| One theory of immune regulation involves homeostasis between T-helper 1 (Th1) and T-helper 2 (Th2) activity. The Th1/Th2 hypothesis arose from 1986 research suggesting mouse T-helper cells expressed different cytokine patterns. Th1 cells produce IFN-γ,IL-2,IL-12,TNF-αand Th2 cells produce IL-4,IL-5,IL-6,IL-10,IL-13 just only and respectively.This hypothesis is adapted to human immunity, with Th1- and Th2-helper cells invoving in different immune response pathways. Th1 cells modulate the type-1 pathway ("cellular immunity") to fight viruses and other intracellular pathogens, kill cancerous cells, and induce delayed-type hypersensitivity (DTH) skin reactions. Th2 cells induce the type-2 pathway ("humoral immunity") and up-regulate antibody production against extracellular organisms;Overactivation of either pattern can cause disease, and either pathway can down-regulate the other.The imbalance of Th1/Th2 involved in the pathological process of many disease ,for example,atopic disease,rheumatic disease, alloimmune response,infection and tumor.Rectifying the imbalance of Th1/Th2 is a new theroputic pathway for all these disease.Cholecystokinin (CCK) is a typical braingut peptide, which was discovered in 1928 by Ivy and Oldferg. cholecystokinin octapeptide (CCK-8) is the major function fraction of CCK.CCK-8 localizes in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localizes in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role. Recent studies have demonstrated the presence and the regulatory function of CCK-8 in the immune system,but all these studies focused on the effection of CCK-8 on the innate immune response,there is rarely studies on its fuction in regulating the adaptive immune response.In our previous studies ,we found that CCK-8 has the function to anti-inflammation and protect endotoxin shock ,in this study we will found if CCK-8 has the function to regulate the imbalance of Th1/Th2.Mitogen-activated protein (MAP) kinases have been implicated in many physiological processes, including cell proliferation, differentiation, and death. These processes are relevant to the development and function of T cells. There 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). These MAP kinases are activated by dual phosphorylation on the tripeptide motif Thr-Xaa-Tyr.The sequence of this tripeptide motif is different in each group of MAP kinases.The ERK, p38, and JNK groups of MAP kinases are present in T cells.A lot of studies have proofed the provitol role of MAP kinases in CD4+ T cells activation and differentiation into Th1/Th2 cells.CCK-8 can activate the three MAP kinases in many tissues and cells,indicated that CCK-8 has a regulation function of MAP kinases.In our study we will investigate the regulation function of CCK-8 on activating MAPK in antigen primed splenocytes and its relationship with Th1/Th2 cytokine production,in order to found the signal transduction mechnisms in the effection of CCK-8 on Th1/Th2 imbalance.Except for CD4+T cells ,CD8+ T cells also can produce a lot of Th1/Th2 cytokines in vitro,but they mostly produce IFN-γin vivo.So, CD4+ and CD8+ T subsets has a nearly correlation with Th1/Th2 balance.In our study,we will investigate the regulation function of CCK-8 on CD4+ and CD8+ T subsets and its effection on Th1/Th2 imbalance synchronizely,in order to found that whether CCK-8 effects Th1/Th2 balance through the regulation of T lymphocyte subsets.When antigen entered the bodies,immuncytes were activated,and involved in immune response,now Th1/Th2 and T subsets equilibrium was important to keep the homeostasis of the immune system and the whole body. CD4+ T cells hyperactivation and Th2 predominance involved in the allergric disease for example asthma.So we will also investigate the effects of CCK-8 on allergric pulmonary injury as well as regulation of Th1/Th2 imbalance in order to probe into the significant of CCK-8 in the treatment of allergric disease.1 KLH induced Th1/Th2 imbalance in splenocytes of Balb/c miceObjective:To make an animal model of Th1/Th2 imbalance.Methods:⑴Female Balb/C mice were divided into different groups immunized with different doses of KLH emulsified in complete Freund's adjuvant once or twice.The mice were sacrificed in indicated times,splenoctes were acquired and incubated in vitro,the production of Th1 cytokines IFN-γ,IL-2,IL-12p40 and Th2 cytokines IL-4,IL-5 in cell culture supernates were detected by ELISA.⑵Female Balb/C mice were immunized subcutaneously on the dorsum with 125μg KLH emulsified in complete Freund's adjuvant on day 1. On day 7,the mice were challenged subcutaneously with the same dosage of KLH﹢CFA.On day 15, The mice were sacrificed and blood serum were acquired,the specific KLH antibody IgG2a and IgG1were detected by ELISA.Results:⑴The spleen of KLH immunized mice appeared hypertrophic generally and the number of splenocytes was manifold((1.68±0.80)×108～(2.36±0.90)×108,P﹤0.01 or 0.05 vs control((0.39±0.13)×108)).⑵Splenoctes derived from KLH immunized mice incubated in vitro without the same antigen restimulation produced undetectable levels of IL-2 and low levels of IFN-γ,IL-5,IL-4 and IL-12p40,but when restimulated with KLH,they can produce abundant IL-5,an elevated IL-4,IL-2 and IFN-γ, but not IL-12p40,the IL-4/ IFN-γratio was elevated.⑶IL-2 production by KLH immunized splenocytes was elevated from incubated for 24 h,and achieved pinnacle at 48h,then descended gradually.IL-4,IL-5 and IFN-γwas elevated from 24 h, and elevated gradually to 96h.IL-12 p40 production was very low at every time points.⑷Using different doses of KLH and immunized once or twice can all lead to the elevated production of IL-2,IL-4,IL-5,IFN-γand lead to the elevation of IL-4/ IFN-γratio,but the group using 125μg KLH and immunized twice was higher than other groups.⑸The level of KLH specific antibody IgG2a and IgG1,especially IgG1 was elevated in blood serum of KLH immunized mice.Conclusions:⑴Female Balb/C mice immunized subcutaneously on the dorsum with different doses of KLH emulsified in complete Freund's adjuvant can induce a Th2 predominance imbalance in splenocytes,but middling doses and immunized twice can help us acquire a more elevated cytokine production.⑵KLH immunized splenocytes must be restimulated with the same kind of antigen for reactivation and differentiation in vitro.⑶KLH immunized splenocytes produced different kind of cytokines in vitro in a time depended manner,the peak value of IL-2 was about 48 h,and that of IL-4,IL-5,IFN-γwas about 96h.2 Cholecystokinin octapeptide modulates Th1/Th2 cell balance in splenocytes of KLH immunized miceObjective: To investigate the effection of CCK-8 on Th1/Th2 cell imbalance in splenocytes of KLH immunized mice.Methods:⑴in vivo experiment:Female Balb/c mice were devided into five groups(n=4):①KLH group,mice were immunized subcutaneously on the dorsum with 125μg KLH emulsified in complete Freund's adjuvant on day 1. On day 7,the mice were challenged subcutaneously with the same dosage of KLH﹢CFA.②KLH﹢CCK 1 nmol group,③KLH﹢CCK 5 nmol group,④KLH﹢CCK 10 nmol group,mice were immunized by the same way as KLH group,and since day 7, CCK-8 1 nmol,5 nmol ,10 nmol or saline were injected intraperitoneally once a day for 7 days.⑤Control group,mice were injected saline every times. On day 15,the mice were sacrificed, splenoctes were acquired and incubated in vitro with KLH restimulation,the production of Th1 cytokines IFN-γ,IL-2 and Th2 cytokines IL-4,IL-5 in cell culture supernates being collected in indicated times were detected by ELISA. IFN-γ,IL-2,IL-4,IL-5mRNA expression was detected by RT-PCT. Blood serum were also acquired,the specific KLH antibody IgG2a and IgG1were detected by ELISA.⑵in vivo experiment:Female Balb/C mice were immunized subcutaneously on the dorsum with 125μg KLH emulsified in complete Freund's adjuvant on day 1. On day 7,the mice were challenged subcutaneously with the same dosage of KLH﹢CFA.On day 15,the mice were sacrificed, splenoctes were acquired and restimulated in vitro with KLH and devided into different groups①KLH group(which was restimulated with KLH alone) and CCK group(which was incubated with CCK-8 1×10-8 mol/L together with KLH restimulation),cell culture supernates were collected at 6h,12h,24h,48h,72h,96h respectively,IFN-γ,IL-2,IL-4,IL-5 production were detected by ELISA.