Regulatory Effects Of Vasoactive Intestinal Peptide On The Migration And Cytokine Production Of Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells playing a key role in the induction of the immune response. They are known to be the primary cells for responsible for activating naive T and B cells. Bone marrow-derived DC precursors reach almost every tissue via the bloodstream, where they become resident immature DC (iDC) surveying for incoming antigens. After capturing antigens they migrate via afferent lymphatics from peripheral tissues to the secondary lymphoid organs to initiate a specific immune response. During migration from peripheral tissue, iDC undergo phenotypical and functional maturation. Notably, the migration capability of DCs is strictly regulated by their response to chemokines that characterize maturation stage and shape functional activity of DCs. iDCs express many inflammatory chemokine receptors, such as CCR1, CCR2, CCR5 and CCR6, allowing that they can respond to many CC and CXC chemokines, such as MIP-1α, MIP-1β, RANTES, and MIP-3a, whereas mature DCs (mDCs) lost their responsiveness to most of these chemokines, as a result of down-regulation of receptor expression or activity, and respond well to MIP-3P and 6Ckine as a consequence of an up-regulation of their receptor (CCR7). Studies have shown that CCR1 and CCR5 and their ligands are more important in the recruitment of iDC into inflammation site.However, studies in knock-out mice for CCR7 have shown the crucial importance of the CCR7/MIP-3p interaction for mDC induction of a primary immune response.Vasoactive intestinal peptide (VIP) is a neuropeptide released by both innervation and immune cells, particularly T helper 2 (Th2) cells, in response to antigen stimulation and under inflammatory/autoimmune conditions. VIP is a potent immunomodulatory agent. It inhibits Thl and promotes Th2 differentiation and survival directly or indirectly through effects on antigen-presenting cells (APCs). Recent researches have shown that VIP can affect DC maturation and function mainly via cell surface receptor VPAC1. It has opposite effects on iDC and LPS-stimulated bone marrow-derived DC (BM-DC) in terms of expression of costimulatory molecules and the capacity to activate CD4+ T cells. In addition, the presence of VIP during early phases of DC differentiation induces the generation of regulatory DCs with the capacity to induce regulatory T cells (Tr) and to prevent autoimmunity. However, the impact of VIP on the expression of chemokine receptors by DCs remains elucidative. Here we report that VIP differentially regulates the expression of CCR1 and CCR7 by mDC and inhibits the secretion of pro-inflammatory cytokines by mDC. Methods1. Preparation and treatment of DCsC57BL/6 mouse derived bone marrow cells were depleted erythrocytes by Tris-NHUCL and washed by phosphate-buffered saline (PBS) then seeded at 3xlO6 cells per well (Corning, 6-well tissue culture plate) in 3 ml of DC culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and 20ng/ml GM-CSF and 20ng/ml IL-4). On day 3, nonadherent cells were removed and 3 ml per well fresh DC culture medium were added to the plate. On day 6, immature DCs were collected and purified by using anti-CDllc microbeads according to the manufacturer's instruction. The purified CDllc+ dendritic cells were seeded in flatbottom 24-well microtiter plates at 1 * 106 cells per well in 1 ml DC culture medium. Cells were stimulated with LPS (200 ng/ml) in the presence or absence of VIP (from 10'12Mtol0"6M)for24h.2. Chemotaxis assayThe migration of DCs in response to chemokines was evaluated using a 24-well chemotaxis chamber. LPS and VIP treated DCs were collected and added to the top chamber at lxlO5 per well in lOOul assay media (fetal bovine serum free DMEM). FCS free DMEM containing chemokines (20ng/ml, 600ul/well) was added to the low wells and the top chambers were placed into the wells and incubated at 37°C. Assay medium was used as basal control. 