vivo administration of VIP in Ag-immunized mice reduces the number of IFN-γ-secreting cells and enhances the number of IL-4-secreting cells [77]. The preferential differentiationinto Th2 effector cells after Ag stimulation inducedby VIP-treated macrophages is mediated throughthe up-regulation of B7.2 expression. As we willdiscuss below, several studies indicate that VIPinhibits IL-12 production in LPS-stimulatedmacrophages [78,79] subsequently regulating IFN-γ production by activated T cells. Thus, theregulatory effect of VIP on IL-12 production could

further promote Th2 differentiation, and inhibitthe initiation of Th1 response [79].

Based in our results we propose the model forVIP modulation of Th1/Th2 differentiationshowed in Fig. (1).

Immunoglobulin Production

The mechanism of VIP modulation onimmunoglobulin production may be complex.Stanisz et al. [62,80] have shown that VIP effectsdiffer both with the isotype of immunoglobulinexamined and the tissue source of thelymphocytes. Thus, VIP increases IgA productionin mesenteric lymph nodes and spleen in Con A-stimulated cultures of murine mononuclear cells,but inhibits IgA synthesis in Peyer’s patches. IgMsynthesis in Peyer’s patches is increased by VIPbut is not affected in spleen and mesenteric lymphnodes. In contrast, Neil et al. [81] find nodetectable effect of VIP in other model ofimmunoglobulin secretion by B cells in mixedmononuclear leukocytes. In humans, the additionof VIP to cultures of intestinal lamina propriamononuclear cells was associated with a significantincrease of IgA production, whereas IgG levelswere reduced [82]; however, VIP enhanced thesecretion of IgA without affecting IgG or IgMproduction by purified tonsillar B cells [73].However, VIP suppresses the production of IgGby human blood mono-nuclear leukocytes [83].

Several investigations by Kimata and coworkershave demonstrated VIP modulation of Igproduction in human tonsillar mononuclear cells[84,85], several B cell lines [73] and fetal B cells[86]. VIP inhibits IL-4-stimulated IgE, IgG2 andIgG4 production in human tonsillar mononuclearcells from non-atopic children [84]. In atopicpatients, VIP inhibits IgE production by humantonsillar mononuclear cells without affecting theproduction of IgM, IgA, IgG1, IgG2 or IgG3, andalso selectively inhibits spontaneous IgE and IgG4production in atopic patients, being both T cellsand monocytes required for the effect of VIP [85].Collectively, these results indicate that VIPdifferentially modulates Ig production dependingon culture systems used. Evidence has beenprovided that the ability of VIP to modulate theproduction of immunoglobulins by tissue andblood mixed mononuclear leukocytes can be linked


Fig. (1). Model for VIP modulation of Th1/Th2 differentiation. Thp, T helper precursors; M f , macrophages.

in part to the effects of VIP on regulatory subsetsof T cells, because a relatively small number ofperipheral blood B cells bear VIP receptors [87]and the modulatory effect of VIP on Ig synthesisseems to be dependent on the presence of T cellsand monocytes in culture [72,83,84].

Phagocytic Cell Functions

The phagocytic process involves severalfunctions such as tissue adherence, mobility, andingestion and digestion of ingested material.Moreover, phagocytes, especially macrophages,play a number of important regulatory andfunctional roles in the immune system. These cellsphagocytize and kill microorganisms, processantigens and present them to T cells, kill tumorcells and serve as accessory cells for lymphocytes.We have investigated the effect of VIP on theadherence of rat peritoneal macrophages, resultingin stimulation of this property in the concentrationrange of 10-12 to 10-7 M through PKC activation[48]. On the contrary, other authors [91] haveobserved an inhibitory effect of VIP on ratperitoneal macrophages, using the same methoddescribed by us [reviewed in Ref. 57] and in asimilar concentration range (10-12 to 10-6 M) ofVIP. A possible explanation could be that wecarried out the adherence test with resting macro-

In vivo studies showed that VIP enhances IgMsynthesis by circulating lymphocytes in humanpatients with pancreatic endocrine tumors [88].

The effects of VIP on human Ig productionwere also studied in human plasma cell linesshowing that it enhanced Ig production and growthin these cell lines [89]. In B cell lines VIP inhibitedPMA-stimulated IgM production by SKW 6.4[90]. In contrast, a stimulation of IgA, IgG andIgM synthesis by VIP was observed by differentcultured lines of human B cells [73,89].Stimulatory effects on these B cell lines occur inthe absence of cAMP generation and may involveprotein kinase C.


phages, obtained immediately after sacrifice of theanimals and without any previous manipulation,whereas Segura and colleagues obtained cells after4 days of 6% sodium caseinate injection.

[99]. A similar effect of VIP was observed in thehuman monoblast cell line, U937, and alveolarmacrophages obtained by bronchoalveolar lavage[99].

Regarding mobility, we have described anincrease of spontaneous mobility and chemotaxisby VIP at the concentration range 10-12 to 10-7 Min rat peritoneal macrophages [48]. On thecontrary, Litwin et al. [92] found an inhibitoryeffect of VIP on rat alveolar macrophage chemo-taxis using a range of concentrations from 10-9 to10-6 M. This discrepancy could be due to thedifferent cellular types studied, as the responsecould vary depending on the protocol used. Inrelation to chemoattractant capacity, VIP is achemoattractant factor for peripheral macrophages[48]. This effect is mediated by PKC activation.Similarly, Sacerdote and collaborators [93] havealso shown that this neuropeptide is active ininducing human monocyte chemotaxis with amaximum effect at 10-10 M .