②KLH group,CCK group which was incubated with different doses of CCK-8 together with KLH restimulation, cell culture supernates were collected at 24h and 96h, IFN-γ,IL-2,IL-4,IL-5 production were detected by ELISA.Results:⑴In vivo experiments:①Splenocytes derived from control mice produced low levels of IFN-γ,IL-2,IL-4,and a comparatively hige level of IL-5.Splenocytes derived from KLH immunized mice produced much higher levels of IL-5,IL-2,IL-4 and IFN-γthan control mice(P<0.01 vs control),IL-4/IFN-γratio was elevated(P<0.01 vs control).(the production of IFN-γ,IL-2,IL-4 and IL-5 in KLH group was (70.12±9.98)ng/L,(264.53±98.77)ng/L,(200.96±20.51)ng/L,(1147.19±271.04)ng/L respectively,the production of IFN-γ,IL-2,IL-4 production in control was undetectable,the production of IL-5 in control was(45.84±12.13)ng/L;IL-4/IFN-γratio of KLH group was 2.91±0.49 , that of control was 0.32±1.55).Splenocytes derived from KLH immunized mice together injected with different doses of CCK-8 in vivo produced much higher IFN-γand IL-2 than KLH group,but IL-4 and IL-5 production was reduced (P<0.01 or 0.05vs KLH).(The production of IFN-γ,IL-2,IL-4 and IL-5 in KLH﹢CCK 1 nmol,5 nmol,10 nmol group was(122.11±57.47)ng/L,(286.11±113.75)ng/L,(112.64±32.80)ng/L,(508.21±139.84)ng/L;(131.11±50.81)ng/L,(330.53±49.15) ng/L,(107.53±33.18) ng/L,(372.73±147.38) ng/L;(155.10±29.99) ng/L , (388.27±35.44) ng/L , (98.77±26.00) ng/L ,(378.80±187.65) ng/L respectively;IL-4/IFN-γwas 1.11±0.55,1.098±1.01, 0.65±0.22 respectively)②IFN-γ,IL-2 mRNA expression in splenocytes derived from control mice was very low,but IL-4 and IL-5mRNA,especially IL-5mRNA was comparatively hige. IFN-γ,IL-2 and IL-4 mRNA expression in splenocytes derived from KLH immunized mice was elevated(P<0.01 or 0.05 vs control),IL-5mRNA was elevated too,but there is no statistics differentia with control mice. IFN-γ,IL-2 mRNA expression in splenocytes derived from KLH﹢CCK groups was much high than that of KLH group,but IL-4,IL-5mRNA expression was much lower than KLH group(P<0.01 or 0.05 vs KLH).③KLH specific IgG2a and IgG1 in blood serum was elevated in KLH immunized mice (P<0.01 vs control),CCK-8 made the level of IgG1 reduce and the level of IgG2a heighten(P<0.01 or 0.05 vs KLH).⑵In vitro experiments:①Splenocytes derived from KLH immunized mice produced a certain level of IFN-γ,IL-2,IL-4 and IL-5 when restimulated in vitro.Being given 1×10-8 mol/L CCK-8 together with KLH restimulation made the concentration of IL-4 in cell culture supernates elevate at 6h,12h and 24h,IFN-γconcentration elevate distinctly at 96h,IL-2 concerntration of KLH﹢CCK groups was higher than KLH groups which was at the same time point(P<0.01 or 0.05vs KLH).There was no distinct effection of 1×10-8 mol/L CCK-8 on IL-5 production at all time points.②CCK-8 stimulation IL-2,IFN-γand IL-4 production in a dose-depended manner and there was no effection of different doses of CCK-8 on IL-5 production.Conclusions:in vivo CCK-8 enhanced Th1 response and reduced Th2 response in KLH immunized mice,in vitro CCK-8 stimulated KLH-immunezed precursor and memory T cells reactivation and differentiation into Th 1 cells,suggested CCK-8 can regulat adaptive immune respons,it maybe involved in immune physiology and pathology and it maybe a useful regulator in regulation of Th1/Th2 imbalance.3 Cholecystokinin octapeptide effects on the process of MAPK signal transductions modulating Th1/Th2 imbalance in KLH immunized splenocytesObjective: To investigate the function of MAPK signal transductions in modulate Th1/Th2 imbalance in splenocytes induced by KLH immunization and investigate the effects of CCK-8 on these processes.Methods:⑴Western Blot detection of MAPK activation in KLH immunized mice:Female Balb/C mice were devided into KLH group and control group,immunal method was identical with that previously described in part two.Then the mice were sacrificed and splenoctes were acquired. Splenoctes derived from KLH group was restimulated in vitro with KLH and devided into different groups:KLH group, KLH﹢CCK group, KLH﹢CCK﹢CR1409 group, KLH﹢CCK﹢CR2945 group, KLH﹢PD98059 group, KLH﹢SP600125 group, splenoctes derived from control group was given PBS at the same time.After incubated for 3h,the cells were collected to extract total cell proteins, p38 MAPK, ERK1/2 and JNK1/2 activation was detected by Western Blot.