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Abstract
Although atopic dermatitis (AD) is characterized by cytokine production predominantly mediated by T helper (Th) 2 cells, AD pathogenesis also involves innate immune and Th1 cells. To optimize the cytokine milieu required for accurate reproduction of AD-related gene expression profile in vitro, we evaluated the expression pattern of CCL22, CCL17, IL5, IL13, IL33, IL25, TSLP, FLG, and LOR in human lesional AD skin and cytokine-stimulated HaCaT cells. An increase in Th2 mediators (IL5, IL13, CCL22, CCL17, IL25, IL33, and TSLP) and a decrease in genes related to cornified cell envelope (filaggrin and loricrin) were observed in human AD lesions. Innate (tumor necrosis factor-α) and/or Th1/Th2 adaptive cytokines (interferon-γ/ IL-4) were required for inducing these inflammatory changes in HaCaT cells, implying that a complex network of innate, Th1, and Th2 cytokines drives AD-like changes. Therefore, stimulation with various combinations of cytokines, beyond Th2 polarization, is necessary when HaCaT cell line is used to study genetic changes implicated in AD pathogenesis.
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Figure 1.
Assessment of HaCaT cell viability following their stimulation with various combinations of Th1 (IFN-γ, 10 ng/ml or TNF-α, 10 ng/ml) and Th2 (IL-4, 50 ng/ml) cytokines for 24 hours. (A) Evaluation of the morphological changes in cytokine-stimulated HaCaT cells using phase-contrast microscopy. Original magnification ×20. (B) The effects of cytokine stimulation on the growth of HaCaT cells were measured using a water-soluble tetrazolium salt assay and the cell viability was determined by measuring the absorbance at 450 nm wavelength. Graphs show the mean±standard error of the mean. *** p<0.001 (Student's t-test).
Figure 2.
Correlation between AD-related gene expression in lesional skin and that observed in cytokine-stimulated HaCaT cells. HaCaT cells were cultured in the presence of IFN-γ (10 ng/ml), TNF-α (10 ng/ml), and/or IL-4 (50 ng/ml) for 24 hours. The expression of CCL22 (encoding macrophage-derived chemokine), CCL17 (encoding thymus and activation-regulated chemokine), IL5, IL13, FLG (filaggrin), LOR (loricrin), IL33, IL25, and TSLP was evaluated by real-time PCR as a fold change normalized to the expression of GAPDH in skin samples from AD patients (left) and cytokine-stimulated HaCaT cells (right). mRNA expression of each gene in cytokine-stimulated HaCaT cells was compared with non-stimulated control. Graphs show the mean±standard error of the mean. MDC, macrophage-derived chemokine. * p<0.05, ** p<0.01, *** p<0.001 (Student's t-test).
Table 1.
Characteristics of patients with atopic dermatitis
| Patient No. | Sex/age | Duration (yr) | Aggravation (mon) | Co-morbidities |
|---|---|---|---|---|
| 1 | M/20 | 10 | 3 | Asthma |
| 2 | M/19 | 12 | 2 | Allergic rhinitis |
| 3 | M/21 | 2 | 2 | Allergic rhinitis |
| 4 | M/13 | 5 | 2 | Urticaria |
| 5 | M/25 | 10 | 1 | – |
| 6 | M/46 | 2 | 3 | – |
Table 2.
Primer sequences for real-time PCR
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