The Mouse Model Of The Allergic Contact Dermatitis Induced By DNFB And FITC And The Study Of Their Pathogenesis

Objectives Allergic contact dermatitis (ACD) is a T cell-mediated cutaneous immune/inflammatory reaction to haptens. Based on their cytokine profiles, T cell reactions could be classified into Th1 type or Th2 type. Type â…  cytokine and Type â…¡ cytokine may play important roles as effectors and regulators in the development of ACD. Type â…  cytokine (IFN-γ, IL-2, TNF) is produced by either Thl cells and Te1 cells. Type â…¡ cytokine (IL-4, IL-10) is secreted by either Th2 cells and Tc2 cells. Some experiments demonstrated that cytokine profiles in ACD were hapten dependent. Previous studies showed that Dinitrofluorobenzene (DNFB) and 2,4,6-trinitrochlorobenzene (TNCB) induced the ACD in BALB/c mice, in which IFN-γ is secreted predominantly. In our primarily investigation it was found that FITC could promote the development of IL-4- but not IFN-γ-secreting cells in draining lymph nodes. Using BALB/c mice we established the ACD animal model induced by topical treatment with DNFB or FITC respectively. And then the expressions of the Thl cytokine (IFN-γ, IL-2) and Th2 cytokine (IL-4) were assessed to study the important roleof Th1/Th2 cytokines in the murine ACD. Methods1. Groups of mice: In test group, mice (n=10) were painted on a shaved abdomen with 20μL 0.5% DNFB in 4:1 acetone/olive oil or with 400 μL 0.5% FITC in 1:1 acetone/dibutylpthalate respectively on day 0 and day 1. Then the skin reactions were challenged on day 5 or 6 by topical applications of 20 μL 0.2% DNFB or 0.5% FITC onto the right ear respectively. As negative-control the left ear was treated only with vehicle alone.2. The ear swelling was determined by the measurement of ear thickness with an engineer's micrometer before and after challenge and reported as the mean change in ear thickness with standard error.3. The ears were removed 24hr after challenge with DNFB/FITC and fixed in 10% formalin. Then 4- μm paraffin sections were cut and stained with haematoxylin eosin.4. Ear tissues embedded in OCT were snap-frozen in liquid nitrogen and kept at -70℃ for later use in immuno-histochemical study. The cytokines were detected by immuno-histochemistry with the rat mAbs which were specific for mouse IL-2 or IL-4.5. Single-cell suspensions were made from mouse draining lymph nodes (LN) by mechanical tissue disaggregation through a sterile stainless steel gauze in PBS. LNCs were washed and resuspended in RPMI Medium 1640 containing 10% fetal bovine serum in a cell concentration of 10⁶ cell/mL. The cells were incubated in 24-well plates (each well containing 1 mL culture medium) at 37℃ with an atmosphere of 5% CO₂ and saturated humidity for 12h or 120h. Supernatants were harvested and stored at -70°C for cytokine analysis by enzyme-linked immunosorbant assay (ELISA).6. The cytokines were detected by using the ELISA method which contained rat mAbs specific for mouse IL-4 or IFN-γ according to the manufacturer's protocol of test kits. Standard curves were made with commercial purified murine IL-4 and IFN-γ.7. Data were analyzed statistically by x² test and t test. P<0.05 was considered significant.Results1. Topical applications of DNFB/FITC on the right ears provoked typical contact dermatitis, in which ear-swelling was significantly greater than that on left ears (negative control) (P<0.05).2. Severe eczematic histopathological changes were observed in ear lesions. Histopathology showed a massive infiltrated inflammatory cells, such as monocytes and neutrophils, and dermis hypertrophy, in the dermis treated with DNFB/FITC treated ears, which was not visible in the vehicle-treated ears. The epidermis showed no significant abnormal alterations on both sides ears.3. Cytokine expression in ear tissue in DNFB and FITC-sensitized BALB/c mice was polarized. Predominantly IL-2 secreting was detected in the ear tissue treated by DNFB, while almost exclusively IL-4 secreting was detected in the ear tissue treated by FITC.4. The LN-cell culture showed that the IFN-γ level secreted in...