NK Cell Activity

NK cells are now appreciated as having abroader role in host defense against invadingpathogens, especially in the earliest phases of hostimmune responses. They also regulate thesubsequent development of specific immunitythrough cytokine secretion. Moreover, NK cellsoperate in the protection against tumors and arealso involved in the regulation of haematopoiesis.NK cell activity was found to be diminished byVIP in human peripheral blood lymphocytes[100,101]. The inhibitory action of VIP on NKcells could be due to an elevation of cAMP levels.However, interestingly, when lymphocytes werepreincubated with VIP, a significant augmentationof NK activity ensued. These studies suggest thatenhanced NK cell activity results from increasedinteraction of target cells with effector cells, andinhibition of NK cell activity by co-incubationwith VIP is mediated by another transductionpathway. Furthermore, VIP is able to decrease NKcell activity of human large granular lymphocytes[101] and of murine spleen, mesenteric lymphnodes and peripheral blood mononuclear cells [64].This inhibitory action was due to a lack ofmicrotubular cytoskeleton polarization in theeffector cell, in order to face the target, uponincubation of conjugates of NK cells and theirtarget with VIP [102].

The phagocytic and digestive capacities ofmacrophages are the most characteristic functionsof these cells. We have demonstrated that theingestion capacity of foreign cells (Candidaalbicans) or inert particles (latex beads) wasincreased by VIP [48]. However, Litwin et al. [92]and Ichinose et al. [94] indicated that VIPsuppresses phagocytosis in a dose-dependentmanner. The differences in the outcomes of thesestudies could be due to the difference betweenprotocols and, moreover by the fact that residentor inflammatory macrophages could have adifferent response to VIP.

In contrast, it has been demonstrated the abilityof VIP in restoring NK cell activity depressed byhepatitis B surface antigen in human lymphocytes[103], and also, VIP was found to stimulate NKactivity against Caco-2 colon carcinoma target cellsfrom lamina propia mononuclear cells forhistologically normal mucosa [104]. In the sameway, VIP increased activity of human CD16+ NKcells modulating the cellular immune responseagainst tumor cells, especially from the colon[104].

The digestion capacity can be analyzed by theproduction of reactive oxygen species (respiratoryburst) with microbicidal activity. The superoxideanion (O-2), the first oxygen metabolite in therespiratory burst of rat peritoneal macrophages,shows an increase in the presence of VIP atconcentrations ranging from 10-12 to 10-7 M[reviewed in Ref. 57]. However, the production ofreactive oxygen compounds in activated humanmonocytes was inhibited by VIP [95-97].Chedeville et al. [98], showed that VIP had noeffect on hydrogen peroxide (H2O2) production inmore differentiated cells such as human alveolarmacrophages. VIP also inhibited the capacity ofO-2 formation in peripheral blood neutrophils andmononuclear cells from healthy human subjects

From all of the findings described above, weconclude that VIP may play a relevant role in thedevelopment of the immune response in vivo. Inthis sense, VIP also exerts its immunomodulatory


Table 2. Cells, Receptors, Intracellular Pathways and Transcriptional Factors Involved in the VIP Action onCytokine Production

Factor Cell Stimulus Action Receptor IntracellularPathways

TranscriptionalFactors Ref.

IL-2 L TCR/Mitog ⇓ VPAC1/VPAC2 PKA

PKA-indep

AP-1, c-Jun, JunB 61,63,72,

106-112

IL-4 L TCR Antigen ⇓

⇑

VPAC1

VPAC1

PKA

PKA

Post-transcriptionalindirectly through IL-2Indirectly through Th2

differentiation

77,106,107,

109,113

IL-5 L S. mansoni ⇑ VPAC1/VPAC2 PKA-indep Post-transcriptional 114,115

IL-6 M LPS (high)None or

LPS (low)

⇓

⇑

PAC1VPAC1 PKCPKA CD14 shedding

ND

121-123

121

IL-8 S Arthritis ⇓ VPAC1 PKA CREB 124

IL-10 L

M

TCR/Mitog

LPS

⇓

⇑

VPAC1

VPAC1

PKA

PKA

ND

CREB

125

126

IL-12 M LPS/IFNγ ⇓ VPAC1>VPAC2 PKA

PKA-indep

IRF-1

NF-kB

41,79,127,

128

TNFα M LPS ⇓ VPAC1>VPAC2 PKA

PKA-indep

CREB, MEKK1, JNK

NF-kB

129-131

IFNγ L LPS

None

⇓

None

VPAC1 PKA Indirectly through IL-12 41

41,61,111,

134

NO M LPS/IFNγ ⇓ VPAC1 PKA

PKA-indep

IRF-1, Jak/STAT-1

NF-kB

127,139

⇓, inhibition; ⇑, stimulation; ND, not determined; M, macrophages; L, lymphocytes; S, synovial cells

action by regulating cytokine production, afunction that will be deeply analyzed in the nextsection.

transducting signal through specific-cell surfacereceptors. In the last years, it has been suggestedthat the phenomena of cytokine pleiotropy andredundancy is also exhibited by cytokine receptors[105].

EFFECTS OF VIP IN CYTOKINE PRO-DUCTION One of the first effects described for VIP in the

immune system was the role played in theproduction of cytokines, which began in 1987 byOttaway demonstrating that VIP downregulatesIL-2 expression in activated T lymphocytes [72].Lately, an increasing number of reports hasdemonstrated that one of the most importantmechanisms by which VIP regulates the immuneresponse is the modulation of cytokineproduction. In the next paragraphs we are going todo a review of the different effects of VIP in theproduction of several cytokines.

Cytokines are proteins which are secreted bycells of the immune system and other nonimmunecells, act generally locally in an autocrine and /orparacrine way and have limited half-lives in thecirculation. It is well known that many cytokinesare pleiotropic referring to the ability of a cytokineto exert many different types of responses, oftenin different cell types. Moreover, cytokines areredundant referring to the fact that many differentcytokines can induce similar signals. Cytokinesexert their actions by interacting with a


Effect of VIP on IL-2 Production inflammatory cytokines generated by activatedTh1 cells. The effect on IL-4 production was firststudied by Ganea and colleagues, who reportedthat VIP inhibits IL-4 production in both spleencells and thymocytes stimulated through the TCR-associated CD3 complex [106,107,109]. AlthoughVIP reduces IL-4 both in terms of protein level andbio-activity, the steady-state IL-4 mRNA level isnot affected, suggesting a post-transcriptionalinhibitory process [106,109]. Since murineperipheral T lymphocytes have been reported tosequentially produce IL-2, IL-4 and IL-5 uponstimulation, it is possible that VIP affects IL-4production indirectly through its effect on IL-2secretion. This is indeed the case, and it seems tobe that IL-2 affect IL-4 production through asustained increase in the stability of the newlytranslated IL-4 protein [113].