⑵Female Balb/C mice were immunezed with KLH as previously described. Then the mice were sacrificed,splenoctes were acquired and restimulated in vitro and devided into different groups:KLH group, KLH﹢CCK group, KLH﹢PD98059 group, KLH﹢SP600125 group, KLH﹢CCK﹢PD98059 group, KLH﹢CCK﹢SP600125 group.After incubated for 48 h and 96h,the cell culture supernates were collected and the concentration of IL-2(48h), IL-4,IFN-γ(96h) was detected by ELISA.Results:⑴There was no distinct diversity within different groups of p38 MAPK.The level of p-p38 MAPK was low in control group,and was elevated in KLH group(about 5.21 fold vs control, P<0.01 vs control),but was reduced in KLH﹢CCK group(about 3.15 fold vs control,P<0.05 vs KLH).In KLH﹢CCK﹢CR1409 group and KLH﹢CCK﹢CR2945 group the level of p-p38MAPK was about 4.82 fold and 3.38 vs control,partly reversed the inhibition effects of CCK-8 on p38 MAPK activation.⑵The level of p-ERK1/2 was low in control group, and was elevated in KLH group(about 7.97 and 6.64 fold for p-ERK1/2 respectively vs control, P<0.01 or 0.05 vs control).In KLH﹢CCK group the level of p-ERK1/2 was much higher than KLH group (about 17.40 and 21.57 fold for p-ERK1/2 respectively vs control, P< 0.05 vs KLH).In KLH﹢CCK﹢CR1409 and KLH﹢CCK﹢CR2945 group,the level of p-ERK1/2 was about 11.75,20.10 and 11.69,14.90 fold vs control group,partly reversed the stimulation effect of CCK-8 on ERK1/2 avtibation.In KLH﹢PD98059 group, the level of p-ERK1/2 was inhibited distinctly about 0.31 and 0.52 fold vs control.⑶The level of p- JNK1/2 was low in control group, and was elevated in KLH group(about 24.21 and 25.25 fold for p- JNK1/2 respectively vs control) In KLH﹢CCK group the level of p- JNK1/2 was much higher than KLH group (about 482.73 and 337.78 fold for p-JNK1/2 respectively vs control, P< 0.01 vs KLH).In KLH﹢CCK﹢CR1409 and KLH﹢CCK﹢CR2945 group,the level of p-JNK1/2 was about 321.28, 273.64 and 74.09, 25.64 fold vs control group,partly reversed the stimulation effect of CCK-8 on JNK1/2 activation.In KLH﹢SP98059 group, the level of p-JNK1/2 was inhibited distinctly about 3.71 and 3.80 fold vs control.⑷Splenocytes derived from KLH immunized mice produced a certain level of IL-2, IL-4 and IFN-γwhen restimulated in vitro, PD98059 and SP600125 inhibited IL-2,IL-4 and IFN-γproduction in KLH immunized splenocytes, SP600125 made the IL-4/IFN-γratio elevate but PD98059 made the IL-4/IFN-γratio reduce.Being given 1×10-8 mol/L CCK-8 together with KLH made the concentration of IL-2 and IFN-γin cell culture supernates elevate,but had little effect on IL-4 production,made the IL-4/IFN-γratio reduce,KLH﹢CCK﹢PD98059 group and KLH﹢CCK﹢SP600125 group produce much higher IL-2,IL-4 and IFN-γthan KLH﹢PD98059 group and KLH﹢SP600125 group ,but lower than KLH﹢CCK group .Conclusions:⑴KLH immunization made p38 MAPK, ERK1/2 and JNK1/2 in splenocytes of Balb/c mice activate,especially p38 MAPK and ERK1/2.ERK , JNK specific inhibitors inhibited IL-2 ,IL-4 and IFN-γproduction in KLH immunized splenocytes, SP600125 made the IL-4/IFN-γratio elevate but PD98059 made the IL-4/IFN-γratio reduce,suggested ERK and JNK signal transduction involved in the Th1/Th2 imbalance induced by KLH immunization.⑵C CK-8 inhibited p38 MAPK activation,and stimulated ERK1/2, JNK1/2 phospholation ;CCK-8 together with KLH made the production of IL-2 and IFN-γelevate,but had little effect on IL-4 production,made the IL-4/IFN-γratio reduce,KLH﹢CCK﹢PD98059 group and KLH﹢CCK﹢SP600125 group produce much higher IL-2,IL-4 and IFN-γthan KLH﹢PD98059 group and KLH﹢SP600125 group ,but lower than KLH﹢CCK group ,suggested that CCK-8 can modulate Th1/Th2 imbalance through MAPK and other signal transductions.