Cholecystokinin Octapeptide Prevents Collagen-induced Arthritis By Modulating Both Dendritic Cell Activity And T Cell Differentiation

The activation and differentiation of antigen-specific T cells requires the help of antigen presenting cells (APCs). Only receiving three signals from APCs, T cells can be fully activated and differentiate into effector T cells. The first signal involves the presentation of antigen on the surface of an MHC class II molecule, which facilitates T cell recognition of the cognate antigen through the TCR. In order for antigen-specific T cells to become activated and this population to expand in number, a second siganl must be generated through the interaction of adhesion and costimulatory molecules present on the APCs, such as CD80 and CD86, with CD28 on the surface of T cells. The third signal is the secretion of cytokines by APCs, which directs the differentiation of activated antigen-specific lymphocyte into an effector T cell subtype. Dendritic cells (DCs) are the most potent professional APCs,only DCs can activate naive T cells and initiate adaptive immune responses. Rissoan considered that DC controled T cell proliferation and effector function by providing costimulation and establishing the cytokines environment at the time of T cell priming. Thus, regulation of DC costimulatory molecules expression and cytokines production may have different consequences on the T cell mediated autoimmune disorders.Cholecystokinin (CCK) is a typical neuropeptide, and it is identified as several different size of the peptide including 4, 8, 33, 39, and 58 amino acid forms. The sulfated cholecystokinin octapeptide (CCK-8) is the biologically predominant active form of endogenous CCK, which exerts a variety of physiological actions in both the peripheral and central nervous system through activating cell surface CCK receptors (CCKR). A series of studies from our laboratory indicate that CCK-8 can act as an immunomodulator with predominant anti-inflammatory effects through the specific receptors. CCK-8 decreases pro-inflammatory cytokines like TNF-α, IL-6 and IL-1β, increases anti-inflammatory cytokines IL-10, down-regulate B7.1 and B7.2 expression and inhibits the costimulatory activity in lipopolysaccharide (LPS)-activated macrophages. In addition, CCK-8 regulates LPS-activated B cells costimulatory molecule expression and cytokines production. DC also expresses CCKR, and CCK-8 increases the production of IL-12 by modulating MAPK-NF-κB signaling pathways through its specific receptor on DC. So we presumed that CCK-8 might have different effect on T-cell mediated diseases via regulating dendritic cells.Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease primarily affecting the peripheral joints. Although the precise pathogenesis of RA remains unknown clearly, most of scientists believe that RA is a T cell-mediated autoimmune disease. Traditionally, RA has been identified as a Th1-mediated autoimmune disorder. However, neutralization or clearance of IFN-γor IL-12 can not prevent the disease. Soon after a subset of interleukin (IL)-17-producing T (Th17) cells distinct from Th1 and Th2 cells was identified by Laurie E Harrington research group and shown to play a crucial role in the induction of autoimmune tissue injury by up-regulating the release of IL-1β,TNF,IL-6,IL-8,G-CSF,PGE2 and VEGF by synoviocytes, macropahges and monocytes. Most of investigators consider that Th17 and IL-17 may play critical role in pathogenesis of RA.Based on these theory and results, we presumed that CCK-8 might regualte antigen-specific CD4+T cell proliferation and differentiation by modulating the co-stimulatory molecular expression and cytokines production of DC, furtherly might have effect on the pathogenesis of RA. In the present study, we investigated the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for CD4+T cell in vitro and in vivo. Furthermore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA.1 Effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for antigen-specific CD4+T cell in vitroObjective: Following maturation, DCs influence T cell proliferation and effector function by providing costimulation signals and establishing the cytokine environment at the time of T cell priming. The regulation of DC costimulatory molecules expression and cytokines production may have different effect on T cell mediated immune response. In this part, we examined the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for antigen-specific CD4+T cell in vitro.Methods: Bone marrow cells were flushed from the tibiae and femurs of DBA/1J mice. The cells were cultured in RPMI-1640 supplemented with heat-inactivated FCS, penicillin, streptomycin and GM-CSF. On day 6~7, the non-adherent cells and loosely adherent cells were harvested and used as BM-DC. (1) The cells were incubated in medium or LPS in the absence or presence of various concentration of CCK-8 for 24 hour, and then simultaneously stained with FITC-conjugated anti-mouse CD80, CD86 or MHC II, and PE-conjugated anti-mouse CD11c. Expression of the costimulatory molecules and MHC II on CD11c+ Cells were analysed by flow cytometry. (2) Purified BM-DC (pBM-DC) was isolated from BM-DC using anti-CD11c mAb immunomagnetic beads. Then the cells were cultured in medium or LPS in the absence or presence of various concentration of CCK-8 for 24 hour.