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Adrenocorticotropin Hormone Stimulates Interleukin-18 Expression in Human HaCaT Keratinocytes
ORIGINAL ARTICLE
Adrenocorticotropin Hormone Stimulates Interleukin-18 Expression in Human HaCaT Keratinocytes Hyun Jeong Park1,5, Hee Jung Kim1,5, Jun Young Lee1, Baik Kee Cho1, Richard L. Gallo2,3 and Dae Ho Cho4 The hypothalamic–pituitary–adrenal (HPA) axis is activated by stress. This involves the production of corticotropin releasing hormone (CRH) with the subsequent release of proopiomelanocortin (POMC) peptides, of which adrenocorticotropin hormone (ACTH) is most important. Although the skin has the capacity to produce CRH and POMC peptides, the immunomodulatory roles of ACTH in skin are yet unknown. IL-18 has been known to affect cells involved in the inflammatory response. In this study, we aimed to identify the regulatory effect of ACTH on IL-18 expression of skin keratinocytes. Exposure of HaCaT cells to ACTH stimulated formation of IL-18 mRNA transcript and its protein products in a dose-dependent manner. Furthermore, we suggest that ACTH-induced IL-18 production is via the caspase-1 activation pathway, as IL-18 production induced by ACTH could be suppressed by caspase-1 inhibitor, and ACTH could increase caspase-1 activity. The effect of ACTH on IL-18 production was blocked by specific inhibitors of p38 kinase (SB203580) or extracellular signalregulated kinase (ERK) (PD98059). In addition, ACTH-induced rapid phosphorylation of p38 kinase and ERK, and ACTH signaling occurred via melanocortin receptor 1 (MC1R) and receptor 2 (MC2R). These results suggest that ACTH stimulates IL-18 expression in human keratinocytes, which provides an insight into the interaction between ACTH and inflammatory mediators. Journal of Investigative Dermatology (2007) 127, 1210–1216. doi:10.1038/sj.jid.5700703; published online 18 January 2007
INTRODUCTION As an adaptive response to systemic stress, the hypothalamic– pituitary–adrenal (HPA) axis is activated (Chrousos, 1995). The process starts with the hypothalamic release of corticotropin-releasing hormone (CRH) which, in turn stimulates production of adrenocorticotropic hormone (ACTH) and other proopiomelanocortin (POMC) peptides via CRH receptor type 1 (CRH-R1). ACTH, a 39-amino-acid peptide, regulates glucocorticoid production through its effect on the adrenal cortex. Steroids are powerful anti-inflammatory agents that counteract stressors and buffer tissue damage 1
Department of Dermatology, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 2Department of Dermatology, University of California, San Diego, California, USA; 3VA San Diego Health Care System, San Diego, California, USA and 4Department of Life Science, Sookmyung Women’s University, Seoul, Korea
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These authors are co-first authors.