IL-2, the major interleukin involved in the clonalproliferation of antigen-specific T lymphocytes, issecreted by activated Th1 cells, and can act both asan autocrine and paracrine growth factor for Tlymphocytes. Based on the initial observationsthat VIP reduces T cell proliferation in response tomitogen stimulation, and on the role described forIL-2 as a T cell growth factor, it was hypothesizedthat VIP could downregulate IL-2 production inactivated T cells. In fact, several reports haveestablished that VIP inhibits IL-2 production inmurine CD4 T lymphocytes stimulated with eithermitogens or through the TCR-associated CD3complex [61,63,72,106,107]. The inhibition of IL-2 production by VIP seems to be mediated throughboth VPAC1 and VPAC2 and the subsequentcAMP induction [107-109], although a cAMP-independent pathway has been also demonstrated[110]. VIP affects both IL-2 mRNA stability andde novo transcription [61,109,111]. Transcriptionof the IL-2 gene is initiated following binding ofmultiple transcriptional factors including Oct 1,NF-kB, AP-1, NF-AT, and CD28RC to the IL-2promoter/enhancer region. Based on a variety ofstudies with cAMP agonists, varioustranscriptional factors such as NF-AT, AP-1 andthe TGGGC-binding factor were involved in theinhibition of IL-2 gene expression by cAMP.Several evidences demonstrate that VIP reducesthe levels of nuclear NF-AT, but not AP-1, NF-kB, or the TGGGC-binding factor [109] in TCR-activated lymphocytes; however, it has beenrecently reported that VIP changes thecomposition of the AP-1 complexes, from c-Jun/Fos to JunB/Fos dimers, with the subsequentdecrease in DNA binding and loss oftransactivating activity, by downregulating c-Junexpression/c-Jun N-terminal kinase (JNK) activityand by upregulating JunB expression [112].Regarding the physiological role played by VIP-mediated IL-2 inhibition, it has been speculatedthat it could mediate the anti-proliferativeresponse described for VIP in T cells.

Contrary to the previously discussed findings,we have recently demonstrated that VIP is able toinduce IL-4 production by antigen-stimulated CD4T cells [77]. This apparent contradiction could beexplained because the two experimental systemsare different. In the first system, Ganea et al.investigated the effect of VIP directly on naive Tcells stimulated with anti-CD3 mAbs or ConA.However, in our system the effect of VIP ismediated through the regulation of thecostimulatory signal of macrophages and indirectlythrough the differentiation of antigen-primed Thcells.

Effect of VIP on IL-5 Production

IL-5 is a key regulator for the generation anddifferentiation of eosinophils which is producedby antigen-stimulated Th2 lymphocytes. Theeffect of VIP on the production of IL-5 has beeninvestigated primarily since 1992 by Weinstockand his group in pathological conditions, i.e., ingranulomas from mice infected with Schisto-somiasis mansoni. Although VIP did not affect IL-5 secretion in resting T lymphocytes or inimmediate mitogen or antigen-stimulated T cells, itstimulated IL-5 production from pre-activatedsplenic T cells and from isolated T cell-enrichedgranuloma cells [114]. The effect of VIP wasexerted at a post-transcriptional level, probablystimulating release of IL-5 stored in pre-activatedT cells, and surprisingly it did not involveintracellular cAMP [115]. Further experiments will


IL-4 is a Th2-derived cytokine whichparticipates in the differentiation and clonalexpansion of Th2 cells and B lymphocytes andalso down-regulates the expression of pro-


confirm whether VIP-stimulated IL-5 productionin normal or pathological conditions participates inthe eosinophilia observed in parasitic infectionsand allergies.

absence of a stimulus or after low doses of LPS,VIP plays a role in immune system homeostasis,whereas in toxicity conditions, induced by highdoses of LPS, VIP regulates the excessive releaseof IL-6 in order to reduce inflammation or shock.

Effect of VIP on IL-6 ProductionEffect of VIP on IL-8 Production

IL-6 is a multifunctional cytokine that isproduced by both lymphoid and non-lymphoidcells and regulates several immune responses,acute-phase reactions, and haematopoiesis, andparticipates as a mediator in the inflammatoryresponse. The production of IL-6 is induced byseveral factors, including tumor necrosis factor α(TNFα), IL-1β, as well as the bacterial endotoxinlipopolysaccharide (LPS). Although stimulatoryeffects of VIP on IL-6 production have beenstudied in several non-immune cells such aspituitary-stellate cells, astrocytes, osteoblasts andbone marrow-derived stromal cells [116-120], datain cells of the immune system are very scarce. Wehave recently demonstrated a dual effect of VIP onthe IL-6 production by murine peritoneal macro-phages depending on the nature and dose of theinflammatory stimuli [121,122]. Whereas VIPinhibits the release of IL-6 from macrophagesstimulated with a LPS dose range from 100 pg/mlto 10 µg/ml, it enhances IL-6 secretion inunstimulated macrophages or macrophagesstimulated with very low LPS concentrations (1-10 pg/ml). These results were also observed invivo. Both stimulation and inhibition of IL-6production by VIP are exerted at a transcriptionallevel, although the involved VIP receptors andintracellular pathways are different. Indeed,whereas VIP inhibits LPS-induced IL-6 productionthrough PAC1 binding and PKC activation, itstimulates IL-6 release mainly through VPAC1 andthe subsequent increase of intracellular cAMPlevels [122]. A recent report has suggested that theinhibitory effect of VIP on IL-6 production bymacrophages could be mediated through theinhibition of LPS binding to its receptor CD14 inmacrophages by inducing the shedding ofmembrane-bound CD14 from LPS-stimulatedmacrophages [123]. Further experiments willidentify the transcription factors involved in thestimulatory effect of VIP on IL-6 production, andwhether other mechanisms, in addition to VIP-mediated CD14 regulation, are implicated in theinhibition of LPS-stimulated IL-6 expression.Based in these findings, we postulate that in the

IL-8 is a chemokine produced by a high varietyof cell types, implicated in chemotaxis,angiogenesis and as a major contributing factor inseveral acute as well as more chronic inflammatorystates. It has been recently reported that VIPinhibits IL-8 production by synovial cells isolatedfrom patients with rheumatoid arthritis [124]. Theinhibitory effect on IL-8 production is exerted at atranscriptional level, and probably mediatedthrough VPAC1; however, a surprising result isthe fact that VIP decreases the intracellular cAMPlevels and the nuclear translocation of thetranscription factor cAMP response elementbinding protein (CREB) in rheumatoid synovialcells [124]. These results suggest that anti-inflammatory neuro-peptides such as VIP could beused as a possible clinical application in somepathophysiological conditions such as rheumatoidarthritis.


