In Vitro The Comparison Of The Effect Of Interleukin 10 Signal Transduction Mechanism On Immune Effector Cells In C57BL/6 And MRL/1pr Mice

IntroductionSystemic lupus erythematosus is a kind of autoimmune disease.Many reseach has confirmed the concentration of IL-10 increases in SLE patients' serium and is related to activity of SLE.IL-10 must combine its special receptor to put into fullplay.During our previous reseach,we found many changes of the sequences in the encoding areas of IL-10RA gene.The change of G1146A codon induces encoding amino acid to change from glycine to glutamic acid.Consequently the motif taking on negative regulation of IL-10 signal transduction and phosphorated by tyrosine-protein kinase(TPK)JAK1 and TYK2 is absent.So,the signal transduction mediated by NZW IL-10R cannot inhibit cellular immune and inflammation but boost up humoral immune.That brings on a series of immunopathologic process.Sequently SLE comes on.We found IL-10RA gene of MRL/lpr^- is the same as NZW's after measuring the 1146^thbase of IL-10RA in many strains of mice with PCR-SSCP.So we presumed some locus polymorphism of IL-10R coding gene induced corresponding amino acid to change,then led to the refashionment of receptor function and influenced signal tranduction system.Ultimately susceptibility of organism to SLE altered.We regard C57BL/6 and MRL/lpr^- mice as reseach subjects and detect the function and activity of their peritoneal exudated macrophages and splenocytes,which lays a foundation for studying the mechanism of IL-10R signal transduction and its role during SLE.Materials and Methods1,Sample C57BL/6 and MRL/lpr^- mice(SPF),female,8～10 weeks old.2,Reagentsthioglycollate broth(BD company);recombinant murine interleukin 10(R&D Systems);BioEasy SYBR Green I Real Time PCR Kit(BIOER Technology CO.,LTD); rabbit anti-mouse IgG(H+L)ads,IgG(H+L)-ads-HRP,goat anti-mouse IgM-ads,IgM-ads-HRP,goat anti-rabbit IgG(H+L)mouse/human ads-HRP(Southern Biotechnology Associates,Inc);rabbit anti-mouse JAK1,TYK2,STAT3(Santa Cruz Biotechnology);rabbit anti mouse p-STAT3,β-actin(Cell Signaling Technology, Inc);Super ECL Plus Detection Reagent(Applygen Technologies Inc.).3,Method(1)Cell Preparation and CulturePeritoneal exudate macrophages and splenocytes were harvested from C57BL/6 and MRL/lpr^- mice primed 3 days earlier by receiving an i.p.injection of 2ml of 4.05% thioglycollate broth.Cell were cultured in 96-well plates and 6-well plates in the presence or absence of IL-10.(2)Proliferation assayThe proliferation of macrophages and splenocytes from C57BL/6 and MRL/lpr^-mice were made by MTT colorimetric technique after incubation for 72h at 37℃in a humidified atmosphere containing 5%CO₂.(3)Real time PCRPeritoneal exudate macrophages were incubated at 37℃for 72h and adherent cells were collected for isolation of total RNA using ISOGEN.First-strand cDNA was synthesized using an Oligo(dT)primer.Amplification ofβ-actin served as a reference to normalized data for the total amount of RNA input.(4)ELISAIgG,IgM secretion was evaluated by sandwich ELISA using commercial antibodies.(5)Western blotProtein concentration of the lysates was measured.Protein was then subjected to Western blot analysis:â‘ electrophoresis;â‘¡transfer from gel to PVDF membrane; â‘¢blocking;â‘£incubation with primary antibody;⑤enzyme conjugate incubation;â‘¥substrate incubation.4,Statistical analysisAll data are shown as mean values±SD unless otherwise indicated.Comparison between groups was made using the one-way ANOVA analyse.Values of p＜0.05 were considered significant.ResultWith augmented concentration of interleukin 10,the proliferation of peritoneal exudated macrophages and splenocytes,the synthesized ability of antibody in cell supernatant and the secretory volume of inflammatory cytokine from C57BL/6 mice present downgrading;the proliferation of peritoneal exudated macrophages and splenocytes,the synthesized ability of antibody in cell supernatant and the secretory volume of inflammatory cytokine from MRL/lpr^- mice display upgrading;JAK1,TYK2 and STAT3 from peritoneal exudated macrophages and splenocytes are both expressed in the two strains of mice,but the phosphorylation degree of STAT3 from MRL/lpr^- is lower than that from C57BL/6 mice.DiscussionSystemic lupus erythematosus is a kind of autoimmune disease.IL-10 concentration in SLE patients' serium is higher than normal persons significantly and is related to the activity of lupus closely.However IL-10 is controversial during the pathogenesis of lupus.Experimentation on animals also demonstrates IL-10 may play an important role in some kinds of autoimmune disease.IL-10 is an important susceptible factor during the pathogenesis of SLE while the activity of IL-10 brings into play by light of IL-10 receptor.So we presume IL-10R play an important part in the pathogenesis of SLE.Recently some studies discover some sites present polymorphism in IL-10 coding gene,while some polymorphism may induce the change of correspongding amino acid,then lead to the refashionment of receptor function and influence signal tranduction system further.Ultimately susceptibility of organism to SLE altered. Up to now,the clearest IL-10 signal transduction pathway is JAK/STAT(Janus Kinase/Singnal Transducer and Activator of Transcription)system.IL-10/IL-10R interaction phosphorylates special tyrosine residue through JAK1,TYK2 protein kinase.Accordingly tyrosine on STAT3 molecule is phosphorylated by JAKs which are combined with receptor in turn,which activates gene transcription.We find phosphorylation degree of proteins from MRL/lpr^- is lower than that from C57BL/6 mice after testing protein of peritoneal exudated macrophages and splenocytes of two strains of mice with western blot.So we presume low phosphorylation degree cannot or cannot totally activate STAT3 to conduct or conduct subsequent signal completely,which induces inhibition effect of IL-10 to die out.IL-10 develops inhibition effect in C57BL/6 mice and develops proliferation effect in MRL/lpr^- mice,which makes us presume mutual IL-10R gene induces subsequent signal to conduct obstructively after IL-10 combines IL-10R,which leads to abnormal function.At last interleukin 10 cannot inhibit cell mediated immunity and inflammatory reaction but boost up humoral immune.That brings on a series of immunopathologic process.Sequently SLE comes on.By the nature of things,the supposition is still await to be demonstrated.ConclusionInterleukin 10 cannot inhibit cell mediated immunity and inflammatory reaction but promote humoral immune in MRL/lpr^- mice,which mediates a series of immunopathologic process then induces SLE.