Journal List > Immune Netw > v.18(2) > 1148263

Kim, Baek, Lee, Roh, and Jung: Optimization of Cytokine Milieu to Reproduce Atopic Dermatitis-related Gene Expression in HaCaT Keratinocyte Cell Line

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).
in-18-e9f1.tif
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).
in-18-e9f2.tif
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
Target gene Primer sequence
CCL22 Forward: 5′-GAA GCC TGT GCC AAC TCT CT-3′
  Reverse: 5′-GGG AAT CGC TGA TGG GAA CA-3′
CCL17 Forward: 5′-CGG ACC CCA ACA ACA AGA GA-3′
  Reverse: 5′-CTC CCT CAC TGT GGC TCT TC-3′
IL5 Forward: 5′-TCT ACT CAT CGA ACT CTG CTG A-3′
  Reverse: 5′-CCC TTG CAC AGT TTG ACT CTC-3′
IL13 Forward: 5′-TGT TTG TCA CCG TTG GGG AT-3′
  Reverse: 5′-TGA GTC TCT GAA CCC TTG GC-3′
FLG Forward: 5′-TGA AGC CTA TGA CAC CAC TGA-3′
  Reverse: 5′-TCC CCT ACG CTT TCT TGT CCT-3′
LOR Forward: 5′-GAG GTG TTT TCC AGG GGC A-3′
  Reverse: 5′-TGG GGT TGG GAG GTA GTT GTA-3′
IL33 Forward: 5′-TGT CAC ATT GGG CAA AGT T-3′
  Reverse: 5′-CAG TAA GCA GTG TTA TCA GGA A-3′
IL25 Forward: 5′- TTG TTT GTT TAC TCA TCA CTC AG-3′
  Reverse: 5′- TCC TCC TCA GAA TCA TCC A-3′
TSLP Forward: 5′-TCC TCT GAA GAC CTG ACC-3′
  Reverse: 5′-TCT CCT TTC TCC CTA ATC CTC-3′
GAPDH Forward: 5′-CTG GGC TAC ACT GAG CAC C-3′
  Reverse: 5′-AAG TGG TCG TTG AGG GCA ATG-3′
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