IL-10, one of the major anti-inflammatorycytokines, was initially described as a Th2 productthat inhibits the secretion of Th1-derivedcytokines through the down-regulation of theantigen-presenting function of professional antigenpresenting cells. In addition to T cells, activatedmonocytes/macrophages serve as a major IL-10source, especially in response to LPS stimulation.The effect of VIP on IL-10 production has beenextensively investigated by our group. We havefound a dual effect depending on the cell nature.Whereas VIP inhibits IL-10 production by CD4 Tcells activated through the TCR-associated CD3complex [125], it stimulates IL-10 expression inLPS-stimulated macrophages in vitro and in vivo[126]. In both cases, stimulation and inhibition ofIL-10 production by VIP are exerted at atranscriptional level mediated through the bindingto VPAC1 and the increase in cAMP levels andPKA activation. In a recent work we havedemonstrated that VIP stimulates IL-10 mRNA


expression in activated macrophages by increasingthe cAMP-dependent phosphorylation/activationof the transcription factor CREB, and itssubsequent nuclear translocation and increase inIL-10 gene transactivation [126]. Although thetranscriptional factors involved in the inhibitoryeffect of VIP on IL-10 production in activatedlymphocytes has not been already identified,probably an increase in the phosphorylation andsubsequent activation of CREB is also involved inthis effect because other cAMP-inducing agentsare able to inhibit IL-10 mRNA expression instimulated lymphocytes [125], demonstrating adifferential regulation depending on the cellularsource. Whereas the VIP-mediated stimulation ofIL-10 production in macrophages is clearly relatedwith its anti-inflammatory function, the roleplayed by VIP-induced IL-10 inhibition inlymphocytes is still unclear, although it has beensuggested that could participate in the IL-10mediated B cell differentiation.

IL-12 inhibition by VIP has been also described byXin et al [127]. IL-12 inhibition is mainly mediatedthrough VPAC1, although in a lesser degreethrough VPAC2 [41]. VIP-mediated IL-12inhibition occurs at a transcriptional level byaffecting both NF-kB binding and the compositionof the Ets-2 binding complex [79]. VIP prevents,through a cAMP-independent pathway, theactivation-induced nuclear translocation of the NF-kB components p65 and c-Rel by inhibiting thephosphorylation of and subsequent reduction incytoplasmic IkBα [79]. Moreover, VIP inhibits, ina cAMP-dependent manner, the synthesis of theinterferon responsive factor-1 (IRF-1) byinhibiting the IFNγ-mediated activation of theJak/STAT1 pathway [128]. The decrease innuclear IRF-1 and c-Rel results in alteration of theEts-2-binding complex. Because IL-12 participatesin T cell activation and cytotoxicity activity andpromotes the differentiation of Th cells into theTh1 subset, VIP might play a significant role in thedown-regulation of cell-mediated immunity invivo, and provides an understanding by which VIPmanipulates Th1 and Th2 responses and therebyalleviates Th1- and Th2-associated diseases.


IL-12, an early proinflammatory cytokinesecreted by macrophages activated by microbialproducts, plays a central role in the regulation ofcell-mediated immunity. IL-12 stimulates theproliferation of activated T lymphocytes andenhances IFNγ secretion by NK cells and Tlymphocytes. Consistent with this latter effect,IL-12 has a pivotal role in the induction of CD4Th1 cell responses, acting in antagonism to IL-4,the major promoter of the Th2 response. In mice,IL-12 plays a decisive role in the protectionagainst intra-cellular pathogens, including parasitesand bacteria. IL-12 is an unique cytokine becauseof its hetero-dimer structure. IL-12 (p70) iscomposed of two disulfite-linked subunits (p35and p40) encoded by two separate genes. Whenboth subunits are produced within the same cell,they assemble into a biologically activeheterodimer. However, although the expression ofthe p35 gene is constitutive in a wide variety ofcells, the p40 gene is highly tissue-regulated, beingrestricted to phagocytic cells with antigen-presenting capability, and is therefore consideredto function as the regulatory component for IL-12expression. We have demonstrated that VIPinhibits bioactive IL-12 production by endotoxin-activated macro-phages, by regulating IL-12 p40gene expression in vitro and in vivo [41]. A similar

Effect of VIP on Tumor Necrosis Factor α(TNFα) Production

TNFα is an important macrophage secretoryproduct that contributes to pathophysiologicalchanges associated with several acute and chronicinflammatory conditions, including septic shock,autoimmune diseases, wasting, rheumatoidarthritis, inflammatory bowel disease, andrespiratory distress syndrome. VIP has been foundto prevent, in vitro and in vivo, the endotoxin-induced production of TNFα by macrophages[129]. VIP-mediated TNFα inhibition occurs at atranscriptional level, primarily through the bindingto VPAC1, which initiates two transductionpathways [130]. The cAMP-dependent pathwayleads to inhibition of the MAP-kinase-kinase 1(MEKK1)/MAP-kinase 4 (MEK4)/cJun-kinase(JNK) pathway [131] and to an increase in CREBphosphorylation [130], resulting in the alterationof the CRE binding complexes from high Jun/lowCREB in LPS-stimulated cells to low Jun/highCREB in LPS plus VIP-treated cells. The cAMP-independent pathway leads to an inhibition of NF-kB nuclear translocation, resulted in reduced NF-kB binding to the TNFα promoter [130]. In


addition, higher amounts VIP-induced CREBcompete with NF-kB for limited amounts of thecoactivator CREB binding protein (CBP) (Delgadoand Ganea, submitted for publication). Thedecrease in c-Jun and NF-kB, and the sequesteringof CBP leads to the inhibition of TNFαtranscription. Because TNFα plays a central rolein various inflammatory diseases such as endotoxicshock, multiple sclerosis, cerebral malaria, andvarious autoimmune conditions, the down-regulatory effect of VIP may have a significanttherapeutic potential.

