| Dendritic cells (DCs) are professional APC playing key roles in capturing, processing and presenting antigens as the most potent professional antigen presenting cells. DCs can activate naive T cells and initiate adaptive immune responses, which plays a pivotal role in controlling and maintaining immune responses. DCs have multiple phenotypes and functions and are widely distributed throughout the body. Immature dendritic cells (iDCs) are characterized by a high capacity for antigen uptake, but they can not induce primary immune responses, because they do not express the requisite co-stimulatory molecules, nor do they express antigenic peptides as stable complexes with MHC molecules. In the presence of inflammatory signals, they rapidly take up foreign antigens and undergo a differentiation/maturation process that down-regulate further antigen-processing capacity, but enhance their expression of major histocompatibility complex class I and II and co-stimulatory molecules(e.g. CD40,CD80/CD86,CD58), and the production of inflammatory cytokines important for successful antigen presentation. They then migrate to the lymphoid organs where they gain a high capacity to present antigens and consequently become mature dendritic cells (mDCs). DCs direct both the quality and the extent of the immune response, but the functional properties of DCs are strictly dependent on their maturation state. From the above, it is very important to manipulate the phenotypcial and functional maturation of dendritic cells.Cholecyetokinin (CCK), a typical braingut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role. CCK is identified as several different size of the peptide, cholecystokinin octapeptide (CCK-8) is the smallest molecular owed full biological function of CCK. Since 1990s, it is found that CCK-8 had the regulatory function on the immune system, for instance CCK-8 can regulate the proliferation, migration and the production of proinflammatory cytokines. Our previous studies showed that CCK-8 down-regulated B7.1 and B7.2 expression and inhibited the costimulatory activity in LPS-activated macrophages by activating cAMP-PKA signaling pathways, and inhibiting IκB phosphorylation and the NF-κB binding activity. These results show the fine prospects of the CCK-8's anti-inflammation and immunomodulation effects.Maturation of DCs can be triggered by multiple stimulus, including bacteria, viruses, proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), and contact allergens. Lipopolysaccharide (LPS) is a potent inducer of DCs maturation. The signaling pathways involved in this process are intricate, which have not been characterized. It has been accepted that MAPK-NF-κB signaling pathways play an important role in the maturation of DCs. Our laboratory previous studies demonstrated that CCK-8 caused an inhibition of LPS-induced p38 MAPK-NF-κB activity in macrophage. Whether CCK-8 regulates the signaling pathway and functional alteration in LPS-activated dendritic cells has not been reported. To elucidate the immunoloregulation mechanism of CCK-8, we isolated mouse bone marrow-derived dendritic cells (BM-DCs) and investigated the effects of CCK-8 on the MAPK—IκB—NF-κB—phenotype and function pathways in LPS-treated DCs.1 The expression of CCK receptor in the mice bone marrow-derived dendritic cellsBone marrow cells were flushed from the tibiae and femurs of C57BL/6 and depleted of red cells with ammonium chloride. The cells were cultured in RPMI-1640 supplemented with heat-inactivated FCS, penicillin, streptomycin and GM-CSF. On day 7, the non-adherent cells and loosely adherent cells were harvested, the cells labeled with bead-conjugated anti-CD11c mAb were purified and then the purity of iDCs populations was identified by flow cytometry. After iDCs were incubated with anti-CCK-1R/2R antibody and anti-goat IgG-FITC, the expression of CCKR was analyzed by flow