①Cell culture supernatants were collected and the content of IL-23 was determined by ELISA kit.②The cultured pBM-DC were collected and treated with 50μg/ml MMC for 1 hour at 37°C, then added to purified CD4+T cells from CII-immunized mice in 96-well plates for 3 days. T-cell proliferation was measured by MTS and the cytokines profile was assayed by ELISA.Results: (1) Bone marrow cells cultured for 6~7 days in the presence of GM-CSF as immature BM-DC expressed low levels of CD80, CD86 and MHC II. In contrast, BM-DC, matured following LPS treatment, express high levels of CD80, CD86 and MHC II, while CCK-8 treatment, concomitant with LPS, inhibited CD80, CD86 and MHC II expression.These results indicated that CCK-8 could inhibit the LPS-induced phenotypic maturation of DCs. (2) The immature CD11c+ DC purified from BM (pBM-DC) produced too little IL-23 to be detected. In contrast, pBM-DC, matured following LPS treatment, produced much higher level of IL-23, while CCK-8 treatment at 10-6 and 10-8 mol/L, concomitant with LPS, decreased the production of IL-23, but CCK-8 treatment at 10-10 mol/L had no this effect.(3) Purified BM-DC matured by LPS treatment as APC induced T cell proliferation, however, treatment with LPS and different concentration of CCK-8 reduced the stimulatory capacity.(4) T cells exposed to LPS-treated pBM-DC produced high levels of IFN-γand IL-17. If the DC were treated with LPS plus CCK-8, IFN-γproduction was significantly increased, while IL-17 production was significantly decreased without significant changes in IL-4 and TGF-β.Our previous study showed that DC, matured following LPS treatment, produced high levels of IL-6,IL-1β,IL-12 and TNF-α, while CCK-8 treatment, concomitant with LPS, significantly decreased the production of IL-6,IL-1βand TNF-α, but increased the production of IL-12. Considering these results, CCK-8 can affect antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production. CCK-8 might promote Th1 profile by increasing IL-12 and inhibit Th17 profile by decreasing IL-6 and IL-23.2 Effect of CCK-8 on DC and T cells in vivoObjective: We have demonstrated that CCK-8 can affect antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production in vitro. In this part, we used collagen-induced arthritis (CIA) to examine the effect of CCK-8 on DC and T cells in vivo.Methods: DBA/1J mice were used to induce CIA. Experimental mice were immunized subcutaneously at the base of the tail with emulsified chicken CII and Freund's complete adjuvant on day 0 and boosted with the same agent on day 21. Treatment with different doses of CCK-8 (1nmol; 5nmol; 10nmol) commenced with the secondary immunization, and it was administered intraperitoneally on 7 consecutive days. The control groups of mice were injected saline alone. On day 28 after the first immunization, draining lymph node (DLN) were isolated from mice. (1) Using flow cytometry to analyze the expression of CD80, CD86 and MHC II on CD11c+ cells. (2) Total RNA was prepared from DLN, and IL-12p35, IL-12p40, IL-23p19, IL-6, T-bet, Gata-3, RORγt, Foxp3, IFN-γ, IL-4, IL-17 and TGF-βmRNA expression were measured by quantitative RT-PCR. (3) CD4+T cells were isolated from DLN cells by magnetic-antibody cell sorting. Then T-cell proliferations specific to CII were measured by MTS assay and cytokine levels in the culture supernatants were determined by ELISA.Results: (1) The expression of CD80, CD86 and MHC II on CD11c+ cells were significantly decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice (P<0.05 or P<0.01). These results indicated that certain concentration of CCK-8 could also inhibit the phenotype maturation of DC in vivo. (2) The expression of IL-12p35 and IL-12p40 mRNA were significantly increased in CCK-8 treated mice at dose of 5 nmol but not 1- and 10nmol, compared to control mice. In contrast, the expression of IL-6 and IL-23p19 mRNA were significantly decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice. IL-12,IL-6 and IL-23 are mainly produced by APCs, and DCs are the most potent professional APCs. Considering the fact that we demonstrated the effect of CCK-8 on DC in vitro at part 1, we presumed that certain concentration of CCK-8 can also medulate the cytokine production of DC in vivo.(4) The expression of T-bet, Foxp3, IFN-γ, IL-4 and TGF-βmRNA were significantly increased, but the expression of RORγt and IL-17 mRNA were obviously decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice. There is no difference of GATA-3 mRNA expression among 4 groups. (4) T cells from control mice showed marked CII-specific proliferation, and they produced high level of IL-17 and low levels of IFN-γand TGF-β. Differently, T cells from CCK-8 treated mice proliferated much less, produced low level of IL-17 and high levels of IFN-γand TGF-β; the amount of IL-4 was not significantly affected.These results indicated that CCK-8 administration to CIA can down-regulate the expression of CD80, CD86 and MHCII on DC. By decreasing the production of IL-6, IL-23, and increasing the production of IL-12, CCK-8 inhibited Th17 profile and promoted Th1 and Treg profile. 