Study On The Role Of IL-31 In Skin Inflammation And Its Mechanism

Objective To clone human IL-31 gene,construct prokaryotic and eukaryotic expression vector,express it in E.coli BL21 and mammalian cell CHO,and study the role of hIL-31 in skin inflammation.Methods Total RNAs were isolated from peripheral blood mononuclear cells after twelve-hour activation with PMA and PHA.Full-length human IL-31 cDNA was amplified by RT-PCR and cloned into pGEM-T and sequenced.The functional fragment of human IL-31 gene without signal peptide was PCR amplified from the pGEM-T vector containing human IL31 cDNA and cloned into the pET28a(+) vector.The recombinant protein was achieved in E.coli through IPTG induction.Meanwhile hIL-31 gene was cloned into eukaryotic expression plasmid pcDNA3.1/myc-His(-)A.The expression of rhIL-31 in CHO cells were analysed by RT-PCR and western-blot.The recombinant protein purified with Ni resin was used to study the role of hIL-31 in skin inflammation and prepare hIL-31 polyclonal antibody.Chemotaxis of supernatant of human epidermal keratinocyte HaCaT cells to PBMC was detected through transwell madreporic plate after HaCaT cells were stimulated by different dose of rhIL-31.The effect of the recombinant protein on the production of MIP-3β,TARC,MDC andⅠ-309 was detected by RT-PCR and real-time RT-PCR.Phosphorylation of STAT1,STAT3 and STAT5 in HaCaT cells was analysed by western-blot.BALB/c mice were injected intradermally with hIL-31.Histopathology of paraffin-embedded skin section from BALB/c mice were stained with hematoxylin and eosin,and observed with microscope.The blood cells were counted.The levels of serum cytokine IL-1β,IL-6 and TNF-αwere detected by ELISA.Cell suspensions from thymus and spleens were stained with fluorescence-labed mAbs to a variety of cells surface,then were analysed by flow cytometry.Results Obtained full encoding sequence of hIL-31 was identical with that included in Genbank,and the prokaryotic expression vector pET28a(+)-hIL31 and eukaryotic expression vector pcDNA3.1/myc-His(-)A-hIL31 were constructed correctly.IL-31 could be expressed in E.coli after IPTG induction.IL-31 could be expressed in CHO cells.The protein was bound by 6×his monoclonal antibody and hIL-31 polyclonal antibody.It was found supernatant of HaCaT cells can chemoattract PBMC.The expression of chemokines and phosphorylation of STAT1,STAT3 and STAT5 were up-regulated in HaCaT cells. Soluble IL-31 could stimulate the expression of MIP-3β,TARC,MDC andⅠ-309 in HaCaT.Mice treated with IL-31 had hair loss,and inflammatory cell infiltration was observed in histopathogy of paraffin-embedded skin section.The number of peripheral white blood cells was increased,with the increased percentage of the neutrophil.The increased serum IL-1β,IL-6 and TNF-αwas detected.The data from flow cytometric analysis indicated that proportion of spleens CD4~+ T lymphocytes was increased.Conclusion Human IL-31 is cloned and expressed successfully in E.coli and CHO cells.Supernatant of HaCaT cells can chemoattract PBMC after being treated with rhIL-31 for 6 hours.IL-31 stimulates the expression of MIP-3β,TARC,MDC andⅠ-309 in HaCaT. IL-31 induces skin inflammation in mice.