cytometry, meanwhile cells stained with the appropriate isotype-matched Ig were used as negative controls. The iDCs on glass slide were fixed with 4% w/v formaldehyde and then the cells were incubated with CCK-1R/2R antibody and anti-goat IgG-FITC, Cells stained with the PBS and anti-goat IgG-FITC were used as negative controls. The expression of CCKR was investigated by immunofluorescence. Results: (1)After the cells were positively sorted with CD11c+ microbeads, the purity of the selected cell fraction was > 90%. (2) CCK-1R and CCK-2R were detected in BM-DCs, which provided direct structural basis for further elucidation of regulatory effects of CCK-8 on DCs under physiological and pathological condition.2 Effects of CCK-8 on the maturation of the mice bone marrow-derived dendritic cells stimulated by LPSDCs undergo the change in phenotype and function during the maturation induced by LPS. In this study, we analyzed the effects of CCK-8 on phenotype and function in LPS-activated DCs. The cells were divided into the following groups: (1) control group; (2) LPS+CCK-8(10-10,10-8,10-6 mol·L-1) group; (3) LPS+CCK-8(10-8 mol·L-1)+CR1409(10-6mol·L-1) group; (4) LPS+CCK-8 (10-8 mol·L-1)+CR2945(10-6 mol·L-1) group; (5) CCK-8 (10-8 mol·L-1) group.The cells stimulated with different treatment were harvested and incubated in staining buffer (1×PBS) containing PE-conjugated anti-CD86, anti-MHCII and FITC-conjugated anti-CD80 antibody. Cells stained with the appropriate isotype-matched Ig were used as negative controls. Stained cells were analyzed using a FACSCalibur flow cytometer. Cells were incubated with fluorescein-conjugated dextran (40,000 molecular mass; Sigma-Aldrich) at a concentration of 1 mg/ml for 1h at 37℃, and then stained DCs were analyzed by a FACSCalibur flow cytometer. The contents of IL-12p40, IL-12p70, TNF-α, IL-6 and IL-1βin DCs culture supernatants were analyzed by ELISA, according to manufacturer's instructions. Responder T cells, used for the allogeneic T-cell reaction, were isolated by being passed through splenocytes from BALB/c mice in a MACS column. They were mainly composed of CD4+ cells. DCs were treated with 50μg/ml mitomycin C for 1 h and added in a 2×104 dose to 2×105 allogeneic T cells in U-bottomed 96-well microtiter culture plates. Cells proliferation was quantified by monotetrazolium (MTT) colourmetric assay. Data were presented as x±s and analyzed with ANOVA and least significant difference (LSD) using SPSS statistical program. A p value of <0.05 was considered to be significant. Results: (1) The expression of CD80, CD86, and MHC-II in LPS group was higher than that of control group (P<0.01). Compared to LPS group, the expression of CD80, CD86, and MHC-II was decreased in LPS+CCK-8(10-10,10-8,10-6 mol·L-1) group, which indicated that CCK-8 dose-dependently inhibited the LPS-induced phenotypic maturation of DCs(P<0.05, P<0.01 ). In contrast to LPS+CCK (10-8 mol·L-1) group, the expression of CD80, CD86, and MHC-II was increased in LPS+CCK+CR1409 group and LPS+CCK+CR2945 group (P<0.05, P<0.01). The expression of CD80, CD86, and MHC II was not affected by the treatment with CCK-8 alone (P>0.05). (2) The cells of control group had higher endocytic capacity for FITC-dextran, and the positive percentage of LPS-stimulated DCs was less than that of untreated DCs(P<0.01). Also, the CCK-8-treated DCs showed a higher endocytic capacity for dextran-FITC than LPS-stimulated DCs, and CCK-8 inhibited the decrease of LPS-induced endocytic capacity in a dose-dependent manner (P<0.01). In contrast to LPS +CCK-8(10-8 mol·L-1) group, the endocytic capacity was decreased in LPS+CCK+CR1409 group and LPS+CCK+CR2945 group. CR1409 and CR2945 partially reversed the inhibitory effects of CCK-8 (P<0.05). CCK-8 alone did not affect the endocytic capacity (P>0.05). (3) In contrast to control group, the concentrations of IL-12p40, p70 in CCK-8 group were no obviously