Experimental Study On Immunoloregulation Mechanisms Of Imiquimod

Background and object:Imiquimod is a potential immunomodulator which has been widely used in the skin diseases caused by viruses and some skin tumors. 5% imiquimod cream has shown a good curative effect clinically, yet its mechanism of anti-viruses and anti-tumor was still not fully understood. In our study, we first observed the effect of 5% imiquimod cream on the mRNA expressions of TNF-a, IL-1β and IL-6 in the skin of BALB/C mouse in vivo.Langerhans cells, as one of the antigen present cells, play an important role in the function of anti-viruses and anti-tumors. But it is rare in the skin — only 2% ~ 5% of the total epidermal cells and hard to be isolated and purified. We used the density gradient centrifugation and magnetic cell sorting simultaneously to separate and purify the Langerhans cells from human epidermis.Most dermatoses which are sensitive to imiquimod, such as Condyloma acuminatum, occur in cutaneous layer. To clarify the effect of imiquimod on the human epidermis, we investigated the IL-6 secretion in human epidermal cell suspension at different time intervals after the application of imiquimod. As we know, the main immune cells in epidermis are Langerhans cells and keratinocytes. We also compared the effect of the imiquimod solution on the secretion and the mRNA expressions of cytokines (TNF-a, IL-1β and IL-6) from human epidermal Langerhans cells and human eternal cell lines—HaCaT keratinocyte cells to investigate that which kind of epidermal cells were involved in the cytokines' up-regulation mechanism by imiquimod. All the abovementioned were to illuminate the immunoloregulation mechanisms ofimiquimod.Materials and methods1. The effect of 5% imiquimod cream on the mRNA expression of TNF-a, IL-ip and IL-6 in the skin of BALB/C mouse in vivo: Twelve BALB/C mice were divided into two groups, 6 in each groups. The left ears of the mice were treated by 5% imiquimod cream each other day, the right ears with matrix as controls. The skin samples were obtained at d2 and d7 after local application for harvesting the total RNA.The mRNA expressions of TNF-a, IL-1|3 and IL-6 were detected by RT-PCR.2. Separation and purification of Langerhans cells from human epidermis: the epidermal cell suspension was prepared from foreskins of normal adults. Density gradient centrifiigation and magnetic cell sorting were combined simultaneously to separate and purify the Langerhans cells from human epidermal cell suspension. The separated cells were specially labelled by monoclonal mouse-anti-human CD la antibody conjugated to FITC. The purification rate was analyzed by flow cytometric analysis fluorography and the cell viability was observed by 0.4% trypan blue staining.3. The time-effect of imiquimod on the cytokine secretion from human epidermal cell suspension: the epidermal cell suspension of foreskins was prepared, and then inoculated into the 24 cell culture plate. The solution of 5(ig/ml imiquimod was added to the experimental group and the culture medium for the control group. The conditioned supernatant was collected lh, 2h, 4h, 8h and 12h after the treatment, the level of IL-6 secretion of each groups was detected by ELISA ( Enzyme-linked immunosorbent assay) .4. The effect of imiquimod on the cytokine secretion of human epidermalLangerhans cells and human epithelial cell lines- HaCaT cells: Langerhans cells and human eternal epithelial cell lines- HaCaT cells were also divided into two groups — experimental group and control group. 5ug/ml of imiquimod solution was added to the experimental group and the culture medium for the control group. The conditioned supernatant was collected 4 hours after treatment and stored at -20 °C. The levels of TNF-a, IL-ip and IL-6 secretion of each groups were detcted by ELISA. 5. The effect of imiquimod on the mRNA expressions of 3 kinds of cytokines from human epidermal Langerhans cells and HaCaT cells: Langerhans cells and HaCaT cells were divided into two groups — experimental group and control group. The solution of 5ug/ml imiquimod was added to...