synthesized by a family of NO synthases (NOS)which can be classified into two major groups:constitutive NOS (neuronal and endothelial) andinducible NOS. Macrophages express atranscriptionally inducible NOS (iNOS)undetectable unless the cells are activated.Although NO can not be considered as a cytokine,induced NO production is one of the principalmechanisms of macrophages cytotoxicity fortumor cells, bacteria, protozoa, helminthes, andfungi. In general, expression of iNOS follows ageneralized or localized inflammatory responseresulting in ischemia or tissue injury. Despite itsbeneficial role in host defense, sustained NOproduction can be deleterious to the host, and NOsynthesis induced by cytokines and/orinflammatory stimuli has been implicated inexperimental arthritis, inflammatory bowel disease,hypotension associated with septic shock, andother types of tissue injury. The functionalrelationship between VIP and NO is rathercomplicated, and it has been extensively studiedby Said´s group [reviewed in Ref. 135]. VIP andNO can be colocalized and coreleased from someneurons and can regulate each other´s release insome tissue. In some physiological outcomes, suchas the relaxation of smooth muscle, VIP and NOcooperate, whereas in others, such as theinflammatory response, they play opposite roles,with NO increasing and VIP defending againsttissue and cell injury. The opposite role played byVIP and NO in an inflammatory process raises thepossibility that VIP may regulate NO productionor activity. Indeed, in injury models that involveneuronal NOS, VIP does not inhibit NO synthesis,but prevents its toxic action [136,137]. Also, arecent report indicates that VIP inhibits iNOSexpression in stomach, spleen, and rectum,whereas augmenting neural NOS in brain [138]. Inthis sense, it has been demonstrated that VIPinhibits in vivo and in vitro NO production byperitoneal macrophages stimulated with LPSand/or IFNγ [127,139]. The inhibitory effect wasexerted by reducing iNOS expression at proteinand mRNA level. VPAC1, and in a lesser degreeVPAC2, mediate the effect of VIP, through twotransduction pathways, a cAMP-dependentpathway that preferentially inhibits IRF-1transactivation and a cAMP-independent pathwaythat blocks NF-kB binding to the iNOS promoter[139]. The inhibition of iNOS transcription byVIP may have therapeutical potential, becauseexcessive NO production has been implicated in

Effect of VIP on IFNγ Production

IFNγ is a CD4 Th1 lymphocyte-derivedcytokine that plays a central role in thedifferentiation and clonal expansion of Th1 subset.It exits apparent contradictory results regardingthe effect of VIP on IFNγ production. Forexample, Muscettola and Grasso [132] and Tayloret al. [133] reported a reduction in IFNγproduction by VIP in human peripheral blood cellsand antigen-stimulated lymph node cells,respectively, whereas we and others[41,61,111,134] could not detect an inhibitory orstimulatory effect on IFNγ production by murinesplenic or peritoneal T cells as well as a Th1 cellclone stimulated directly with anti-CD3 ormitogenic lectins. In addition, we have recentlydemonstrated that VIP inhibits the production ofIFNγ by peritoneal T lymphocytes indirectly,through the inhibition of IL-12 production byLPS-stimulated macrophages [41]. In contrast,Goetzl´s group has described an stimulation ofIFNγ production by VIP in antigen-stimulatedcytokine differentiated lymph node Th1 cells [77].Although these results may be attributed tospecies differences or to the experimental modelsused, another factor to be considered is thepossible involvement of IL-12. If IL-12 producedby macrophages is the major factor which mediatesthe stimulation of IFNγ production by T cells,IFNγ secretion in mitogen or anti-CD3 stimulatedT cells, which presumably does not involve IL-12,could be indeed resistant to VIP.

Effect of VIP on Nitric Oxide (NO) Production

NO, an unstable free radical gas that mediatesmany physiological and toxic functions, is


the tissue injuries characteristic for severalinflammatory and autoimmune diseases.

hypokalemia (WHDA syndrome) [142]. AlthoughVIPomas are frequently malignant, they can beeffectively suppressed by therapy with octreotide,a synthetic analog of somatostatin (whichreceptors are often present in these tumors) thatreduces circulating VIP levels by potentlyinhibiting its release from tumor tissue [143].Surgical excision must be employed to completethis treatment with successful results [144].VIPoma spread do not always occur, as long asmetastatic disease to lymph nodes is just reportedin half of the cases. Its metastases are often limitedto the liver, but hepatectomy and livertransplantation are suitable alternatives tocompletely eliminate the tumor [145].

Conclusions

In this section, we have seen that VIP is amultifunctional neuropeptide that regulate theproduction of a high variety of cytokines producedby several immunocompetent cells, and throughthis cytokine regulation VIP may exert, at leastpartially, its immunomodulatory function affectingseveral immunological aspects, including theinflammatory response, differentiation and clonalexpansion, and amelioration of some injurydiseases. It is important to evidence that, althoughmost of the immunomodulatory functions of VIPare in agreement with an in vivo anti-inflammatoryrole, recent observations suggest a more complexpattern of immune regulation, depending on thecell type, development stage, and overall activationstate of the cellular targets, and therefore, a moreaccurate description of the physiological role ofVIP is their participation in the control of immunehomeostasis. Therefore, the physiologicalconsequences of the VIP presence in the immunemicroenvironment could depend on the timing oftheir release and the activation stage of theneighbouring immune cells.