Correspondence: Dr Hyun Jeong Park, Department of Dermatology, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. E-mail: [email protected] and Dae Ho Cho, Department of Life Science, Sookmyung Women’s University, Chungpa-Dong 2-Ka, Yongsan-Ku, Seoul 140-742, South Korea. E-mail: [email protected] Abbreviations: ACTH, adrenocorticotropin hormone; CRH, corticotropin releasing hormone; ERK, extracellular signal-regulated kinase; HPA, hypothalamic–pituitary–adrenal; MAPK, mitogen-activated protein kinase; MCIR, melanocortin receptor 1; PBS, phosphate-buffered saline; POMC, proopiomelanocortin Received 6 July 2006; revised 27 October 2006; accepted 7 November 2006; published online 18 January 2007
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(Chrousos, 1995). Recent evidence suggests that ACTH is produced outside the pituitary tissue, which includes the skin (Chakraborty et al., 1996; Wakamatsu et al., 1997; Slominski et al., 2000a). Slominski et al. have acknowledged the skin as a cutaneous equivalent of the central HPA (Slominski and Mihm, 1996; Slominski et al., 2000b, 2001, 2004a). Although similar to its systemic equivalent, the cutaneous HPA axis is responsive to local stressors (solar, thermal, chemical, biological, etc.) and resultantly activates the neuronal, endocrine, and immune systems in the skin. A variety of cytokines (e.g., IL-1b, IL-6, and TNF-a) are upregulated by stress, suggesting their role in mediating stress responses (Dugue et al., 1993). However, the molecular mechanism of stress-induced cytokine in impairing host defenses is not well understood. IL-18 was originally discovered as IFN-g-inducing factor (Okamura et al., 1995; Ushio et al., 1996). It is normally expressed in its precursor form (24 kDa), but can be activated (18 kDa) by caspase-1 (IL-1b-converting enzyme) (Ghayur et al., 1997; Gu et al., 1997). IL-18 performs multiple biological activities, which include Fas (CD95, APO-1) ligand induction, enhancement of T-cell cytolytic activity, and production of T helper type 2 (Th2) cytokines (Hoshino et al., 1999; Yoshimoto et al., 2000; Nakanishi et al., 2001). IL-18 is expressed in a wide range of cells, including Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, and adrenal cortex cells. It plays an important role in the Th1 response to toxic shock and shares functional similarities with IL-12 (Dinarello, 1999). As a & 2007 The Society for Investigative Dermatology
HJ Park et al. IL-18 Expression in Human HaCaT Keratinocytes
proinflammatory cytokine, overproduction of IL-18 induces severe inflammatory disorders. Thus, it is important to know how IL-18 expression is regulated in inflammatory response and in maintaining the balance of immune system function. Recently, IL-18 by keratinocytes was reported to play an important role in response to cutaneous inflammation (Mee et al., 2000). In this study, we investigated the effect of ACTH on IL-18 expression of human HaCaT keratinocytes, and also examined its regulatory mechanism. RESULTS Effect of ACTH on IL-18 production in HaCaT cells
To measure the effect of ACTH on IL-18 expression in HaCaT cells, we treated the cells with the fixed concentrations of ACTH, and then assessed IL-18 mRNA and its protein expression by reverse transcriptase-PCR and ELISA, respectively. IL-18 mRNA expression was increased in a dosedependent manner by ACTH, with the peak response at 109 M (Figure 1a). IL-18 assessed by ELISA was also increased in a dose-dependent manner (Figure 1b). As shown
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Because caspase-1 is required for cleaving pro-IL-18 into its mature form, its involvement in IL-18 production was examined by pretreating HaCaT cells for 2 hours with the caspase-1-specific inhibitor Ac-YVAD-CHO before stimulation with ACTH. We observed Ac-YVAD-CHO to suppress the production of IL-18 (Figure 2a). Next, we investigated the molecular mechanism underlying ACTH-induced IL-18 production by performing caspase-1 colorimetric assay. Caspase-1 activity in HaCaT cells were elevated 10-fold by ACTH (Figure 2b). The results indicate that ACTH is involved in IL-18 production of HaCaT cells probably via a caspase-1 activation pathway. In a previous report, caspase-1 was activated by caspase-11 (Wang et al., 1998). It has also been reported that caspase-11 can be activated by cathepsins B (Schotte et al., 2001). To understand the involvement of caspase-11 in the ACTH-induced caspase-1 activation, we examined it by using Z-FA-FMK, an inhibitor of cathepsins B. In our observation, Z-FA-FMK suppressed the production of IL-18 induced by ACTH, indicating that caspase-11 is also involved in the ACTH-induced IL-18 production in HaCaT cells (Figure 2c). Effect of mitogen-activated protein kinase inhibitors on IL-18 expression
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Figure 1. Effects of ACTH on the mRNA expression and production of IL-18 in HaCaT keratinocytes. (a) HaCaT cells were harvested after ACTH treatment for 6 hours. Total RNA was extracted, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to b-actin by densitometry. (b) Cells were treated with indicated concentration of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control.