6h later, cells that transmigrated into the lower chamber were collected and counted by flow cytometry. Results are shown as migration index (mean ± SD of three experiments), which represents the ratio between cells that migrated to the lower chamber in the presence of the chemokine and cells that migrated in response to medium alone.3. Flow cytometry analysisDCs were harvested and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated fetal calf serum (wash buffer). Cells were blocked with 10% fetal calf serum for 20 minutes at 4°C, then incubated with either primary antibody (anti-CCRl or anti-CCR7) for 30 minutes at 4°C, followed by appropriate FITC-conjugated secondary antibody at 4°C for 30 minutes at a final concentration of 2.5 H-g/ml. Appropriate isotype-matched antibodies were used as negative controls. After extensive washing, stained DCs were analyzed on a FACScan flow cytometer. Fluorescence data were expressed as mean channel fluorescence (MCF) and/or as percentage of positive cells after subtraction of background isotype-matched values.4. Semiquantitative RT-PCR analysisTotal RNA was extracted from purified CDllc+ DCs cultured at a concentrationof 1X 106 cells/ml for 24 h in the presence or absence of VIP and LPS (200ng/ml) by Trizol Reagent according to the instructions of the manufacturer. From each sample lug total RNA was reverse-transcribed into cDNA in a total volume of 20ul. The cDNA as readout of the mRNA was quantitated in PCR using specific primers for CCR1, CCR7 and P-actin, respectively.PCR products were visualized by electrophoresis in a 1% agarose gel containing 0.5 ug/ml ethidium bromide. Densities of the amplified p-actin, CCR1 and CCR7 were analyzed using Kodak EDAS120 digital imaging system version 3(Gibco-BRL). Results were expressed as a ratio of quantified CCR1 or CCR7 product over P-actin product.5. Cytokines detectionIL-lp and IL-12p70 were detected by using a sandwich ELISA according to the manufacturer's instruction.Results1. VIP differentially regulates migration of mDC in response to RANTESand MIP-3pchemotaxis assay showed that iDCs sensitively migrate in response to RANTES while don't migrate in response to MIP-3P compaired with control. In contrast with iDCs, LPS treated-mDCs sensitively migrate in response to MIP-3P while don't migrate in response to RANTES. VIP treated iDCs showed a similar chemotaxis acvtivity with iDC cultured in medium in response to RANTES and MIP-3P. However, VIP treated mDCs showed an activity of migration in response to RANTES while the activity of migration in response to MIP-3P was inhibited. These regulatory effects can be partly reversed by VIP-A. The Effect of VIP promotion/inhibition mDC migration in response to RANTES/MIP-3P is dose-dependent.2. VIP down-regulates CCR7 and up-regulates CCRl expression on mDCs Flow cytometry analysis showed that VIP does not affect the expression of CCRland CCR7 on iDCs, but it can remarkably down-regulate CCR7 and up-regulate CCRl expression on mDCs. This effect can be partly reversed by VIP-A. Differential effects of VIP on CCRl and CCR7 expression of mDCs are dose-dependent.3. VIP differentially regulates CCRl and CCR7 mRNA expression by mDC In accordance with previous report, bone marrow derived CD1 lc+ iDCs expresshigh level of CCRl mRNA but low level of CCR7 mRNA. However, LPS-stimulated DCs (mDC) express low level of CCRl mRNA but high level of CCR7 mRNA. VIP does not affect the expression of CCR7 mRNA and CCRl mRNA by iDCs, but it can remarkably down-regulate CCR7 mRNA expression and up-regulate CCRl mRNA expression by mDCs. Again, this effect can be partly reversed by VIP-A. Differential effects of VIP on CCRl mRNA and CCR7 mRNA expression of mDCs showed a dose-dependent manner.4. Effects of VIP on cytokines secretion by mDCVIP does not affect the secretion of IL-ip and IL-12p70 by iDCs, but it can remarkably, in a dose dependent manner, down-regulate IL-lp and IL-12p70 by mDCs. This effect can be partly reversed by VIP-A. ConclusionIt has been known that VIP plays anti-inflammatory function through many ways including macrophage/dendritic cell/microglia deactivation, and support for Th2 effector differentiation and survival. Our findings provide novel evidence implying that VIP serves as key endogenous anti-inflammatory agents through inhibiting migration of mDCs to draining lymph node and secretion of pro-inflammatory cytokines by mDCs, hereby preventing the induction of inflammatory immune response.