Study On The Effect Of MMPs And Antigen Peptides Presented By HLA-B*1301in TCE Induced Skin Hypersensitivity

Occupational dermatitis medicamentosa-like of trichloroethylene (DMLT) has been becoming an occupational disease which seriously affected the health of workers who were exposed to TCE during work. DMLT is characterized by generalized sever dermatitis, often accompanied by systemic symptoms or signs such as fever, lymphadenopathy, abnormal liver function and nephritis. Specific preventive and treatment is absent for DMLT, because it’s mechanism is unclear until now.It has been shown that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) play critical roles in the induction and/or progress of inflammatory skin diseases such as drug eruptions. Skin patch test performed in cured patients with DMLT gave the evidence that metabolites produced in the oxidative metabolism of TCE play important roles in the induction of DMLT. Based on these data, the first section of this paper will concentrate on the effect of tricholoehanol (TCOH) and trichloroacetic acid (TCA), which are two major oxidative metabolites of TCE in vivo, on the expression of MMPs and TIMPs in HaCaT cell, an immortalized keratinocyte cell line.Our team’s previous work, a case-control study performed in TCE-exposed workers, found that human leukocyte antigen (HLA)-B*1301is strongly associated with the DMLT susceptibility and indicated that HLA-B*1301is crucial in the pathogenesis of DMLT. So the second section of the paper will focus on the molecular biological method to clone HLA-B*1301and HLA-B*1302, which has only six basic difference and3amino acidic difference with HLA-B*1301, to establish C1R-B*1301and C1R-B*1302cell lines which highly express HLA-B*1301and HLA-B*1302, respectively and to isolate and purify HLA-B and it’s associated intrinsic antigenic peptides. 1. Effects of TCOH or TCA on the expression and/or gelatinolytic activity of MMPs and TIMPs in HaCaT cellsThe HaCaT cells were treated with different concentrations of TCOH or TCA (0,0.25,0.5,1.0,2.5,5.0mM) up to144hours. The geltin-zymography was used to assay the effect of TCOH or TCA on the geltinolytic activity of HaCaT cell at different time points. Real-time PCR was employed to determine the influence of TCOH or TCA on the mRNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-2in HaCaT for24hours treatment. And western blotting was adopted to detect the MMP-9and TIMP-1protein expression alterations in HaCaT cells after treatment with TCOH. After TCOH treatment, the HaCaT cells expressed higher level of MMP-9and TIMP-1mRNA and protein, and a significant dose-effect relationship between TCOH concentration and MMP-9or TIMP-1mRNA and protein was observed. At the meantime, TCOH induced more MMP-9release to the culture media, which was evidenced by the higher gelatinolytic activity after treatment of HaCaT cells with TCOH. However, we did not observe such effects with TCA in HaCaT cells. Those data indicate that TCOH might play important roles in TCE induced skin hypersensitivity by up-regulation of MMP-9and TIMP-1in skin keratinocytes.2. The construction of C1R-B*1301and C1R-B*1302cell lines and the purification of HLA-B*1301and HLA-B*1302We amplified HLA-B cDNA of DMLT patient who is HLA-B*1301allele carrier by application of HLA B locus sequence specific polymerase chain reaction (PCR). We cloned the HLA-B*1301cDNA into the eukaryotic expression vector of pcDNA3.1(+) by using the gene recombination technique. And we got pcDNA3.1-B*1302using pcDNA3.1-B*1301as template by using site-direction mutagenesis method. The pcDNA3.1(+), pcDNA3.1-B*1301FLAG and pcDNA3.1-B*1302were transfected into C1R cell using Lipofectin liposome mediated transfection method. HLA-B*1301and HLA-B*1302protein were purified using the immunoaffinity chromatography which is based on linkage of W6/32monoclonal antibodies to the protein A sepharose CL-4B beads. W6/32mAb is antibody raised against HLA ABC nonpolymorphic region. The purified the HLA-B molecules released associated intrinsic antigens following acidic and healing treatment and the latter were collected by ultracentifugation with a3kD cut-off ultracentifugal device. The method established in this section provide methodology basis for research the associated peptide sequence of HLA-B*1301and HLA-B*1302. Significant difference of preference amino acid in major anchoring position (P2and P9) of antigenic peptides presented by HLA-B*1301was observed, in comparison with ones of HLA-B*1302. For instance, the preference amino acid in P2and P9of antigenic peptides presented by HLA-B*1301are lysine and glutamine (P2) and lysine, phenylalanine and isolucine, respectively. And the ones for HLA-B*1302are serine and leucine (P2) and lysine and valine. Moreover, the preference amino acid in minor achoring position for HLA-B*1301and HLA-B*1302are also different markedly. All these data indicate that the molecular bais underlying the strong association between trichloroethyelene induced hyperrespontivity reaction and HLA-B*1301might lie on antigenic peptide sequence characteristics presented by HLA-B*1301. Comparing the preference amino acid in major anchoring position of antigenic peptides presented by other HLA-B molecules, we found that the difference is not significant, suggesting that the interaction and joint action between major and minor anchoring position of antigenic peptides presented by HLA-B*1301might play a crucial role in the pathogeneisis of occupational TCE-induced skin hypersensitivity reaction.In summary, the effect of TCOH or TCA on the expression and activity of MMP-2, MMP-9, TIMP-1and TIMP-2in HaCaT cells was studied. And TCOH could upregulation of TIMP-1and MMP-9in HaCaT cell in a dose-effect manner. Taking the patch test results in vivo together, TCOH might play an important role in TCE induced skin hypersensitivity. Secondly, in the present study, we construct C1R-B*1301and C1R-B*1302cells which highly express HLA-B*1301and HLA-B*1302respectively and the immunoaffinity purification of HLA-A*1301and HLA-B*1302was also established. Coparing the sequenses of antigenic peptides presented by HLA-B*1301and HLA-B*1302, we observed that the prefence amino acids in major and minor anchoring positions are different significantly. Preliminary evidence was given for strong assotion between HLA-B*1301and DMLT.