There are many other diseases which show arelation with the existence of VIP. That is the caseof multihormonal tumors like testicular carcinoid[146], neuroendocrine carcinoma of skin [147],breast cancer [148], carcinoma of the vulva [149]or acinic cell carcinomas of salivary gland [150]where many VIP-positive cells have beendescribed besides other neuropeptides likesubstance P between others. However, thepresence of VIP is not only limited to cancer. Ithas been also implicated in numerous pathologieswith very different etiologies. That is the case ofcholelitiasis, where hyperplasic VIP-ergicinnervation in the gallbladder causes relaxation,stasis and mucosal fluid imbalance, thuscontributing to gallstone formation [151]. It hasbeen also reported an elevation of VIP levels inamygdala in Type I schizophrenics, where theimbalance of several neuropeptides in the limbicsystem may be important in thepathophysiological basis of the disease [152].Recently, it has been seen that the presence of highamounts of VIP- and SP- immunoreactive nervefibers in the mucosal layer of the appendix inpatients of non-acute appendicitis may beinvolved in the development of acute rightabdominal pain in the absence of an acuteinflammation of the appendix [153]. In addition,there are some other diseases that show increasedlevels of VIP but a defective action of thisneuropeptide on its main targets. That is the caseof impotence in diabetes mellitus. In diabetic rats,the VIP content of the major pelvic ganglion andpenis is markedly increased, but a defect of itsreceptor or of the associated G-protein produces afailure of intracavernous VIP to produce erection[154].

VIP AND PATHOLOGY

It has been longly known that abnormal levelsof VIP or its receptors are related to the beginningand the development of several pathologies. Aslong as it plays an important role in the regulationof the body function, an excessive or deficientproduction of this neuropeptide can lead to severediseases. In some of them, VIP misregulation isclearly the cause of pathology. That is the case ofVIPomas (also called Verner-Morrison syndromeor pancreatic cholera), neuroendocrine tumors ofVIP-producing cells from very different origins,such as pancreatic islet-cell adenoma, islet-cellhyperplasia, bronchogenic carcinoma,pheocromocytoma, ganglioneuroblastoma,medullary thyroid carcinoma and retroperitonealhistiocytoma [140,141]. In these kind of tumors,high levels of VIP are released, thus causingimportant alterations in its main systemic targets,like the gastrointestinal tract, where it generateswatery diarrhea, hypochlorhydria and


There is also a clear association betweendecreased levels of VIP and pathology. In thiscase, reduction of this neuropeptide innervationgenerates disturbances in intestinal motility suchas constipation. Innervation can be damaged forseveral reasons. Sometimes, the disease isprimarily due to a neuropathy, as it occurs infamilial amyloidotic neuropathy [155], sclerosis[156], or idiopatic chronic constipation [157]. Inother situations, secondary causes can lead to thedestruction of VIP-innervation. That is the case ofneonatal necrotizing enterocolitis [158], Crohn'ssyndrome [159] or diseases caused by the invasionof parasites, such as Trypanosoma cruzi in Chagasdisease [160], or infection by Hymenolepisdiminuta [161]. Finally, the reduction of VIP levelswith aging is closely related to the gastrointestinaldysfunction in the elderly human [162].

sensory nerves are very important in the regulationof the cutaneous immune system [173]. On thatscore, it has been seen that dermal papillae ofmature psoriatic plaques have more VIP-containingnerves than controls [174], but also that thecontact between them and mast cells is decreased[175]. Imbalance of the levels of differentneuropeptides could lead to this kind ofinflammatory disease.

As the elevation of VIP levels is a failedattempt to regulate immune response in pathology,exogenous administration of this neuropeptidecould be an interesting option to avoid disease.

There are also some diseases that show normallevels of VIP, but an altered expression of itsreceptors. In breast cancer, high levels of VPAC1receptor have been [176,177], and it has beendemonstrated that VIP elevates cAMP andstimulates nuclear oncogene expression, beingclearly implicated in the pathophysiology of thedisease. The presence of VIP binding sites can beuseful to detect cancer nodules by employingradiolabeled derivatives such as 123I- or 99mTc-VIP[178,179]. In allergic contact dermatitis, thenumber of dermal mononuclear cells showingVPAC2 receptor immunoreactivity is reducedwhen compared to controls [180], thus suggestingthe implication of VIP in the development ofcutaneous contact hypersensitivity.

Although VIP is clearly related to thedevelopment of many systemic diseases, there aremany others, where the implication of VIP isanother evidence of the interrelation that existsbetween the neuroendocrine and the immunesystems. For example, Schistosomiasis, is aparasitic disease in which focal inflammatoryresponses called granulomas develop in the liverand intestines. The inflammatory cells contained inthese granulomas produce VIP, that acts as animmune modulator by suppressing T cellproliferation and IL-2 and IL-5 production fromgranuloma T cells, which express VPAC1 andVPAC2 receptors [163].

All in all, VIP seems to be a very importantpeptide to keep the systemic homeostasis(including the immune function) as themodification of its production or action over itsreceptors produces critical alterations in theindividuals.

Sometimes, the augmentation of VIP levels is aresult of feed back regulation in order to ameliorateharmful immune responses. Thus, some authors ofthe 80's described high levels of VIP in patientsand animals suffering endotoxemia, and showed adirect association with the disease degree,suggesting VIP as a cause of the disorder. Tenyears later, some groups, in which we are included,suggested that the increased VIP levels in thosedisorders is not probably a cause but aconsequence of the disease, and that VIP could besynthesized in response to the injury to try toregulate the deleterious effects of the excessiveimmune action [164-171]. It occurs the same inarthritis, where presence of VIP in synovial fluid isa manifestation of self regulation of the immunefunction in relation with disease [172]. Anotherdisease that has been related to the increase of VIPis psoriasis. Neuropeptides released from the

