High Calcium, ATP, and Poly(I:C) Augment the Immune Response to β-Glucan in Normal Human Epidermal Keratinocytes  Carren Sy Hau, Yayoi Tada, Sayaka Shibata, Hideya Uratsuji, Yoshihide Asano, Makoto Sugaya, Takafumi Kadono, Naoko Kanda, Shinichi Watanabe, Kunihiko Tamaki, Shinichi Sato  Journal of Investigative Dermatology  Volume 131, Issue 11, Pages 2255-2262 (November 2011) DOI: 10.1038/jid.2011.201 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 β-Glucan stimulation induces the production of IL-8, IL-6, and IL-1α in NHEKs. Cells were cultured in the presence or absence of β-glucan (20 μg ml-1). Supernatant samples were collected at 6, 12, 24, 36, and 48 hours incubation and analyzed by ELISA. Data show mean±SEM (n=3) of three separate experiments using cells from three donors. Significant differences compared with medium only: *P<0.05. NHEK, normal human epidermal keratinocyte. Journal of Investigative Dermatology 2011 131, 2255-2262DOI: (10.1038/jid.2011.201) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 High calcium, ATP, and poly(I:C) augment cytokine and chemokine production by NHEKs. (a) NHEKs were incubated with 0.05 or 1.3 mM calcium for 24 hours before stimulation with β-glucan (20 μg ml-1). (b) Stimulation with ATP (250 μM) and β-glucan (20 μg ml-1). (c) Stimulation with poly(I:C) (25 μg ml-1) and β-glucan (20 μg ml-1). Supernatant samples were collected at 12, 24, and 48 hours and analyzed for their IL-8, IL-6, and IL-1α levels through ELISA. Data show mean±SEM (n=3) of three separate experiments using cells obtained from three donors. Significant differences compared with medium or low calcium only: *P<0.05. Significant differences compared with medium with compound or high calcium: **P<0.05. Significant differences compared with medium or low calcium with β-glucan: †P<0.05. ATP, adenosine 5′-triphosphate; NHEK, normal human epidermal keratinocyte. Journal of Investigative Dermatology 2011 131, 2255-2262DOI: (10.1038/jid.2011.201) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Dectin-1 is expressed on the cell surface of NHEKs, with downregulation noted in cells treated with β-glucan. (a) RT-PCR of dectin-1 in NHEKs and THP-1 cells, with human cDNA as positive control. (b) RT-PCR of dectin-2 in NHEKs and THP-1 with or without β-glucan (20 μg ml-1) stimulation for 24 hours. (c) NHEKs were incubated with β-glucan (20 μg ml-1) or an equal amount of 0.3 M NaOH alone for 24 hours before staining, with unstimulated cells serving as control. Cells were then stained with anti-dectin-1 Ab or isotype control. Cells were also treated with 0.025% trypsin for 5 minutes at 37 °C, washed, and stained. Dashed line: isotype control. Solid line: dectin-1. Data are representative of three independent experiments using cells from three donors. NHEK, normal human epidermal keratinocyte; RT-PCR, reverse transcription-PCR; THP-1, human acute monocytic leukemia cell line-1. Journal of Investigative Dermatology 2011 131, 2255-2262DOI: (10.1038/jid.2011.201) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Glucan triggers the activation of the p44/42 MAPK (ERK1/2) and p38 MAPK signaling pathways in NHEKs. (a) Protein extracts of NHEKs stimulated with β-glucan (20 μg ml-1) for the indicated time periods were analyzed for ERK and p38 MAPK phosphorylation by immunoblotting using anti-phospho-ERK and anti-phospho-p38 MAPK Abs. Equivalent protein loading was confirmed by reprobing with anti-total ERK and anti-total p38 MAPK Abs. (b) NHEKs were pre-treated with 75 μM PD98059 or 20 μM SB203580 for 30 minutes before β-glucan stimulation. EGF served as positive control for ERK. (c) NHEKs were pre-incubated with 75 μM PD98059 or 20 μM SB203580 for 30 minutes before adding β-glucan. NHEKs incubated with DMSO were used for comparison. Culture supernatants were collected after 48 hours and IL-8 levels were measured using ELISA. Data show percentage modulation mean±SEM (n=3) of three separate experiments using cells from three donors. Significant differences compared with medium only: *P<0.05. Ab, antibody; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; NHEK, normal human epidermal keratinocyte. Journal of Investigative Dermatology 2011 131, 2255-2262DOI: (10.1038/jid.2011.201) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions