Study On Cytokine Expression And The Exploration Of A Transgenic Mouse Model Of TCE Induced Skin Hypersensitivity

Hypersensitivity dermatitis induced by occupational exposure to TCE has become one of the serious occupational diseases and affects the health of workers who are susceptible to TCE seriously in China. Up to now, according to its epidemiological, clinical features, most dermatologists and physicians in occupational medicine categorize this disorder as a delayed type hypersensitivity reaction. However, the detailed mechanisms of TCE induced skin hypersensitivity is still not clear due to the complexity of pathogenesis in hypersensitivity reaction. Meanwhile, the study on the mechanisms of TCE induced skin hypersensitivity is restricted by the absence of appropriate animal model. Cytokine play a critic role during the induction and activation phase of hypersensitivity by promoting T cell activation and proliferation. The expression of cytokine has been widely used to the study of drug-induced hypersensitivity syndrome (DIHS). In the present study, we first evaluate the effects of TCE and its two main in vivo metabolites, TCOH and TCA, in TCE induced skin hypersensitivity by the examination of the production of IL-1β, IL-6, IL-8and TNF-α. Our previous work, a case-control study performed in TCE-exposed workers, found that human leukocyte antigen (HLA)-B*1301is strongly associated with the TCE induced skin hypersensitivity susceptibility. So we establish the transgenic HLA-B*1301and02M mouse by the method of microinjection and explore the transgenic mouse model of TCE induced skin hypersensitivity to provide clue for the study of pathological mechanism of TCE induced skin hypersensitivity. Cytokine expression, T cells activation and proliferation were used as the biomarkers of a successful mouse model of TCE induced skin hypersensitivity.1. Study on cytokine expression induced by TCE and the mechanism of regulation.28TCE-induced hypersensitivity dermatitis patients,22TCE exposed workers and22non-exposed controls were enrolled in the study. The serum levels of interleukin (IL)-1β, IL-6, IL-8and tumor necrosis factor (TNF)-α were analyzed using a magnetic colorbead-based multiplex assay. The patients showed significantly higher levels of serum IL-1β (p=0.033and p=0.015), IL-6(p<0.001), IL-8(p<0.001and p=0.002) and TNF-α (p=0.009and p=0.005) than the TCE exposed workers and non-exposed controls. There was a significantly positive correlation among these cytokine concentrations, but no significant correlation was found between these cytokine concentrations and the disease duration in patient group. We further compared the effects of trichloroethanol (TCOH) and trichloroacetic acid (TCA), two major metabolites of TCE, on cytokine expression in keratinocyte cell line (HaCaT). IL-la, IL-6, IL-8and TNF-α concentrations were tested using enzyme-linked immunosorbent assay (ELISA) after HaCaT cells were treated with different concentrations of TCOH or TCA for24h. We found that TCOH, but not TCA, increased the levels of IL-la and IL-6in a dose-dependent manner. We also found that TCOH activated the nuclear factor kappa B (NF-κB) pathway. Bay11-7082(NF-κB inhibitor) significantly attenuated the TCOH-induced production of IL-6in HaCaT cells, but IL-la production was not affected.2. Establishment of human HLA-B*1301/β2M double transgenic mouse and exploration of a transgenic mouse model of TCE induced skin hypersensitivityWe cloned the HLA-B*1301andβ2M cDNA into the eukaryotic expression vector of pUbC by using the gene recombination technique, respectively. The linearized recombinant vectors were introduced into the fertilized eggs of C57BL/6mice by micro injection. Those survived embryos were transferred into the oviducts of pseudopregnant ICR female mice. DNA was isolated from the pups after they were weaned at21days of age. The DNA preparations were analyzed for the presence of the HLA-B*1301and β2M gene using PCR. The PCR-positive transgenic mice were mated to normal mice for generating transgenic mouse lines and DNA samples isolated from tail of the offspring were identified using PCR. RT-PCR was used to evaluate mRNA expression of F1、F2generation mice and the founder mice with high mRNA expression of HLA-B*1301and β2M were screened. HLA-B*1301/p2M double transgenic mouse were established using hybridization between HLA-B*1301and β2M transgenic mouse. The expression of HLA-B*1301protein were determined by flow cytometry. Wild type and HLA-B*1301/β2M double transgenic mouse were exposed to TCE by intraperitoneal injection and back skin exposure. The immunological effects were