changed (P>0.05). The concentrations of IL-12p40, p70 in LPS group were higher than that of control group, and the concentrations were even higher in DCs pretreated with LPS+CCK-8 (P<0.01). In contrast to LPS +CCK-8(10-8 mol·L-1) group, the concentrations of IL-12p40, p70 in LPS+CCK+CR1409 group and LPS+CCK+CR2945 group were decreased (P<0.05, P<0.01). Little TNF-α, IL-6 and IL-1βspontaneous production was detected in untreated DCs. Compared with control group, LPS-treated DCs secreted higher concentrations of TNF-α, IL-6 and IL-1β, and additional treatment with CCK-8 reduced the concentrations. CR1409 and CR2945 partially reversed the inhibitory effects of CCK-8 (P<0.05, P<0.01). (4) The ability of stimulating the proliferation of allogenic T cells by DCs was detected by measuring A values in MTT. LPS-stimulated DCs enhanced proliferative responses more effectively than untreated DCs (P<0.01), whereas CCK-8 dose-dependently impaired these effects of allogeneic T cells by LPS-induced DCs (P<0.05, P<0.01). CR1409 and CR2945 partially reversed the inhibitory effects of CCK-8 (P<0.05, P<0.01). Above observation indicated that CCK-8 inhibited the LPS-induced phenotypic and functional maturation of DCs, except that CCK-8 increased the expression of IL-12 in LPS-induced DCs.3 Effects of CCK-8 on MAPK activity in the mice bone marrow-derived dendritic cells stimulated by LPSThere are three major groups of MAP kinases in mammalian cells, the extracellular signal-regulated protein kinases (ERK), the p38 MAP kinases, and the c-Jun NH2-terminal kinases (JNK). LPS stimulation has been shown to activate MAPK signal pathways in DCs. To elucidate the molecular machenism and the signal pathway, we determined whether CCK-8 had regulatory effects on the p38, JNK, ERK phosphorylation in LPS-stimulated DCs. After the purified cells were treated with different reagent (the same groups as that of the second part), the cells were collected to extract cytoplasma proteins, p38 MAPK, ERK1/2 and JNK1/2 activation was detected by Western Blot. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1)No significant difference in p38 expression was found among all groups (P>0.05). The level of p-p38 protein was low in control group, CCK-8 alone had no effect on the expression of p-p38 MAPK (P>0.05). The p-p38 expression was elevated in LPS group(about 1.54 fold vs control, P<0.01), The p-p38 expression of LPS+CCK(10-10,10-8,10-6 mol·L-1) groups increased further (about 1.19, 1.31 and 1.43 fold vs LPS group, P<0.05, P<0.01). In contrast to LPS+CCK (10-8 mol·L-1) group, CR1409 and CR2945 inhibited the effects of CCK-8 by 18.31%, 15.49% respectively (P<0.05, P<0.01). (2) There was no significant difference in JNK1/2 expression among all groups (P>0.05). The p-JNK1/2 protein levels were significantly higher in DCs stimulated with 1mg/L LPS in comparison with unstimulated cells (about 5.30 and 5.16 fold respectively vs control, P<0.01), and additional treatment with CCK-8 markedly reduced the level in a dose-dependent manner. CCK-8 at the concentrations of 10-10 mol·L-1, 10-8 mol·L-1, and 10-6 mol·L-1 inhibited LPS-induced p-JNK1/2 expression by 46.41%,44.69%;54.49%,54.06%;64.67%,63.13% respectively (P<0.01). The effects of CCK-8 were attenuated by CR1409 and CR2945, the levels of p-JNK1/2 protein were about 1.22, 1.25 fold and 1.28, 1.28 fold vs LPS+CCK (10-8 mol·L-1) group (P<0.01). (3) There was no significant difference in ERK1/2 expression among all groups (P>0.05). The level of p-ERK1/2 protein was low in control group, CCK-8 alone had no effect on the expression of p-ERK1/2 (P>0.05). The p-ERK1/2 expression was elevated in LPS group (about 3.79 and 4.36 fold vs control, P<0.01). CCK-8 had no significant effect on the promoting role of LPS (P>0.05). These data showed that CCK-8 increased the expression of p-p38, inhibited the expression of p-JNK, but CCK-8 had no obvious effect on