Doina Ganea

The Significance of Vasoactive Intestinal Peptide in Immunomodulation

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

The Proinflammatory Effect of Prostaglandin E2 in Experimental Inflammatory Bowel Disease Is Mediated through the IL-23→IL-17 Axis

Although Crohn’s disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE2, a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE2 exacerbate the inflammatory process in inflammatory bowel disease through the IL-23→IL-17 axis. We assessed the effects of PGE2 on IL-12, IL-27, and IL-23 and found that PGE2 promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE2 are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4+IL-17+ T cells in the colonic tissue. These studies suggest that high levels of PGE2 exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.

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.

Prostaglandin E2 induces IL-23 production in bone marrow-derived dendritic cells.

Interleukin‐23, a recently described cytokine produced by activated antigen‐presenting cells, including dendritic cells, is a p19/p40 heterodimer. The p40 subunit is shared with IL‐12, the major Th1‐driving cytokine, while p19 is distantly related to IL‐12 p35. IL‐23 has pro‐ inflammatory actions, inducing IL‐17 secretion from activated CD4+ T cells, and stimulating the proliferation of memory CD4+ T cells. Here, we examined the effects of PGE2, a well‐known immunomodulator, on the production of IL‐23 by bone marrow‐ derived dendritic cells (BM‐ DCs). Our results indicate that PGE2 increases the production of functional IL‐23 from immature BM‐DCs in a time‐ and dose‐dependent manner. PGE2 induces both the expression of p19 and p40, without affecting p35 expression. The effect of PGE2 is mediated through the specific receptors EP2/4 and is mimicked by cAMP‐inducing agents, such as forskolin and dbcAMP. Although PGE2 also induces IL‐1β and IL‐6 expression in non‐stimulated DCs, the stimulatory effect of PGE2 on IL‐23 production is not mediated through IL‐1β or IL‐6. GM‐CSF, the pro‐ inflammatory cytokine required for the generation of BM‐DCs, amplifies the IL‐23 inducing activity of PGE2 in a synergistic manner. Recent studies described both pro‐ and anti‐ inflammatory effects of PGE2, and our results suggest an additional mechanism for its pro‐ inflammatory role, particularly significant for autoimmune diseases, such as rheumatoid arthritis.

In Vitro Differentiation of Dendritic Cells in the Presence of Prostaglandin E2 Alters the IL-12/IL-23 Balance and Promotes Differentiation of Th17 Cells

PGE2, an endogenous lipid mediator released in inflammatory conditions, affects both dendritic cell (DC) differentiation and maturation. Whereas the effect of PGE2 on fully differentiated DC was studied extensively, little is known about its effects on DC differentiation. In this study, we show that bone marrow-derived DC generated in the presence of PGE2 (DCp) acquire a proinflammatory profile; produce higher levels of proinflammatory cytokines/chemokines; express higher levels of MHC class II, costimulatory molecules, and TLRs; and exhibit increased activation of the NF-κB-signaling pathway. In addition, DCp exhibit a different IL-12/IL-23 profile than DC generated in the absence of PGE2. The low IL-12 and high IL-23 production in LPS-stimulated DCp is associated with the down-regulation of p35 and the up-regulation of p19 expression, respectively. In agreement with the DCp proinflammatory phenotype and especially with the altered IL-12/IL-23 balance which strongly favors IL-23, DCp also affect T cell differentiation. In contrast to DC which favor Th1 differentiation, DCp promote Th17 and inhibit Th1/Th2 differentiation, in vitro and in vivo. Previous in vivo studies indicated that PGE2 had a proinflammatory effect, especially in models of autoimmune diseases. Our results suggest that the proinflammatory effects of PGE2 could be mediated, at least partially, through effects on differentiating DC and subsequent alterations in CD4+ T cell differentiation, resulting in the preferential development of pathogenic autoimmune Th17 cells.

