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VIP/PACAP preferentially attract Th2 effectors through differential regulation of chemokine production by dendritic cells

MARIO DELGADO^*, ELENA GONZALEZ-REY^* and DOINA GANEA^,1

^* Instituto de Parasitologica y Biomedicina, CSIC, Granada, Spain; and
Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA

¹Correspondence: Rutgers University, Dept. Biological Sciences, 101 Warren St., Newark, NJ 07102, USA. E-mail: dganea{at}andromeda.rutgers.edu

SPECIFIC AIMS

Vasoactive intestinal peptide (VIP) and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) act as potent anti-inflammatory agents. In addition to direct suppressive effects on activated macrophages/microglia, neuropeptides also promote Th2-type responses. VIP, produced and secreted by Th2 cells following antigen stimulation, participates in a Th2 autoregulatory loop. VIP/PACAP affects differentiation of CD4⁺ T cells directly and indirectly through antigen presenting cells, and promotes proliferation and/or survival of Th2 effectors. Th1 and Th2 effectors express different chemokine receptors that control migration in response to various chemokines. In this study we investigated effects of VIP/PACAP on the production of CXCL10 (a Th1 chemokine) and of CCL22 (a Th2 chemokine) by bone marrow-derived dendritic cells. We found that VIP and PACAP inhibit CXCL10, while promoting CCL22 production, and that the effects are mediated through the VPAC1 receptor and involve cAMP/PKA as intracellular messengers. Induction of CCL22 and suppression of CXCL10 in VIP/PACAP-treated dendritic cells, results in preferential chemoattraction of Th2 effectors both in vivo and in vitro.

PRINCIPAL FINDINGS

1. VIP and PACAP differentially regulate CXCL10 and CCL22 production by dendritic cells
Fully differentiated Th1 and Th2 effectors were reported to express different chemokine receptors that induce migration in response to CXCL10 and CCL22, respectively. Activated dendritic cells are major producers of CXCL10 and CCL22. To determine the effect of VIP and PACAP on CXCL10 and CCL22 production, murine bone marrow-derived dendritic cells were activated with the inflammatory stimuli LPS and/or IFN (a model of bacterial stimulation), or through CD40 (a model of T cell-antigen presenting cell interaction) in absence or presence of various doses of VIP or PACAP, and the amounts of CXCL10 and CCL22 released in the culture supernatants were assayed by ELISA (Fig. 1 ). Unstimulated dendritic cells produce very low amounts of CXCL10 and CCL22, and dendritic cell activation results in a significant increase. VIP and PACAP inhibit CXCL10 production by activated dendritic cells. In contrast, production of CCL22 by both unstimulated and activated dendritic cells is enhanced by both neuropeptides. The effects on both CXCL10 and CCL22 are dose-dependent, with maximal effects for 10^–8 and 10^–6 M VIP/PACAP (Fig. 1C ). Northern blot analysis demonstrated that VIP/PACAP affects CXCL10 and CCL22 expression at the transcriptional level.

View larger version (41K): [in this window] [in a new window]   Figure 1. VIP and PACAP differentially regulate CXCL10 and CCL22 production by dendritic cells. Dendritic cells were incubated with medium, LPS, IFN, or anti-CD40 in the presence or absence of different concentrations of VIP or PACAP. The amounts of CXCL10 and CCL22 were determined by ELISA in culture supernatants.

The effect of VIP and PACAP on CXCL10 and CCL22 production was also evaluated in an antigen-specific model of T cell stimulation. We used PCCF-TCR transgenic (PCCF-Tg) mice whose CD4+ T cells recognize a fragment of pigeon cytochrome (PCCF) in the context of I-E^k. Dendritic cells cocultured with PCCF-Tg CD4+ T cells and PCCF produced high levels of CXCL10 and low levels of CCL22. Addition of VIP or PACAP suppressed CXCL10 release, and induced high levels of CCL22 (not shown). The fact that similar effects were observed when dendritic cells were pretreated with VIP/PACAP, washed extensively, and then cocultured with PCCF-Tg CD4+ T cells and PCCF, suggests that VIP/PACAP affect dendritic cells directly.

2. The effect of VIP/PACAP on CXCL10 and CCL22 production is mediated through binding to VPAC1 and cAMP/PKA activation
Next, we investigated whether the inhibitory effect of VIP/PACAP is mediated through specific receptors. VIP and PACAP act on three distinct receptors (i.e. VPAC1 and VPAC2 that bind VIP and PACAP with equal affinity, and PAC1, the PACAP-preferring receptor). Dendritic cells express VPAC1/2, but no PAC1. By using specific receptor agonists and antagonists we demonstrated that VIP/PACAP affect CXCL10 and CCL22 production through binding to VPAC1. The VPAC1 receptor signals primarily through the cAMP/PKA pathway, and the cAMP/PKA pathway was shown to be involved in regulating the expression of CXCL10 and CCL22. By using calphostin C (a PKC inhibitor), H89 (a PKA inhibitor), and PGE₂ (a cAMP inducing agent), we demonstrated that the effects of VIP/PACAP are mediated through increases in intracellular cAMP and PKA activation.