3 Effect of CCK-8 on the pathogenesis of CIAObjective: Many investigations report that Th17 cell plays a critical role in the pathogenesis of RA by producting IL-17. We have demonstrated that CCK-8 could regualte antigen-specific CD4+T cell proliferation and differentiation by inhibiting the stimulatory or co-stimulatory molecular expression on DC or altering the cytokines production, especially could suppress Th17 cell reaponse. These suggest that CCK-8 may be a potential therapeutic agent for RA. Therefore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA.Methods: The induction of CIA and CCK-8 treatment were the same as part 2. After the second immunization, Mice were analyzed every other day and monitored for signs of arthritis onset using two clinical parameters, incidence and severity. On day 35 after the first immunization, (1) hind paws from mice were collected to examine histopathological changes; (2) protein extracts were isolated by homogenization of joints to determination of cytokines in joints; (3) serum samples were collected to determine the levels of anti-CII IgG, IgG1 and IgG2a Abs. Moreover, on day 35 after primary immunization, synoviums in knee joints were isolated from CIA mice, synovial cells were stimulated with inactivated CII in the absence or presence of different concentrations of CCK-8, and cytokine levels in supernatants were determined.Results: (1) Delayed onset, lower incidence and decreased severity of CIA were observed in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice, as assessed by clinical score. Because we observed few differences between the 5- and 10-nmol doses CCK-8 administrations, all further experiments were carried out using 5nmol dose. (2) The histologic examination of the joints showed even and clearly joint space and smooth articular cartilage in normal DBA/1J mice, while severe articular cartilage and bone erosion as well as massive inflammatory cell infiltration and obvious intra-articular exudates in the control mice. The CCK-8 treated mice exhibited only mild synoviocyte hyperplasia and inflammatory cell infiltration and the degree of arthritis was significantly reduced compared with the control ones. (3) CCK-8 treatment significantly reduced the production of pro-inflammatory cytokines (IL-17, IL-23, IL-6 and TNF-α) and chemokines MCP-1 in the joints of arthritic mice, while upregulated the production of IFN-γand TGF-β. No difference was observed in the levels of IL-4 and IL-10 in the joints between the two groups. (4) CII-specific IgG level, particularly that of IgG2a, in sera from CCK-8 treated mice was significantly lower than those from the control mice, whereas the levels of IgG1 were not different between the two groups. (5) CCK-8 inhibited the production of pro-inflammatory mediators (IL-17, IL-23, IL-6 and TNF-α) and chemokines MCP-1, while increased the production of IFN-γand TGF-βin culture supernatant of synovial cells isolated from mice with CIA on restimulation with CII in vitro.These results suggested that CCK-8 could effectively inhibit the progression of CIA by reducing the incidence of arthritis, delaying the onset, improving symptoms and preventing occurrence of joint damage. These therapeutic effects are brouhgt about through inhibition of Th17 mediated autoimmune and inflammatory responses.CONCLUSIONSIn present study, we firstly investigated the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for CD4+T cell in vitro and in vivo. Furthermore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA. The conclusions were as follows: 1 CCK-8 could modulate antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production in vitro. CCK-8 might promote Th1 polarization by increasing the production of IL-12 and inhibit Th17 polarization by decreasing the production of IL-6 and IL-23.2 CCK-8 administered intraperitoneally to CIA could down-regulate the expression of CD80, CD86 and MHCII on DC. CCK-8 also could inhibit Th17 polarization and promoted Th1 and Treg polarization, by decreasing the production of IL-6, IL-23, and increasing the production of IL-12.3 CCK-8 could effectively inhibit the progression of CIA by reducing the incidence of arthritis, delaying the onset and preventing occurrence of joint damage.In summary, the present study indicates that treatment of CIA mice with CCK-8 has great benefit at the clinical and pathological levels. The effect of CCK-8 was exerted at multiple targets, mainly being associated with down-regulation of Th17-mediated autoimmune and inflammatory responses by modulating DC costimulatory molecules expression and cytokines production. These findings provide a preliminary rationale for studies assessing the efficacy of CCK-8 as a novel therapeutic approach to the treatment of RA.