the the expression of p-ERK in LPS-induced DCs. These results demonstrated that CCK-8 had various effects on MAPK signaling pathways in LPS-induced DCs, which might be relevant to the inhibitory effects of CCK-8 on the phenotypical, most functional maturation and the promoting effects on IL-12 secretion. In addition, these results suggested that other signaling pathways might be involved in the regulation of CCK-8.4 Effects of CCK-8 on NF-κB binding activity in the mice bone marrow-derived dendritic cells stimulated by LPSWe have demonstrated that CCK-8 regulated the activity of MAPK in DCs induced by LPS; NF-κB is one of the downstream transcription factors in MAPK signaling pathways. It is well known that transcriptional factor NF-κB plays a pivotal role in the maturation of LPS-stimulated DCs. The effect of CCK-8 on NF-κB binding activity in DCs was analyzed by electrophoretic mobility shift assay (EMSA) and the IκBαprotein level in the cytoplasma was detected by Western blot. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) The NF-κB binding activity was significantly higher in DCs stimulated with 1mg/L LPS in comparison with unstimulated cells (P<0.01), and additional treatment with CCK-8 markedly reduced the binding activity in a dose-dependent manner. CCK-8 at the concentrations of 10-10 mol·L-1, 10-8 mol·L-1, and 10-6 mol·L-1 inhibited LPS-induced NF-κB binding activity by 20.27%, 35.37% and 47.26% respectively (P<0.01). The effect of CCK-8 was attenuated by CR1409 or CR2945 (P<0.01). CCK-8 alone had no effect on the NF-κB binding activity (P>0.05). The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors. (2) There was no significant difference in IκB expression among all groups (P>0.05). Compared to control group, CCK-8 alone had no effect on the p-IκB protein level (P>0.05). The p-IκB protein level was markedly increased 45 min after incubation with 1mg/L LPS (about 5.2 fold vs control, P<0.01). CCK-8 at the concentrations of 10-10 mol·L-1, 10-8 mol·L-1, and 10-6 mol·L-1 inhibited LPS-induced p-IκB expression by 15.38%, 34.62% and 44.23% respectively (P<0.01). The effect of CCK-8 was attenuated by CR1409 or CR2945 (P<0.01). These results showed that CCK-8 inhibited NF-κB activity through CCK receptors and inhibiting IκB phosphorylation in DCs.CONCLUSIONSIn the present study, we first systematically investigated the modulatory effect of CCK-8 on the phenotypic and functional maturation in mice bone marrow-derived dendritic cells stimulated by LPS. Furthermore, the signal transduction mechanisms including receptors, protein kinase and transcription factors were discussed. The conclusions were as follows:1 CCK-1R and CCK-2R were expressed in DCs, which provided the basis to further study the regulation of CCK-8 on DCs.2 CCK-8 inhibited the phenotypical and functional maturation of DCs, but increased the expression of IL-12, which indicated CCK-8 had the two-way modulation on DCs. CCK-8 might become the potential treatment factor for the autoimmune disease with over-activation of DCs.3 CCK-8 promoted the production of IL-12 in LPS-treated DCs, which might be related to the up-regulation of p-p38 expression and inhibition of JNK activation.4 CCK-8 inhibited the maturation of DCs induced by LPS by inhibiting the NF-κB activity.This study first demonstrated that CCK-8 inhibited the phenotypical and functional maturation of DCs, while increased the expression of IL-12, which might be mediated through MAPK—NF-κB signaling pathways. Taken together, CCK-8 might be a potential therapeutic agent for autoimmune diseases with excessive activation of DCs. However, the maturation process of DCs are intricately and efficiently regulated, which accompanied by manifold signaling pathways, the effects of CCK-8 on other functions of DCs and the related signaling pathways remain to be further studied.
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