⑶Specific CCK receptor inhibitors reversed CCK-8 effects on MAPK activation,suggested CCK-8 can regulate MAPK activation through its receptors.4 Cholecystokinin octapeptide modulates T-lymphocyte subsets in KLH immunized miceObjective:To study the effects of CCK-8 on T-lymphocyte subsets in KLH immunized mice.Methods: Female Balb/c mice were devided into five groups(n=4):①KLH group,mice were immunized subcutaneously on the dorsum with 125μg KLH emulsified in complete Freund's adjuvant on day 1. On day 7,the mice were challenged subcutaneously with the same dosage of KLH﹢CFA.②KLH﹢CCK1 nmol group,③KLH﹢CCK5 nmol group,④KLH﹢CCK10 nmol group,mice were immunized by the same way as KLH group,and since day 7, CCK-8 1 nmol,5 nmol ,10 nmol or saline were injected intraperitonealy once a day for 7 days.⑤Control group,mice were injected saline every times. On day 15,the mice were sacrificed, splenoctes were acquired and incubated in vitro with KLH restimulation,the production of Th1 cytokines IFN-γand Th2 cytokines IL-4 in cell culture supernates being collected in indicated times were detected by ELISA. IFN-γand IL-4 mRNA expression was detected by RT-PCT. Positive percent of CD4+and CD8+ T-cell in peripheral blood or splenocytes were measured using flow cytometer. In addition,lung tissue sections were made and stained with HE.Results:⑴KLH immunization upregulated the positive percent of CD4+and CD8+ T lymphocytes both in peripheral blood and in splenocytes,resulted in the elevation of CD4+/CD8+ ratio. (The positive percent of CD4+and CD8+ T lymphocytes in peripheral blood was as follows:KLH CD4 + :(70.51±1.75)%,control:CD4+:(35.28±3.65)%; KLH CD8+: (20.13±1.02)%,control CD8+:(14.43±2.02)%. The positive percent of CD4+and CD8+ T lymphocytes in splenocytes was:KLH CD4+:(70.95±4.97)%,control:CD4+:(29.89±1.83)%;KLH CD8+:(16.80±1.55)%),control CD8+:(12.09±1.19)%.CD4+/CD8+ ratio in peripheral blood was:KLH :3.50±0.59,control :2.46±0.29,in splenocytes was:KLH :4.24±0.41,control :2.49±0.33.P<0.01 vs control).CCK-8 downregulated the positive percent of CD4+and CD8+ T lymphocytes both in peripheral blood and in splenocytes in KLH immunized mice,resulted in the reduction of CD4+/CD8+ ratio.( The positive percent of CD4+and CD8+ T lymphocytes in peripheral blood was as follows:1 nmol CD4+:(58.22±4.82)%, 5 nmol CD4+:(45.00±3.47)%, 10 nmol CD4+:(39.13±2.64)%;1 nmol CD8+:(18.31±0.79)%,5 nmol CD8+:(15.06±0.86)%;10 nmol CD8+:(14.95±1.40)%, in splenocytes was :1 nmol CD4+:(52.35±3.37)%,5 nmol CD4+:(41.44±6.54)%, 10 nmol CD4+:(34.42±2.98)%;1 nmol CD8+:(15,96±01.3)%,5 nmol CD8+:(15.19±1.45)%,10 nmol CD8+:(13.44±1.57)%.CD4+/CD8+ ratio in peripheral blood was:1 nmol :3.18±0.23,5 nmol:3.03±0.41,10 nmol :2.61±0.30;in splenocytes was:1 nmol :3.30±0.83,5 nmol :2.82±0.81,10 nmol :2.59±0.43. P<0.01 or P<0.05 vs KLH).⑵CCK-8 improved the production of IFN-γ, elevated IFN-γgene transcription,whereas cut down the production of IL-4,decreased IL-4 gene transcription. CCK-8 lightened the pulmonary inflammation induced by KLH.Conclusion:CCK-8 inhibited CD4 + T lymphocytes activation, Th2 cytokine mRNA expression and protein production in KLH immunized mice,indicating CCK-8 could modulate adaptive immune response.CONCLUSIONSIn the present study, we first systematically investigated the modulatory effects of CCK-8 on Th1/Th2 imbalance induced by KLH immunization.We found that CCK-8 can modulate Th1/Th2 imbalance through its specific receptors and MAPK signal transduction was involved; CCK-8 can modulate T subsets imbalance and protect the allergric injury in pulmonary induced by antigen,suggested CCK-8 can regulate adaptive immune response,it maybe involved in immune physiology and pathology and it maybe a useful regulator in regulation of Th1/Th2 imbalance.
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