compared between wild type and transgenic mouse after exposure for one week and two weeks including cytokine expression, T cell proliferation and activation.The results were showed as follows:1) After exposure to TCE for one week, the concentrations of IFN-γ, ILIL-6, TNF-4, and IL-1β in serum samples were detected. The TCE exposed mice showed significantly higher concentrations of IL-6than the non-exposed controls in both wild type and transgenic mice (p<0.05). The serum IFN-y, IL-4, TNF and IL-1β levels were similar in both TCE exposed mice and the controls. After exposure to TCE for two weeks, the TCE exposed wild type mice still showed significantly higher concentrations of IL-6than the non-exposed controls (p<0.05), but there was no significiantly difference between TCE exposed mice and the controls in serum IL-6level of transgenic mice.2) After exposure to TCE for one week, mouse splenocytes were isolated and cultured for48hours treated with TCE (0.1mM), TCOH (0.2mM) and ConA (5μg/ml). T cell proliferation was determined with CCK-8kit. The results showed that T cell proliferation was enhanced treated with ConA in wild type mice, but there were no significantly differences between TCE exposed and control groups. Significantly differences were found between TCE exposed and control groups in transgenic mice (p<0.05). Treated with TCOH in vitro, we also found that T cell proliferation was notably elevated in transgenic mice exposed to TCE than control group (p<0.05). T cell proliferation was also examined using the method of EdU incorporation, treated with ConA in vitro, T cell proliferation was markedly enhanced in both wild type and transgenic mice exposed to TCE than control group (p<0.05), but similar results were not found if the splenocytes were treated with TCOH in vitro.Exposed to TCE for two weeks, mouse splenocytes were treated with ConA in vitro, there were no significantly differences between TCE exposed and control groups in both wild type and transgenic mice.3) T cell activation was further evaluated after exposure to TCE for one week in both wild type and transgenic mice using the cell surface markers of CD3, CD4, CD8, CD25and CD69. Results showed that the proportion of T cell were similar between TCE exposed and control groups in two kinds of mice. Exposed to TCE for two weeks, mouse splenocytes and lymph-node cells were isolated and cultured for48hours treated with TCE (0.1mM), TCOH (0.2mM) and ConA(5μg/ml), respectively. Results showed that the proportion of CD3+, CD3+CD4+, CD3+CD8+cells from lymph node of TCE exposed wild type mice decreased than control group. Treated with ConA in vitro, the proportion of CD3+cells from lymph node of TCE exposed wild type markedly decreased than control group(p<0.05). The proportion of CD3+CD3+CD4+, CD3+CD8+cells from lymph node of TCE exposed transgenic mice notablely decreased than the control group (p<0.05). Treated with ConA in vitro, the proportion of CD3+CD25+and CD3+CD69+cells from lymph node of TCE exposed transgenic mice markedly decreased than the control group(p<0.05). Similar results were not found among T cells from splenocytes of TCE exposed wild type and transgenic mice.4) Histopathological examination (HE staining) of the liver and back skin from the wild type and transgenic mice showed obvious granulocyte and lymphocyte infiltration in the skin of one mouse out of four TCE exposed mice. Cellular edema was also observed in the liver from TCE exposed transgenic mouse accompanied with skin injurys. Similar pathological changes were not found in the control group.In summary, TCOH induced IL-6expression through activation of the NF-κB pathway in HaCaT cells and may play an integral role in TCE-induced skin hypersensitivity. The expression of IL-1、IL-6、IL-8and TNF-α may be a possible marker for the TCE induced hypersensitivity dermatitis. These cytokines can be used to evaluate immunology effects of mouse model of TCE induced hypersensitivity dermatitis. The altered cytokine concentrations found in this study could lead to further discovery regarding the pathogenesis of the disease. After exposure to TCE, the immunology effects were compared between wild type and transgenic mice. Results showed that TCE exposure enhance the immune response to immunogen. Compared with wild type mice, the transgenic mice are more susceptibility to immunotoxicity of TCE. Exposured to TCE for two weeks may induce immunosuppression compared to one week. In present study, we had not established the mouse model of TCE induced hypersensitivity dermatitis, but our exploration had provided some evidences to improve method for the future study.