VIP AS A SURVIVAL FACTOR

As we discuss in the present work, it has beenreported increased VIP levels in several pathologicconditions such as asthmatic disorders, septicshock, other acute inflammatory diseases, andautoimmune conditions. A growing body ofevidences has demonstrated that one of the majorphysiological role of VIP is to act as survivalfactor. The majority of the works have beencarried out in nervous system, mainly by thegroups leadered by Gozes and Brenneman, clearlydemonstrating, by using different experimentalmodels, that VIP is a neuroprotective molecule,


acting in the normal neurodevelopment [reviewedin Refs. 181 and 182], and acting as an essentialsurvival factor for neurons after different injuryconditions, including HIV gp120-induced neuronaldeath [183], exocytotoxic cell death [181,182,184],withdrawal of growth factors [181,182], and it hasbeen suggested its implication for early treatmentof Alzheimer’s disease [181].

destroy no longer needed or even potentiallydamaging T cells, but also to allow the survival of asmall number of activated T cells that differentiateinto memory cells. Although all CD4 T cellsexpress Fas, naive T cells are apoptosis resistantand gradually switch to an apoptosis-sensitivephenotype during the proliferative stage followingactivation. Through its effect on FasL expression,VIP might favor the local generation of memory Tcells, allowing the surviving cells to switch backfrom an apoptosis-sensitive to an apoptosis-resistant phenotype. In addition, it has beenreported that VIP also prevents Fas/FasL-dependent cytotoxicity [190,191], another area ofpossible physiological relevance for VIP,especially since Fas/FasL-dependent cytotoxicityagainst autologous and bystander targets occurs inseveral specific autoimmune and inflammatorydiseases.

At the end of 1995, we and others suggested bythe first time the possibility of the inhibition ofprogrammed cell death or apoptosis as the majormechanism by which VIP exerts its cytoprotectiveeffect [185-187]. Flaws and coworkers [185]reported that treatment of follicles with VIPinhibits apoptosis in the ovary. At the same time,we [186] and Mutt´s group [187] demonstrated aVIP-mediated protection of thymocytes fromglucocorticoid-induced apoptosis, relating thiseffect with a possible role of VIP in thedifferentiation and positive selection of doublepositive thymocytes. Later, this finding wasconfirmed in thymocytes by Pankhaniya et al.[188], and other authors identified the VIP-mediated inhibition of apoptosis as one of themechanism by which VIP exerts itsneuroprotective effect [189].

Another important area in which VIP has beenlately studied as survival factor, with surprisingresults, has been inflammation. Said and coworkershave been working in the protective effect of VIPon lung inflammation from the beginning of 90´s.They have shown that VIP antagonizes the effectsof several inflammatory mediators and attenuatesplatelet-activating factor-induced injury and edemain rat lung [192,193], and reduces or preventoxidant injury in the lungs [135-137,193]. Inaddition, VIP has been shown to prevent tissueischemia-reperfusion in heart [194]. Finally, weand others have provided the basis for clinicaltrials with VIP or VIP-analogues, which mightdemonstrate the role of VIP in inflammation andsepsis, indicating a new therapeutic approach tothese diseases. In 1994, Suzuki et al. [195]reported a PACAP-mediated increase in somehemodynamic constants, such as mean arterialpressure and cardiac output, in endotoxemic dogs,and suggested a possible role of VIP on survival inseptic conditions. At the end of 1998, our ownresults [171] and the work reported by Tunçel etal. [196] described by first time that VIP had aprotective effect against lethal high endotoxemia ina murine model for septic shock. Whereas Suzukiand Tunçel suggested that the protective VIPeffects are mediated through a control of serumlevels of hormones which control hemodynamicconstants, such as adrenaline and cortisol, andalthough we do not discharge this mechanism, wehave demonstrated that the anti-shock effect ofVIP is mediated through a generalized inhibition of

Lymphocyte apoptosis is recognized as a majorelement in the control of the immune response. Tlymphocytes undergo apoptosis in response to avariety of stimuli, including antigenic stimulation,withdrawal of growth factors, glucocorticoids,irradiation, or cytotoxic agents. The antigen-induced cell death (AICD) in mature T cells isthought to be a major mechanism for themaintenance of peripheral tolerance and forlimiting an ongoing immune response. AICD isinitiated by antigen re-engagement of the TCR andis mediated through Fas/Fas ligand (FasL)interactions. In addition to the effect of VIP onglucocorticoid-induced apoptosis of thymocytes[186,187], it has been recently shown that thisneuropeptide prevents antigen-induced apoptosisof mature T lymphocytes by inhibiting the FasLexpression in activated CD4 T cells [42].Moreover, VIP prevents T cell death induced byUV radiation or several cytotoxic drugs byinhibiting FasL-induced apoptosis [42]. At thispoint we have planned the following question,what could be the physiological significance of theanti-apoptotic effect of VIP? During an immuneresponse, mechanisms must operate not only to


the production of pro-inflammatory macrophage-derived cytokines, and the up-regulation of otheranti-inflammatory agents such as IL-10. Thesevere pathological consequences of the septicshock syndrome result from a hyperactive andout-of-control network of endogenous pro-inflammatory cytokines. Since VIP appears toaffect multiple cytokines and inflammatoryfactors, it might provide a more efficienttherapeutical alternative to the use of specificcytokine antibodies or antagonists.

constriction of airway smooth muscle andmicrovascular plasma leakage that ultimately causeairway edema and pulmonary accumulation ofinflammatory cells. Chronic inflammation is animportant aspect of asthma. These chronicinflammatory changes are mediated by thesecretion of distinct mediators, cytokines beingrecognized as important targets for its treatment.IL-2, IL-6, and TNF cause an inflammatoryresponse around airways with significant increasein eosinophils, lymphocytes, and mast cells. Thesecytokines are increased in bronchoalveolar lavagefluid from patients with asthma. IL-4 has a crucialrole in the development of asthma and IL-4-knock-out mice do not develop bronchialhyperesponsiveness [207,208]. Since it is unlikelythat blockade of a single mediator will be entirelyeffective in controlling asthma, inhibitors thatblock several inflammatory mediators should be aninteresting focus of attention. In this sense, VIPappears to be a suitable therapeutic candidate. VIPrelaxes the bronchial smooth muscle, is vasodilator,causing bronchodilatation in asthmatic patients[209], and exerts cellular effects in phagocytes,lymphocytes and mast cells. O’Donnell et al.[197] described that the VIP analog Ro25.1553prevented antigen-induced contraction of thetrachea as well as lung resistance, and also inhibitbronchoconstriction, vasoconstriction andedemagenic responses in sensitized guinea pigs.Antigen-induced lung inflammation, as assessed byleukocyte accumulation, was prevented bypretreatment with such analog.