Neuroprotective effect of vasoactive intestinal peptide (VIP) in a mouse model of Parkinson’s disease by blocking microglial activation

Parkinsons disease (PD) is a common neurodegenerative disorder with no effective protective treatment, characterized by a massive degeneration of dopaminergic neurons in the substantia nigra (SNpc) and the subsequent loss of their projecting nerve fibers in the striatum. To elucidate PD pathogenic factors, and thus to develop therapeutic strategies, a murine PD model based on the administration of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) has been used extensively. It has been demonstrated that activated microglia cells actively participate in the pathogenesis of MPTP‐induced PD through the release of cytotoxic factors. Because current treatments for PD are not effective, considerable research focused lately on a number of regulatory molecules termed microglia‐deactivating factors. Vasoactive intestinal peptide (VIP), a neuropeptide with a potent anti‐inflammatory effect, has been found to be protective in several inflammatory disorders. This study investigates the putative protective effect of VIP in the MPTP model for PD. VIP treatment significantly decreases MPTP‐induced dopaminergic neuronal loss in SNpc and nigrostriatal nerve‐fiber loss. VIP prevents MPTP‐induced activation of microglia in SNpc and striatum and the expression of the cytotoxic mediators, iNOS, interleukin 1β, and numor necrosis factor α. VIP emerges as a potential valuable neuroprotective agent for the treatment of pathologic conditions in the central nervous system, such as PD, where inflammation‐induced neurodegeneration occurs.

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages.

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia‐reperfusion injury. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. We have investigated the effects of VIP and PACAP38 on the production of interleukin‐6 (IL‐6), a proinflammatory cytokine, by endotoxin‐activated murine macrophages. Both neuropeptides exhibit a dual effect on the IL‐6 production by peritoneal macrophages. Whereas VIP and PACAP inhibit with similar dose‐response curves the release of IL‐6 from macrophages stimulated with a LPS dose range from 100 pg/mL to 10 μg/mL, both neuropeptides enhance IL‐6 secretion in unstimulated macrophages and in macrophages stimulated with very low LPS concentrations (1‐10 pg/mL). The inhibition on LPS‐induced IL‐6 production is specific, presumably mediated through a subtype of the PACAP‐R. VIP and PACAP regulate the production of IL‐6 at a transcriptional level. These results were correlated with an inhibition on both IL‐6 expression and release in endotoxemic mice in vivo. These findings support the idea that in the absence of stimulation or in the presence of low doses of LPS, VIP and PACAP could play a role in immune system homeostasis. However, under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of IL‐6 in order to reduce inflammation or shock. J. Leukoc. Biol. 63: 591–601; 1998.

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) as Modulators of Both Innate and Adaptive Immunity

The structurally related neuropeptides VIP and PACAP are released within the lymphoid organs following antigenic stimulation, and modulate the function of inflammatory cells through specific receptors. In activated macrophages, VIP and PACAP inhibit the production of pro-inflammatory agents (cytokines, chemokines, and nitric oxide), and stimulate the production of the anti-inflammatory cytokine IL-10. These events are mediated through the VIP/PACAP effects on de novo expression or nuclear translocation of several transcription factors, i.e., NFkappaB, CREB, c-Jun, JunB, and IRF-1. The in vivo administration of VIP/PACAP results in a similar pattern of cytokine and chemokine modulation, which presumably mediates the protective effect of VIP/PACAP in septic shock. In addition, VIP/PACAP reduce the expression of the co-stimulatory molecules B7.1/B7.2, and the subsequent stimulatory activity of macrophages for T-helper cells. In T-cells expressing specific VIP/PACAP receptors, VIP and PACAP inhibit the expression of FasL through effects on NFkappaB, NFAT, and Egr2/3. The reduction of FasL expression has several biological consequences: inhibition of antigen-induced cell death in CD4 T-cells, inhibition of the FasL-mediated cytotoxicity of CD8 and CD4 effectors against direct and bystander targets, and promotion of long-term memory Th2 cells, through a positive effect on the survival of Th2, but not Th1, effectors. The various biological effects of VIP and PACAP are discussed within the range of a general anti-inflammatory model.

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.