The mitogen-activated protein kinases (MAPKs) control a wide variety of cellular events, which include very complex cellular programs such as cell differentiation, proliferation and apoptosis to processes involved in immune responses. To identify its involvement in ACTH-induced IL-18 expression, HaCaT cells were treated with 10 mM of p38 MAPK inhibitor (SB203580) or extracellular signal-regulated kinase (ERK) inhibitor (PD98059) for 2 hours before adding ACTH (1 nM). We then assessed the expression of IL-18 mRNA transcripts and its products. As shown in Figure 3, both SB203580 and PD98059 had an inhibitory effect over ACTH on IL-18 mRNA expression and its production. To further identify the role of MAP kinase signaling pathways in ACTH-induced IL-18 expression, HaCaT cells were treated with ACTH, and the phosphorylation status of p38 kinase and ERK were analyzed by immunoprecipitation and Western blotting using commercially available antibodies specific for the active forms of the two kinases. ACTH induced transient phosphorylation of both p38 MAP kinase and ERK. The maximal activation of p38 kinase and ERK by ACTH was observed after 5 minutes (Figure 4a and b). These data demonstrate that ACTH stimulates IL-18 production through p38 kinase and ERK signaling pathways. Melanocortin receptor 1 and MC2R expression in HaCaT cells
To study whether melanocortin receptor 1 (MC1R) and/or MC2R are involved in the mediating ACTH-induced IL-18 production in HaCaT cells, the cells were treated with ACTH and then MC1R and MC2R were analyzed by Western blotting. www.jidonline.org 1211
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Figure 2. Caspase-1 is essential for the ACTH-induced IL-18 production in HaCaT keratinocytes. (a) HaCaT cells were pretreated with 40 mM of the caspase-1 inhibitor Ac-YVAD-CHO for 1 hour, and then challenged with 1 nM ACTH. The IL-18 concentration in the culture supernatants was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer’s instruction. The data normalized to the negative control are shown as fold increases in caspase activity. (c) Effects of Z-FA-fmk on ATCH-induced IL-18 production. HaCaT cells were pretreated with 20 mM of Z-FA-FMK for 2 hours, and then stimulated with 1 nM ACTH. The IL-18 concentration in the supernatant was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
We determined that MC1R and MC2R were both expressed on HaCaT cells (Figure 5a). Next, we used anti-MC1R and antiMC2R antibody to examine whether they could neutralize the ACTH-induced IL-18 production. Anti-MC2R antibody was more effective and almost reduced it to the control level (Figure 5b). These results suggest that the induction of IL-18 by ACTH may be mainly mediated by MC2R. Effect of ACTH on IL-18 production in human primary keratinocytes
To see whether ACTH regulates IL-18 production in primary human epidermal keratinocytes, we assessed IL-18 production by ELISA. As shown in Figure 6a, IL-18 production was significantly stimulated by ACTH treatment in human primary keratinocytes. Next, we observed that caspase-1 activity in primary human epidermal keratinocytes was stimulated by ACTH (Figure 6b). Ac-YVAD-CHO, a caspase-1 inhibitor, effectively suppresses IL-18 production by ACTH in primary human epidermal keratinocytes, as shown in the result from human keratinocyte cell line HaCaT (Figure 6a). In the end, we showed that SB203580 and PD98059 could suppress the ACTH-induced IL-18 production, suggesting that p38 kinase and ERK were both involved in the signaling pathway in primary human epidermal keratinocytes (Figure 6c). DISCUSSION In this study, we investigated the regulatory effect of ACTH on expression of IL-18 in HaCaT and normal keratinocytes. 1212 Journal of Investigative Dermatology (2007), Volume 127
Our results show that ACTH stimulates human keratinocytes to secrete IL-18 through MC1R and MC2R. Furthermore, ACTH activates the MAPK p38 and ERK pathways that are further required for IL-18 expression. In response to systemic stress, CRH stimulates the pituitary CRH-R, which leads to production and secretion of POMCderived peptides, including melanocyte-stimulating hormone (MSH) and ACTH (Chrousos, 1995). Recent evidence suggests that CRH stimulation of corticosteroids production in melanocytes and dermal fibroblasts is mediated by ACTH (Slominski et al., 2005a, b). CRH, CRH-R, and POMC genes are expressed in human skin as well as in cultured keratinocytes and melanocytes (Slominski et al., 1993, 1995, 2001, 2004a, b). Slominski et al. (2000b, 2001) proposed that an equivalent of HPA axis, composed of the CRH-CRHR-POMC loop, is conserved in the skin that may be activated in a skin stress response system. Recently, MSH and ACTH act as regulators of skin pigmentation and inhibitors of the skin immune systems (Luger et al., 1998; Slominski et al., 2004a, b). In this study, ACTH induced IL-18 production, which is known as an inflammatory cytokine. However, it is also known that the closely related POMC peptide alphaMSH exhibits anti-inflammatory effects through MC1R in keratinocytes (Pavey and Gabrielli, 2002). To date, five melanocortin receptors are known, and they have a variety of important biological functions (Bohm et al., 2006). To understand the difference between ACTH and alpha-MSH, we detected the expression of MC receptors in HaCaT cells.
