| Cholecystokinin (CCK) being a kind of neuropeptide presents extensively in many systems of the body, with a broad spectrum of biological functions, such as regulating ingestion, anxiety, memory and so on. Our research have found that CCK-8 has some immunomodulatory and antiinflammatory functions in one of our National Nature Scinece Foundation (CCK receptor and anti-inflammatory intracellular signaling mechanisms activated by CCK-8, No. 30270529), CCK-8 inhibits activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity, and then inhibits the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, which shows that CCK-8 can modulate natural (or innate) immunity. But there are still no reports about how CCK-8 modulating acquired immunity. It has been reported that CCK2 receptor and mRNA express on human Jurkat T lymphocytes and CCK mRNA expresses on human T lymphocytes. CCK inhibits the proliferation of human peripheral blood lymphocytes induced by PHA (phytohemagglutinin) and so do the murine lymphocytes. Furthermore, it also inhibits the mobility of murine lymphocytes, but enhances its spontaneous hyperplasia and adhension. The existence and expression of specific CCK receptor on lymphocytes indicates CCK functioning as modulators of T lymphocytes by autocrine or paracrine secretion and affecting acquired immunity.Upon antigenic stimulation, CD4+Th cells can differentiate into two distinct types of effector cells, Th1 and Th2 subsets, each producing its own set of cytokines and mediating separate functions. Th1 cells secrete cytokines (IL-2, IFN-γ, and TNF-β, IL-12) critical for the generation of a cellular immune response, thereby activating CTL (cytotoxic T lymphocytes) and macrophages, inducing delayed-type hypersensitivity (DTH) responses, and stimulating IgG2a Ab production in mice. Th2 cells produce IL-4, IL-5, IL-13 and IL-10, which are critical for IgG1 and IgE Ab production and immunity against helminthic parasites, and inhibit macrophage, B cell and eosinophilic granulocyte activation and Ag presentation, thereby down-regulating the cellular immune responses. It is very important that Th1/Th2 balance maintain the immunostasis of the body. In the current research it has been found that Th1/Th2 balance relates with many diseases and participates in the initiation, development and prognosis of infective diseases (endotoxemia, SIRS, tuberculosis, parasitic infection et al.), hypersensitivity diseases (asthma et al.), the autoimmune diseases (RA, uveitis et al.). A series of studies in our laboratory has been focused on the effect of CCK-8 against endotoxin shock (ES) and releasing rheumatoid arthritis, but we still do not know whether this effect is related to the regulation of CCK-8 on Th1/Th2 balance? Focus on the modulating fuctions and its mechanism of CCK-8 on Th1/Th2 balance will conduce to interpreting the physiological and pathology functions of neuroimmune network, and presenting theory for clinical therapies of Th1/Th2 unbalance diseases.Na?ve CD4+T lymphocytes become activated and undergo clonal expasion, then differentiate into Th0 lymphocytes. Th0 cells have the potential ability of differentiating into Th1 or Th2 phenotype cells. Determining factors include the nature of the APC, the nature and amount of Ag, and the genetic background of the host, with the cytokine microenvironment as the dominant factor. Cytokine microenvironment is the dominant factor on regulating the differentiation of precursor Th cells (Th0). Increasing evidence demonstrates that IL-12 and, to a lesser extent IFN-γ, direct CD4+T cells to differentiate into Th1. In contrast, IL-4 is necessary to induce the development of Th2. When both IL-4 and IL-12 are added to in vitro cultures, IL-4 dominates over IL-12, driving naive CD4+T cells toward the Th2 phenotype. The activation of naive CD4+T lymphocytes requires two signals delivered by APCs, leading to enhanced cytokine production and proliferation and produce effective immune response. The first signal, which confers specificity, is provided by the interaction of the antigenic peptide/MHC complex with the T cell receptor. The second signal is provided by costimulatory molecules expressed on APCs. Among the accessory molecules, the B7 family appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7.1 (CD80) and B7.2 (CD86), both of which can interact with their counterreceptors, CD28 and CTLA-4, on T cells. B7.1 and B7.2 appear to be equivalent in the costimulation of T cell proliferation. However, B7.1 and B7.2 