These findings clearly indicate that VIP may beconsidered as a cytoprotective molecule, that actsat multiple levels, mainly by regulating apoptosisand inflammation.

THERAPEUTICAL APPLICATIONS:INFLAMMATORY AND IMMUNEDISORDERS

VIP has been suggested as a potentialtherapeutic agent for several diseases, includingbronchial asthma [197], sexual impotence[198,199], brain damage [200-202], andgastrointestinal motility disorders [203]. Inaddition, the fact that a number of tumor cellsexpress extensive quantities of VIP receptors, andalso that immune and inflammatory cells areregulated by VIP, suggest that this neuropeptideshould be used for diagnosis and treatment of aseries of tumors, inflammatory diseases andimmune disorders.

The use of VIP as a therapeutic agent has beenimpaired by its short life in vivo due to itsdegradation by several peptidases [204]. Differentstrategies has been developed to overcome thisproblem. Strategies includes the conjugation ofdifferent sequences comprising the active site ofVIP with other peptides less susceptible todegradation, the development of cyclic derivatives,the attachment of lipids and the protection of thenative peptide by negatively charged liposomes[197,205,206]. Some of the developed derivativesare specific for different VIP receptors, thoseimproving the selectivity of their actions. We willfocus in the possible therapeutic use of VIPderivatives in asthma, as well as in otherinflammatory and immune disorders.

Tang et al. have shown that VIP and Ro25-1553inhibit IL-2 production [111]. The chronicinflammation of asthma is unique in that airwaywall is infiltrated by T lymphocytes of the Th2pheno-type, eosinophils, macrophages and mastcells [210]. Interaction of co-stimulatory moleculeson the surface of antigen-presenting cells, inparticular B7.2/CD28 interaction, may lead toproliferation of Th2 cells, thus perpetuating mastcell activation and eosinophilic inflammation. Asdiscussed above, investigations performed in ourlaboratory indicate that VIP, upregulating B7.2expression, stimulates the induction of Th2responses inducing the production of IL-4. In thisway, VIP and analogs, although ameliorating thesymptoms of asthma, may lead to the productionof specific IgE by B lymphocytes, priming mastcells for activation by antigen. So, further researchis needed to elucidate the role of different

Asthma is an inflammatory disease in whichantigen-induced hypereactivity leads to


cytokines and mediators in asthma, the effects ofVIP on them, and the participation of eachreceptor type on each process.

type of response [212]. As described above, wehave pointed out that VIP, by regulating theexpression of the co-stimulatory molecules B7.1and B7.2 on macrophages, inhibits the stimulationof lymphocytes by activated macrophages andalso drives a Th2-response by naive macrophagesand antigen primed lymphocytes, inhibiting Th1responses [76,77].

Besides the properties of VIP as neuro-modulator, the presence of receptors in cells of theimmune system indicated its possible role asimmunomodulator and their anti-inflammatoryproperties was soon pointed out. In theinflammatory states VIP regulates a variety ofimmune functions, such as leukocyte trafficking,lymphocyte proliferation and cytokineproduction, as described above. Whereasinflammatory response is essential for the ultimateelimination of antigens, its intensity and durationhave to be strictly controlled to avoid extensivetissue damage. An insufficient inflammatoryresponse could compromise the survival of theorganism, but an excessive response, that could bedue to shortcoming in the deactivation mechanismsof the process, could develop into acute or chronicinflammatory diseases. At sites of inflammation,cytokines could be mediators of a process thateventually could result in local antigenpresentation and the development of autoimmunediseases. The implication of VIP in thesepathological states should be indicated by thevariations in its levels and the changes in VIP-ergicinnervation reported in the affected organs[124,164].

The effects of VIP and analogs have been testedin several inflammatory disorders such asrheumatoid arthritis and endotoxic shock. Takebaet al. [124] have shown that VIP inhibit theproliferation of, and the secretion ofproinflammatory cytokines and metalloproteinasesby rheumatoid arthritis synovial cells.Investigations performed in our laboratory haveclearly indicated that VIP protects mice fromendotoxic shock in an in vivo model of lethalendotoxemia [171]. VIP is being evaluated in aphase I clinical trial of septic shock. The results ofthis study will give us a first impression of towhat extent the therapeutic use of VIP can beapplied in clinics. Since considerable progress hasbeen made in the understanding of inflammatorydiseases and the mechanisms of the anti-inflammatory effects of VIP, experimentalautoimmune and inflammatory states mediated byTh1 responses, such as inflammatory boweldisease, experimental allergic encephalomyelitis,non-obese diabetes, or skin contact allergy, aresuitable models to perform preclinical studiesabout the therapeutic potential of VIP in humandiseases such as rheumatoid arthritis, Crohn’sdisease, multiple sclerosis or atopic allergy.

Different cytokines and mediators have beenidentified as the causative agents in such diseases.Macrophages play a central role in the regulationof immune and inflammatory responses and are themain targets for the anti-inflammatory effects ofVIP, reducing the levels of proinflammatorycytokines and radicals such as TNFα, IL-6, IL-12and nitric oxide and also enhancing the productionof the anti-inflammatory cytokine IL-10[41,122,126,129]. TNFα and IL-6 contribute tothe pathophysiological changes associated withseveral acute and chronic inflammatory diseases. Ithas also been pointed out the importance of Th1-driven immune response in the development ofchronic intestinal inflammation, characterized byIL-12-driven high IFNγ production [211].Macrophages also participate in the antigenspecific immune response playing a role as antigenpresenting cells and supplying also the co-stimulatory molecules to immunocompetent cells.Co stimulatory signal delivered by naive orstimulated macrophages determine the Th1/Th2

ACKNOWLEDGMENTS

This work has been supported by GrantPM98-0081 from the Spanish Ministry ofEducation and Science.

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