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Figure 3. Effects of the p38 MAPK inhibitor SB203580, or the ERK inhibitor PD98059 on ACTH-induced IL-18 expression in HaCaT cells. HaCaT cells were pretreated with SB203580 (10 mM) or PD98059 (10 mM) for 2 hours before addition of 1 nM of ACTH. (a) Total RNA was extracted at 6 hours following ACTH treatment, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to b-actin by densitometry. (b) After cells were harvested for 24 hours, IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
Figure 5. Involvement of MC1R and MC2R in ACTH-induced IL-18 production. (a) HaCaT cells were treated with 1 nM of ACTH. After that, cells were harvested for 24 hours. The expression level of MC1R and MC2R were analyzed by Western blot. (b) Cells were pretreated with anti-MC1R (2 mg/mL) or anti-MC2R antibody (2 mg/mL) for 1 hour and then incubated with 1 nM of ACTH for 24 hours. The supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
Figure 5 showed that ACTH signals through MC1R and MC2R. This might be an explanation for the differential effects of ACTH and alpha-MSH in inflammation as MC1R and MC2R have different signal pathways (Bohm et al., 2006). IL-18 has multiple and complicated functions as it participates in innate immunity and in Th1- and Th2mediated responses. During inflammation, the activation of the stress system, through induction of a Th2 shift, protects the organism from systemic ‘‘overshooting’’ with Th1/proinflammatory cytokines. Interestingly, it has been reported that the expression of IL-18 is markedly increased in suprabasal keratinocytes in psoriatic lesions (Naik et al., 1999; Ohta et al., 2001), raising the possibility that IL-18 expression is upregulated in differentiated keratinocytes. Therefore, ACTH induced IL-18 expression by keratinocytes may provide new insights into the mechanism underlying the pathogenesis of skin disorders.
