Mario Delgado

VIP and PACAP inhibit IL-12 production in LPS-stimulated macrophages. Subsequent effect on IFNγ synthesis by T cells

Since IL-12 plays a central role against intracellular pathogens, and contributes to the pathogenesis of immune diseases, its regulation is essential. This study examines the effect of two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), on interleukin-12 (IL-12) production. VIP/PACAP inhibit IL-12 dose-dependently. Type 1 VIP receptor (VPAC1), and to a lesser degree type 2 VIP receptor (VPAC2), mediate the inhibition of IL-12, primarily through the cAMP/PKA pathway. VIP/PACAP inhibit the production of IL-12, IL-6, tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) in vivo in endotoxemic mice. The presence of VIP/PACAP in the lymphoid organs and the specific effects on cytokine production offer a physiological basis for their immunomodulatory role in vivo.

The Neuropeptide Vasoactive Intestinal Peptide Generates Tolerogenic Dendritic Cells

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs in vitro and in vivo. The VIP/PACAP-generated DCs are CD11clowCD45RBhigh, do not up-regulate CD80, CD86, and CD40 following LPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptor and protein kinase A, and correlates with the inhibition of IκB phosphorylation and of NF-κBp65 nuclear translocation. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previously described Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensitivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege.

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit the production of inflammatory mediators by activated microglia

Microglia play a central role in the regulation of immune and inflammatory activities, as well as tissue remodeling in the central nervous system. However, activation of microglia is a histopathological hallmark of several neurodegenerative diseases. Pathological microglial activation is believed to contribute to progressive damage in neurodegenerative diseases through the release of proinflammatory and/or cytotoxic factors, including tumor necrosis factor α (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐12, and nitric oxide (NO). Hence, it is important to unravel mechanisms regulating microglia activation of inflamed brain parenchyma to provide insights into efficient therapeutic intervention. This study examines the role of two anti‐inflammatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase‐activating polypeptide (PACAP) on the production of various proinflammatory factors by endotoxin‐stimulated miroglia. VIP and PACAP inhibit TNF‐α, IL‐1β, IL‐6, and NO production by lipopolysaccharide (LPS)‐activated microglia. The specific type 1 VIP receptor mediates the inhibitory effect of VIP/PACAP, and cyclic adenosine monophosphate is the major, second messenger involved. VIP and PACAP regulate the production of these proinflammatory factors at a transcriptional level by inhibiting p65 nuclear translocation and nuclear factor‐κB‐DNA binding. This effect is mediated, as neuropeptides stabilize the inhibitor IκB by inhibiting LPS‐induced IκB‐kinase activity. Therefore, the inhibitory effects on the production of proinflammatory mediators define VIP and PACAP as “microglia‐deactivating factors” with significant, therapeutical potential for inflammatory/degenerative brain disorders.

Vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide inhibit chemokine production in activated microglia

Microglia react to even minor disturbances in CNS homeostasis and function as critical regulators of CNS inflammation. Activated microglia secrete inflammatory mediators such as cytokines and chemokines, which contribute to the pathophysiological changes associated with several neuroimmunologic disorders. Microglia‐derived inflammatory chemokines recruit various populations of immune cells, which initiate and maintain the inflammatory response against foreign antigens. Entry and retention of activated immune cells in the CNS is a common denominator in a variety of traumatic, ischemic, and degenerative diseases. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are two structurally related neuropeptides that function as potent anti‐inflammatory factors in the periphery. Here we investigated the effects of VIP and PACAP on chemokine production by activated microglia. VIP and PACAP inhibit the expression of the microglia‐derived CXC chemokines MIP‐2 and KC, and of the CC chemokines MIP‐1α, ‐1β, MCP‐1, and RANTES. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. The VIP/PACAP inhibition of both chemokine production and of NFkB binding is mediated through the specific receptor VPAC1 and involves a cAMP‐dependent intracellular pathway. Of biological significance is the fact that the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the chemotactic activity generated by activated microglia for peripheral leukocytes, i.e., neutrophils, macrophages, and lymphocytes. Because reduction in the number and activation of infiltrating leukocytes represents an important factor in the control of inflammation in the CNS, VIP and/or PACAP released by neurons during an inflammatory response could serve as neuronal survival factors by limiting the inflammatory process. GLIA 39:148–161, 2002.

Inhibition of Endotoxin-Induced Macrophage Chemokine Production by Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide In Vitro and In Vivo

Inflammatory chemokines recruit various populations of immune cells that initiate and maintain the inflammatory response against foreign Ags. Although such a response is necessary for the elimination of the Ag, the inflammation has to be eventually resolved in a healthy organism. Neuropeptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released after antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines macrophage inflammatory protein-2 and KC (IL-8), and of the CC chemokines MIP-1α, MIP-1β, monocyte chemoattractant protein 1, and RANTES in vivo and in vitro. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NF-κB binding and transactivating activity. The VIP/PACAP inhibition of both chemokine production and of NF-κB binding and transactivating activity is mediated through the specific VIP receptor VPAC1, and involves both cAMP-dependent and -independent intracellular pathways. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of polymorphonuclear cells, macrophages, and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.

Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo

CD4+CD25+ regulatory T (Treg) cells control the immune response to a variety of antigens, including self‐antigens, and several models support the idea of the peripheral expansion of CD4+CD25+ Treg cells. Although hormones such as estrogen and α‐melanocyte‐stimulating hormone have been recently reported to expand the CD4+CD25+ Foxp3‐expressing Treg cell compartment, little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+CD25+ Treg cells. In this study, we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg cells in vivo. The administration of VIP together with specific antigen to T cell receptor (TCR)‐transgenic (Tg) mice results in the expansion of the CD4+CD25+, Foxp‐3/neuropilin 1‐expressing T cells, which inhibit responder T cell proliferation through direct cellular contact. In addition to the increase in the number of CD4+CD25+ Treg cells, VIP induces more efficient suppressors on a per‐cell basis. The VIP‐generated CD4+CD25+ Treg cells transfer suppression, inhibit delayed‐type hypersensitivity in TCR‐Tg hosts, and prevent graft‐versus‐host disease in irradiated hosts reconstituted with allogeneic bone marrow.

Cutting Edge: Is Vasoactive Intestinal Peptide a Type 2 Cytokine?

A component of the chemical language shared by the immune and nervous system is the expression of neuropeptides by immune cells. Vasoactive intestinal peptide (VIP) was shown to be produced by T lymphocytes. Here we investigate whether T cell subsets differentially express VIP. Our studies indicate that, upon specific Ag stimulation, Th2 and T2 cells, but not Th1 and T1 cells derived from TCR transgenic (Tg) mice, express VIP mRNA and protein, and secrete VIP. Following immunization with the specific Ag, significant levels of VIP are present in the serum of syngeneic, non-Tg hosts that receive Th2, but not Th1 Tg cells. Th2 Tg cells recovered from the non-Tg hosts immunized with the specific Ag, but not with an irrelevant Ag, express intracellular VIP. Because VIP is produced by Ag-stimulated type 2 T cells, and differentially affects Th1 and Th2 cells, could VIP be viewed as a type 2 cytokine?

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit Expression of Fas Ligand in Activated T Lymphocytes by Regulating c-Myc, NF-κB, NF-AT, and Early Growth Factors 2/3

Activation-induced cell death in T cells, a major mechanism for limiting an ongoing immune response, is initiated by Ag reengagement and mediated through Fas/Fas ligand interactions. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two multifunctional neuropeptides, modulate innate and adaptive immunity. We reported previously that VIP/PACAP protect T cells from activation-induced cell death through down-regulation of Fas ligand (FasL). In this study, we investigate the molecular mechanisms involved in the protective effect of VIP and PACAP. VIP/PACAP reduce in a dose-dependent manner anti-CD3-induced apoptosis in 2B4.11 T cell hybridomas. The protective effect is mediated through the specific type 2 VIP receptor, and the cAMP/protein kinase A pathway. A functional study demonstrates that VIP/PACAP inhibit activation-induced FasL expression. VIP/PACAP inhibit the expression and/or DNA-binding activity of several transcriptional factors involved in FasL expression, i.e., c-myc, NF-κB, NF-ATp, and early growth factors (Egr) 2/3. The inhibition of NF-κB binding is due to the stabilization of I-κB (inhibitory protein that dissociates from NF-κB), through the inhibition of I-κB kinase α activity. Subsequently, p65 nuclear translocation is significantly reduced. The inhibition in NF-ATp binding results from a calcineurin-independent reduction in NF-ATp nuclear translocation. VIP/PACAP inhibit the expression of Egr2 and 3, but not of Egr1. The effects on the transcriptional factors are mediated through type 2 VIP receptor with cAMP as secondary messenger.

Inhibition of IFN-γ-Induced Janus Kinase-1-STAT1 Activation in Macrophages by Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-γ-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-κB nuclear translocation through the stabilization of IκB and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-γ-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-γ is a major stimulator of innate immune responses in vivo, the down-regulation of IFN-γ-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.

VIP/PACAP oppositely affects immature and mature dendritic cell expression of CD80/CD86 and the stimulatory activity for CD4+ T cells

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) released within lymphoid organs from nerve terminals and/or immune cells play a significant, anti‐inflammatory role by inhibiting macrophage‐induced inflammatory reactions and promoting T helper cell type 2 (Th2) responses. However, dendritic cells (DC) and not macrophages often are the major antigen‐presenting cells and link between innate and adaptive immunity. The role of VIP/PACAP in DC development and function is mostly unknown. Here, we report that bone marrow‐derived DC express VIP/PACAP receptors and that VIP and PACAP exert a differential effect on immature DC (iDC) and lipopolysaccharide (LPS)‐treated DC. In iDC, VIP/PACAP up‐regulates CD86 expression and enables them to stimulate T cell proliferation and differentiation into Th2 effectors in vivo and in vitro. In contrast, VIP/PACAP down‐regulates CD80/CD86 expression in LPS‐stimulated DC and strongly reduces their capacity to stimulate T cell proliferation and secretion of Th1 and Th2 cytokines. The VIP/PACAP effects on iDC and LPS‐stimulated DC are mediated primarily through the VIP receptor 1. These results indicate that neuropeptides such as VIP and PACAP can differentially affect the function of iDC and mature DC. In the absence of an ongoing immune response, VIP/PACAP contributes to the initiation of Th2‐type immunity, whereas in the presence of a full‐blown, inflammatory reaction, VIP/PACAP act as anti‐inflammatory agents.