3. VIP and PACAP differentially affect dendritic cell-induced Th1 and Th2 chemotaxis
We confirmed previous reports that Th1 and Th2 effectors migrate in response to CXCL10 and CCL22, respectively (Fig. 2 A). Since VIP and PACAP regulate CXCL10 and CCL22 production by dendritic cells in an opposite manner, we investigated whether the VIP/PACAP treatment of dendritic cells affects Th1/Th2 chemotaxis. Culture supernatants from unstimulated dendritic cells did not induce chemotaxis of either Th1 or Th2 cells (Fig. 2B ). However, conditioned medium from dendritic cells stimulated with LPS, IFN, or antigen, preferentially attracted Th1 cells (Fig. 2B, C ). The treatment of stimulated dendritic cells with VIP/PACAP significantly reduced Th1, and increased Th2 chemotaxis (Fig. 2B, C ). VIP/PACAP pretreatment of dendritic cells 12h before Ag stimulation led to similar results (i.e., a decrease in Th1 and an increase in Th2 chemotaxis) (Fig. 2D ). The VIP/PACAP-induced changes in chemotaxis are presumably due to the effects on CXCL10 and CCL22 release from dendritic cells. This was confirmed by the fact that the addition of neutralizing anti-CCL22 antibody and of recombinant CXCL10 reversed the effects of VIP/PACAP on Th1/Th2 chemotaxis (Fig. 2E ).

View larger version (38K): [in this window] [in a new window]   Figure 2. VIP and PACAP alter the chemotactic activity of dendritic cells for Th1/Th2 effectors. A) Th1 and Th2 effectors migrate differentially in response to CXCL10 and CCL22. The results are expressed as migration index (number of cells that migrated in response to CXCL10 or CCL22/number of cells that migrated in response to medium alone). B) Dendritic cells were incubated with medium alone, LPS or IFN in the presence or absence of VIP or PACAP. Culture supernatants were collected and assayed for chemoattraction of Th1 and Th2 effectors. C) Dendritic cells were incubated with purified PCCF-Tg CD4+ T cells in the presence of PCCF, with or without VIP or PACAP. Serial dilutions of culture supernatants were assayed for chemoattraction of Th1 and Th2 effectors. D) Dendritic cells were preincubated with medium alone, or with different concentrations of VIP or PACAP. After washing, the dendritic cells were added to purified PCCF-Tg CD4+ T cells in the presence of PCCF. Supernatants were assayed for chemoattraction of Th1 and Th2 effectors. E) Dendritic cells were incubated with PCCF-Tg CD4+ T cells in the presence of PCCF, with or without VIP. Culture supernatants were supplemented with anti-CXCL10, anti-CCL22, a control Ab (IgG), recombinant CXCL10, or recombinant CCL22 and assayed for chemoattraction of Th1 and Th2 effectors.

To determine whether VIP/PACAP affect CXCL10 and CCL22 production and Th1/Th2 chemotaxis in vivo, we cultured dendritic cells with PCCF in the presence or absence of VIP or PACAP prior to their i.p. inoculation in PCCF-Tg mice. 48h later we determined the levels of CXCL10 and CCL22 in the peritoneal fluid. Mice injected with dendritic cells treated without VIP/PACAP produced high levels of CXCL10 and low levels of CCL22. In contrast, high levels of CCL22 and reduced levels of CXCL10 were observed in mice injected with dendritic cells pretreated with VIP or PACAP. To evaluate the presence of Th1/Th2 effectors, peritoneal exudate cells were restimulated in vitro, and the CD4+ T cells were analyzed in terms of intracellular IFN and IL-4 protein expression by FACS. Th1 effectors predominate in mice injected with DC treated without VIP/PACAP, whereas the inoculation of DC pretreated with VIP/PACAP results in a predominant Th2 presence.

CONCLUSIONS AND SIGNIFICANCE

VIP and PACAP are potent immunomodulatory agents. They function as general suppressors of macrophage/microglia activation, and promote the differentiation and survival of Th2, as opposed to Th1 effectors. We have found that VIP, produced and secreted by Th2 cells following antigen stimulation, participates in a Th2 auto-regulatory loop, promoting Th2-type responses through multiple non-excluding and probably interrelated mechanisms. The present study adds a previously unreported mechanism for the preferential VIP-induced Th2 response, by committing dendritic cells to specifically recruit Th2, and prevent Th1 effectors from inflammatory foci and sites of antigen presentation (Fig. 3 ). The VIP concentrations efficient in regulating the chemokine profile in dendritic cells are achieved in vivo in highly inflammatory conditions, and antigen-stimulated Th2 cells are capable to secrete similar VIP amounts. VIP and PACAP are abundant in immune privileged sites such as brain and the anterior chamber of the eye, where their role in promoting Th2 differentiation and chemotaxis at the expense of Th1 cells is highly relevant for maintaining the anti-inflammatory environment. In addition, VIP has been shown to be beneficial in models of inflammatory and autoimmune diseases with a prevalent Th1 background, such as endotoxic shock, Parkinson’s disease, brain trauma, rheumatoid arthritis and Crohn’s disease. In this respect, the data reported in this study offer an additional mechanism for the beneficial effect of VIP in Th1-autoimmunity.

View larger version (47K): [in this window] [in a new window]   Figure 3. Schematic diagram. Additional mechanism in the VIP/PACAP-induction of Th2 responses: preferential attraction of Th2 cells to sites of antigen presentation and inflammatory foci by modulating the production of Th1/Th2-recruiting chemokines by activated dendritic cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1548fje;

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