may differ in the signals provided for T cell differentiation, with B7.2 favoring Th2 development. As a general rule, B7.2 is induced earlier during the activation process and at higher levels than B7.1. Therefore, we will investigate the role of CCK-8 on macrophage B7.1 and B7.2 expression and costimulatory function for Ag-primed CD4+T cells, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo.Some researchers have proved that the activation of Toll/NF-κB results in induction of cytokines and costimulatory molecules― B7.1(CD80) and B7.2(CD86). It is well known that CCK exerts a variety of physiological actions through its cell surface receptors, which have been pharmacologically classified into two subtypes CCK1R and CCK2R according to their affinity to the peptide agonist sulfated carboxyl-terminal octapeptide (sCCK-8) and gastrin. CCK receptors belong to G protein-coupled receptor (GPCR) superfamily and distribute extensively in mammalian body. Our current research suggested that CCK-8 inhibited activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity through its receptors in rat PIMs. To elucidate the intracellular signaling mechanism of CCK-8 regulating macrophage B7.1 and B7.2 expression, we performed a series of studies on mouse peritoneal macrophages, from the effect of CCK-8 on cAMP-PKA pathway, and transcription factors NF-κB which involved in regulating B7.1 and B7.2 expression, and the costimulatory activity on macrophages induced by LPS, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo. 1 Effects of CCK-8 on B7.1 and B7.2 expression and costimulatory activity of macrophageAim: To investigate in vitro effects of CCK-8 on the expressions of B7.1 and B7.2 and on the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Mouse peritoneal macrophages were isolated and incubated with CCK-8(10-12 mol/L 10-6 mol/L), or with LPS and/or CCK-8(10-12～10-6)mol/L for indicated times. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry. CD4+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, or with LPS, CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, for 24h. ConA was added into the culture medium to stimulate CD4+T cell proliferation. The proliferation was determined by measuring [3H]-TdR incorporation in aβ-scintillation counter. Results: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10-7 mol/L to 10-9 mol/L. Anti-B7.2 antibody, but not anti-B7.1 antibody, reduced the modulatory role of CCK-8 on costimulatory activity. LPS-induced B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were inhibited by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10-7 mol/L to 10-9 mol/L. Anti-B7.1 antibody and anti-B7.2 antibody inhibited the modulatory role of LPS on costimulatory activity. Conclusions: CCK-8 enhanced macrophage costimulatory activity by upregulating B7.2 expression, and inhibited LPS-induced macrophage costimulatory activity by down-regulating B7.1 and B7.2 expressions.2 CCK-8 regulated macrophage B7.1 and B7.2 expression by cAMP-PKA-NF-κB signaling pathwayDocuments reported that NF-κB induces the expression of costimulatory molecule, B7.1 (CD80) and B7.2 (CD86). Macrophages are the most important inflammatory and immune effector cells, while LPS is the most effective and intensive activator inducing macrophages to produce and release mediators of inflammation. Some studies proved that cAMP-PKA pathway and an inhibitory signaling are involved in activation of macrophages by LPS. This signaling can down-regulate cytokine production of macrophages, such as TNF-αand IL-1β. Our current studies demonstrated that CCK-8 inhibited LPS-induced NF-κB binding activity via activation of cAMP-PKA signaling pathway and inhibition of PKC activity, and then inhibited the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6. However, there are still no reports about whether CCK-8 regulates B7.1 and B7.2 expression through CCKR-cAMP-PKA-NF-κB signaling pathway? Thus, we will focus on the signaling pathway relating to the B7.1 and B7.2 expression in resting and LPS-inducecd macrophage.Methods:①Mouse peritoneal macrophages were isolated and incubated with LPS and NF-κB inhibitor Gliotoxin, or with CCK-8 at different concentration (10-12mol·L-110-6mol·L-1), Fsk and/or H89, or with CCK receptor antagonists (10-9 mol·L-110-5mol·L-1) for 10 min prior to addition of LPS (5mg·L-1) and CCK-8 (10-8mol·L-1) for indicated time. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry (FACS). CD4+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages, which were pretreated with the superior reagents. ConA was added into the culture medium to stimulate CD4+T cell proliferation. The proliferation was determined by measuring [3H]-TdR incorporation in aβ-scintillation counter representing the assay of macrophage costimulatory activity.②Mouse peritoneal macrophages were isolated and stimulated with LPS in the absence or presence of CCK-8 and NF-κB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) 1h after stimulation. The protein levels of IκB-αand p-IκB-αin the cytoplasma were detected by Western blot 30 min after stimulation. Data were presented as x±s and analyzed by one way ANOVA and least significant difference test using SPSS 11.5 statistical program. A level of P<0.05 was considered statistically significant.Results: (1). Involvememt of NF-κB in CCK-8 regulation of B7.1 and B7.2 expression in mouse macrophages①Gliotoxin, a NF-κB inhibitor, inhibited B7.1 and B7.2 expression and costimulatory activity in LPS-activated macrophages in a dose-dependant manner, which shows that the NF-κB binding activity was related to the B7.1 and B7.2 expression in macrophage, and NF-κB was involved in regulating the costimulation of macrophage.②The NF-κB binding activity was significantly higher in mouse peritoneal macrophages stimulated with 5mg/L LPS for 1h in comparison with unstimulated cells (P<0.01), and additional treatment with CCK-8 (10-6 mol/L, 10-8mol/L, 10-10mol/L) markedly reduced the binding activity in a dose-dependent manner, the inhibition ratio was 56.58%,37.26%,15.59% respectively (P<0.05 or P<0.01). CCK-8 alone markedly increased the NF-κB binding activity at the concentrations of 10-6 mol/L and 10-8 mol/L (P<0.05), while CCK-8 had no effect on it at the concentrations of 10-10 mol/L (P>0.05). The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors.③The IκB-αprotein level in mouse peritoneal macrophages was markedly decreased while p-IκB-αprotein level increased 30 min after incubation with 5mg/L LPS (P<0.01). When added with different concentrations of CCK-8 (10-10 mol/L10-6 mol/L) in macrophages stimulated by LPS, IκB-αprotein level was increased obviously and p-IκB-αprotein level was decreased (P<0.05), leading to decrease the ratio of p-IκB-αprotein level/IκB-αprotein level in a dose-dependent manner (P<0.05). CCK-8 alone increased p-IκB-αprotein level and decreased IκB-αprotein level at the concentrations of 10-6mol/L and 10-8mol/L, then leading to increase the ratio of p-IκB-α/IκB-αprotein level (P<0.05), but with no effect on the IκB-αand p-IκB-αprotein level at the concentrations of 10-10mol/L (P>0.05). These results showed that NF-κB had correlated with the macrophage B7.1 and B7.2 expression and costimulation, CCK-8 induced IκB-αphosphorylation and enhanced NF-κB activity in unstimulated macrophage, and at the same time, inhibited NF-κB activity and IκB-αdegradation in LPS-activated mouse peritoneal macrophage, which might be the upstream mechanism of the regulative effect of CCK-8 on B7.1 and B7.2 expressions.(2). CCK-8 regulated NF-κB binding activity and B7.1 and B7.2 expression on mouse macrophages through cAMP-PKA signaling pathway①Stimulating mouse peritoneal macrophages with 5mg·L-1 LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01). CCK-8 didn't affect cAMP content and PKA activity at low concentration (10-10mol·L-1) (P>0.05), but led to a significant increase of them at high concentration (10-8mol·L-110-6mol·L-1) (P<0.05) in LPS-activated macrophage when compared with that of LPS group, suggesting that CCK-8 affect cAMP content and PKA activity in a dose-dependent manner. CCK-8 alone (10-10mol·L-1,10-8mol·L-1 and 10-6mol·L-1) increased cAMP content and PKA activity in a dose-dependent manner in unstimulated macrophages. These results demonstrated that CCK-8 activated cAMP-PKA signaling pathway at high concentration, and had a synergistical effect with LPS at high concentration on cAMP content and PKA activity in mouse peritoneal macrophages, which might be one of the immunomodulatory molecular mechanisms activated by CCK-8.②Similar to CCK-8, Fsk could increase cAMP content and PKA activity in unstimulated macrophages when compared with control group (P<0.05). Both CCK-8 and Fsk could markedly increase cAMP content and PKA activity compared with CCK-8 group (P<0.05). Stimulating mouse peritoneal macrophages with 5mg·L-1 LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01), and with additional CCK-8 or Fsk, also increase cAMP content and PKA activity (P<0.05, P<0.01). However, there was no difference in cAMP content (P>0.05), and the PKA activity was decreased (P<0.05) when LPS+H89 group, CCK-8+H89 group and LPS+CCK-8+H89 group compared respectively with LPS group, CCK-8 group, LPS+CCK-8 group. These results showed that Fsk activated cAMP and increased PKA activity, while H89 had no influence on cAMP content and inhibited PKA activity.③The NF-κB binding activity and p-IκB-αprotein level were significantly higher in mouse peritoneal macrophages stimulated with 5mg·L-1 LPS in comparison with unstimulated cells (P<0.01), of which were too little to be detected, and additional treatment with CCK-8 or Fsk markedly reduce the binding activity and p-IκB-αprotein level (P<0.05). But CCK-8 alone increased the NF-κB binding activity and p-IκB-αprotein level at the concention of 10-6 mol/L and 10-8mol/L (P<0.05). Fsk alone could increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05) which was similar to CCK-8 alone group. Compared with control group, LPS+Fsk group and CCK-8+Fsk group also increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05). When CCK-8 incubated with LPS, the NF-κB binding activity and p-IκB-αprotein level were lower than LPS group (P<0.05). H89, a PKA inhibitor, co-incubating with CCK-8 and LPS, resulted in obvious increase of NF-κB binding activity and p-IκB-αprotein level (P<0.01) when compared with LPS+CCK-8 group. These results showed that CCK-8 could further activated cAMP-PKA and inhibited the NF-κB binding activity in LPS-induced macrophages.④Fsk (forskolin, a cAMP-inducing agent) had similar effects of CCK-8 with the maximal effect at the dose of 10-6mol/L, which increasing B7.2 expression but having no effect on B7.1 expression in resting macrophages, and inhibiting B7.1 and B7.2 expression in LPS-induced macrophages. In compared with CCK-8 alone group, H89 (a PKA inhibitor) inhibited the B7.2 expression in macrophages activated by CCK-8 in a dose-dependent manner (P<0.05), on the other hand, it could reverse the effects of CCK-8, enhancing macrophage B7.1 and B7.2 expression in a dose-dependent manner (P<0.05), and had the highest effect at the dose of 10-8mol/L on LPS-activated macrophages (P<0.05). The results showed that CCK-8 modulated B7.1 and B7.2 expression through cAMP-PKA signaling pathway. All in all, the results of this part revealed that CCK-8 increased B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages and activating cAMP-PKA pathway. However, CCK-8 down-regulated B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and decreasing IκB-αdegradation in macrophages stimulated by LPS and cAMP-PKA signaling pathway being involved in the regulation of NF-κB binding activity by CCK-8 at least in part, which might be one of immunomodulatory mechanisms activated by CCK-8.(3). Effects of CCK-8 were mediated through CCK receptors①CCK-8 increased resting macrophage costimulatory activity and inhibited that of LPS-induced macrophage by mainly up-regulating B7.2 expression and down-regulating B7.1 and B7.2 expression respectively, which was mediated by CCK1R and CCK2R. However, CCK1R and CCK2R reversed the effects of CCK-8. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.②CR1409 and CR2945 could increase NF-κB binding activity and IκB-αdegradation in dose-dependent manner, which reversed the effects of CCK-8 inhibiting NF-κB activity and IκB-αdegradation in LPS-induced mouse peritoneal macrophage. The effect of CR2945 was a little lighter than CR1409. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating NF-κB activity in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.③The effect of CCK-8 on cAMP content was abrogated to different degree by CR1409 and CR2945 accompanying with the increase of their concentration. CR1409 and CR2945 did not affect the CCK-8-resulted increase of cAMP content and PKA activity at low concentration (10-9 mol·L-1), but decreased them significantly when the concentration reached to 10-7 mol·L-1～10-5 mol·L-1 compared with that of LPS+CCK-8 group (P<0.05), but the effect of CR1409 was more powerful than CR2945 (P<0.05). The inhibitory effects of CR1409 and CR2945 on the CCK-8-resulted increase of cAMP content and PKA activity were dose-dependent. The effects of CCK-8 on cAMP content and PKA activities were attenuated by CR1409 and CR2945 significantly at 10-5 mol·L-1 compare with that of CCK+LPS group (P<0.05 or P<0.01) and the order of potency of different antagonist was as follows: CR1409>CR2945. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating cAMP content and PKA activities in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.Conclusions: All the upper results suggested that CCK-8 enhanced B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages, then leading to activate cAMP-PKA signaling pathway, which were mediated through CCK receptors. However, CCK-8 decreased B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and IκB-αdegradation in LPS-induced macrophages, then leading to further activate cAMP-PKA signaling pathway, , which were mediated through CCK receptors, CCK1R and CCK2R, but CCK1R might play a main role in this process.3 CCK-8 regulated macrophage B7.1/B7.2 expression and costimulatory function in vivoAim: To investigate in vivo effects of CCK-8 on the expressions of B7.1 and B7.2 and the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Several BALB/c mice were randomly assigned to 8 groups (n=4) injected i.p. with N.S. alone, or with LPS (100μg/mouse), in the presence or absence of CCK-8 (5 nmol/mouse) and CR1409 (100μg/mouse), CR2945 (100μg/mouse). After 12h, macrophages were purified from the peritoneal exudate as indicated above. The B7.1/B7.2 expression on purified macrophages was analyzed by flow cytometry and, alternatively, purified macrophages were assayed for macrophage costimulatory activity as described above. ConA was added into the culture medium to stimulate CD4+T cell proliferation. The proliferation was determined by measuring [3H]-TdR incorporation in aβ-scintillation counter.Results and Conclusions: The in vivo administration of CCK-8 resulted in increase of B7.2 expression, but without any influence on B7.1 expression. Peritoneal macrophages harvested from CCK-8-injected mice also exhibited increased costimulatory activity for CD4+T cells. We concluded that, similar to the in vitro experiments, the in vivo administration of CCK-8 stimulated B7.2 expression and induced the costimulatory activity of peritoneal macrophages. Second, we assessed the in vivo effects of CCK-8 and CCKR antagonists (CR1409 and CR2945) in LPS-injected mice. BALB/c mice were injected i.p. with LPS (100μg/mouse), with or without CCK-8 (5nmol/mouse), CR1409 (100μg/mouse), CR2945 (100μg/mouse). Macrophages harvested 12h later were analyzed in terms of B7.1/B7.2 expression and costimulatory activity. In vivo CCK-8 administration reduced both B7.1 and B7.2 expression. Also, CCK-8 completely abolished the costimulatory activity. These effects are consistent with the in vitro effects of CCK-8 on LPS-stimulated macrophages. In vivo modulation of CCK-8 on costimulation was mediated by CCK receptors and CCK1R might be the major receptor, which was in concordance with the in vitro results.CONCLUSIONSIn the present study, we first systematically investigated the modulatory effects of CCK-8 on B7.1 and B7.2 expression and the costimulatory activity in resting and LPS-activated mouse peritoneal macrophages, in vitro and in vivo, at multi-levels from receptors to cAMP-PKA signaling pathway and transcription factors. Then we first studied the intracellular signaling mechanisms of CCK-8 in the regulation of B7.1 and B7.2 expression and its correlation with the costimulatory function of macrophages.1 The present studies for the first time demonstrated that, in unstimulated macrophages, CCK-8 up-regulated B7.1 and B7.2 expression, but mainly B7.2 expression at protein level in vitro and in vivo, and induced the costimulatory activity. In constrast, in LPS-activated macrophages, CCK-8 down-regulated the expression of both B7.1 and B7.2 expressions, and abolished the costimulatory activity. The effects of CCK-8 on the costimulatory activity for T cells correlated closely with the expression of B7.1/B7.2, and might be mediated through the modulation of B7 expression.2 CCK-8 up-regulated B7.1 and B7.2 expression and increased costimulatory activity by activating cAMP-PKA-NF-κB signaling pathway in macrophages. CCK-8 down-regulated B7.1 and B7.2 expression and inhibited its costimulatory activity in LPS-activated macrophages by activating cAMP-PKA signaling pathway, and inhibiting IκB-αphosphorylation and the NF-κB binding activity through CCK receptors, and CCK1R might play a main role in this process.In conclusion, this study firstly demonstrated that CCK-8 regulated the function of macrophages through the modulation of B7.1 and B7.2 expression, which might be one of the mechanisms of CCK-8 on regulating immunomodulatory functions.
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