Ann Dermatol.  2012 Nov;24(4):398-405. 10.5021/ad.2012.24.4.398.

Interleukin-17 and Interleukin-22 Induced Proinflammatory Cytokine Production in Keratinocytes via Inhibitor of Nuclear Factor kappaB Kinase-alpha Expression

• Cho KA
• Kim JY
• Woo SY
• Park HJ
• Lee KH
• Pae CU

• KMID: 2266033
• DOI: http://doi.org/10.5021/ad.2012.24.4.398

Abstract

BACKGROUND
The pathogenesis of psoriasis may involve the interleukin (IL)-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22, which mediates dermal inflammation and acanthosis.
OBJECTIVE
As inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKalpha.
METHODS
We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching to the high concentration (2.8 mM) media with IL-17 or IL-22, then compared the IKKalpha expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies.
RESULTS
Elevated calcium concentration induced IKKalpha expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKalpha in HaCaT cells and reconstituted human epidermis. IKKalpha induction was also noted, following the injection of IL-17 and IL-22 into mice ears.
CONCLUSION
Although the induction of IKKalpha was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to the inflammation occurring via the induction of IKKalpha from keratinocytes or skin layers.

Keyword

Cell differentiation; IKK alpha; Inflammation; Interleukin-17; Interleukin-22; Keratinocytes

MeSH Terms

Animals
Calcium
Cell Culture Techniques
Cell Cycle
Cell Cycle Checkpoints
Cell Differentiation
Ear
Epidermis
Humans
I-kappa B Kinase
Inflammation
Interleukin-17
Interleukins
Keratinocytes
Mice
Psoriasis
Skin
Th17 Cells
Calcium
I-kappa B Kinase
Interleukin-17
Interleukins

Figure

• 

  Fig. 1 IKKα expression is increased under a calcium concentration of 2.8 mM, according to the immunoblot results. HaCaT cells in 0.06 mM of calcium medium were induced to differentiation by replacement with high calcium medium (2.8 mM), and samples were collected every 24 hours for protein extraction. Involucrin, cytokeratin 5, and cytokeratin 10 were detected by the immunoblotting technique described in the Materials and Methods section. Beta-actin blotting was conducted as a loading control.

• 

  Fig. 2 Cell cycle analysis demonstrates that HaCaT cells cultured by switching into high calcium concentrations (Ca 2.8 mM) evidenced an elevated proportion of G0/G1 phase, regardless of IL-17 and IL-22 treatment. By way of contrast, HaCaT cells maintained at low calcium concentrations (Ca 0.06 mM) evidenced more cells in S- or G2/M phase compared with those transferred to a medium containing 2.8 mM of calcium. The data are expressed as the means±standard error of the mean and the statistical differences were analyzed between low- and high-calcium treated groups in each of the experiments. PI=(S+G2/M)/(G0/G1+S+G2/M). NS: not significant, PI: proliferation index. **p<0.01; *p<0.05.

• 

  Fig. 3 The effects of IL-17 and IL-22 treatment on the expression of IKKα and phosphorylated IκB in HaCaT cells. Cells were treated with IL-17 and IL-22 up to a concentration of 100 ng/ml in a calcium concentration of 2.8 mM and cultured for 24 hours. (A) The immunoblot shown is representative of three independent experiments. (B) The pixel densities are divided by β-actin densities to correct the values, and the data are expressed as the means±standard error of the mean at three independent experiments. *p<0.05.

• 

  Fig. 4 IL-17 and IL-22 induce pro-inflammatory cytokine production in HaCaT cells. HaCaT cells were cultured under 100 ng/ml of IL-17 or IL-22 for 48 hours, with or without preincubation of PS1145, at a concentration of 10µM for an hour and collected culture supernatant for ELISA. The data are expressed as the means±standard error of the mean at three independent experiments. NS: not significant. *p<0.05.

• 

  Fig. 5 Both IL-17 and IL-22 induce an increase in IKKα expression in Neoderm. Neoderm tissues were cultured under 100 ng/ml of IL-17 or IL-22 for 48 hours and fixed for H&E and immunohistochemical staining assays. The results shown are representative of the three independent batches of Neoderm experiments.

• 

  Fig. 6 Elevated IKKα levels in inflammatory mice ears, evoked by IL-17 and IL-22 injection. Skin inflammation, edema, and acanthosis, which were involved in the infiltration of neutrophils and dendritic cells into the ear skin of C57BL/6 WT mice, but less profound inflammatory changes, were noted in the sham controls. Each ear of C57BL/6 WT mice were injected intradermally with 3µg of IL-17 and IL-22 for two consecutive days in a total volume of 30µl. The control group of mice was injected with the same volumes of phosphate-buffered saline (PBS) via an identical route.

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