In a previous report, cytokines IL-1b and IL-6 were able to stimulate CRH secretion from the hypothalamus (Navarra et al., 1991), and IL-18 was originally identified as a member of the IL-1 family (Dinarello, 1999) and expected to be able to stimulate CRH production. Interestingly, recent evidence documented that IL-18 inhibited basal and stimulated CRH secretion from isolated rat hypothalami, and reduced CRH gene expression (Tringali et al., 2005). We have recently reported that CRH inhibits IL-18 expression in HaCaT cells, suggesting that IL-18 and CRH act antagonistically to each other (Park et al., 2005). Conti et al. (2000) have shown that the production of IL-18 in the adrenal cortex is stimulated by ACTH and cannot be inhibited by the direct action of corticosterone. In this study, we also showed that IL-18 mRNA expression and its production were significantly stimulated by ACTH in a dose-dependent manner in HaCaT keratinocytes (Figure 1). The above findings suggest that IL-18 may play an www.jidonline.org 1213
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Figure 6. Effects of ACTH on the production of IL-18 in primary human epidermal keratinocytes. (a) Primary human epidermal keratinocytes were pretreated with or without Ac-YVAD-CHO (40 mM) for 2 hours before addition of 1 nM of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer’s instruction. The data, normalized to the negative control, are shown as fold increases in caspase activity. (c) Cells were treated with or without SB203580 (10 mM) or PD98059 (10 mM) for 2 hours before addition of 1 nM of ACTH for 24 hours. Supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
important role on the negative feedback loop, CRH upregulating POMC and, subsequently, ACTH leads to the induction of IL-18, and then the downregulation of CRH. Sugama et al. (2000) have reported differential IL-18 promoter usage in the adrenal gland and immune cells with adrenal gland-specific expression of IL-18 mRNA by ACTH, suggesting that IL-18 may be induced in the adrenal gland during stress. Besides, Sekiyama et al. (2005) demonstrated that ACTH can enhance IL-18 production through superoxide-mediated caspase-1 activation and p38 MAPK kinase. In these studies, animal models were used to show the systematic effects of ACTH by injecting ACTH into the animals. In this study, the aim was to test the direct effect of ACTH in keratinocytes and to compare the difference between the adrenal glands and keratinocytes. The results showed for the first time that ACTH can directly regulate immune functions of keratinocytes, and that the mechanism was the same as that of the adrenal gland. These findings suggest that caspase-1 activation and p38 MAPK kinase pathways are commonly involved in ACTH-induced IL-18 production. In conclusion, we demonstrated that ACTH stimulates IL18 expression by phosphorylation of p38 and ERK MAPK signaling pathway. Therefore, our findings may provide an insight into cellular interactions between ACTH and inflammatory mediators. Because IL-18 is involved in both Th1 and Th2 functions, the ability of ACTH to induce the production of inflammatory mediators by keratinocytes provides a new mechanism for the involvement of ACTH in innate and 1214 Journal of Investigative Dermatology (2007), Volume 127
adaptive immunity and its role in the pathogenesis of skin disorders. MATERIALS AND METHODS Cell culture The human keratinocyte cell line HaCaT was cultured in RPMI 1640 (Gibco-BRL, Gaithersburg, MD) medium supplemented with 2mM L-glutamine, antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL). This cell line was used for experiments while in their log phase of growth. Primary cultures of normal human epidermal keratinocytes were prepared from foreskin of three children who underwent circumcision. The ethical committee of the Catholic University of Korea approved the study, and all patients provided an informed consent. Our experiments adhered to the Declaration of Helsinki Principles. Skin specimens were cut into small pieces and incubated overnight in dispase II (2.4 U/ml; Roche Applied Science, Mannheim, Germany) at 41C. The epidermis was detached from the dermis with fine forceps and incubated in Hank’s solution with 0.25% trypsin (Gibco-BRL) for 20 minutes at 371C. Trypsin activity was stopped by adding FBS. The cell suspension passed through a 40 mm sterile gauze and the keratinocytes obtained were washed twice. Freshly isolated keratinocytes were cultured in keratinocyte basal medium (BioWhittaker, Heidelberg, Germany) with full supplements (0.1 ng/ml human epidermal growth factor, 0.5 mg/ml hydrocortisone, 5 mg/ml insulin, 7.5 mg/ml bovine pituitary extract, 50 mg/ml gentamicin, 50 ng/ml amphotericin-B, and 0.15 mM calcium) according to the manufacturer’s instruction. All cells were incubated in a humidified
HJ Park et al. IL-18 Expression in Human HaCaT Keratinocytes
atmosphere containing 5% CO2 at 371C and were used at the passage two to five.
Cell treatment with inhibitors HaCaT cells were seeded at a density of 1 106 cells/100-mm dishes. After 48 hours, the cells were washed with a serum-free medium and then placed at least 16 hours before experiments in a media without FBS. The cells were pretreated with 10 mM SB203580 (Sigma, St Louis, MO), a p38 MAPK inhibitor, 10 mM PD98059 (Sigma), an ERK inhibitor, 40 mM Ac-YVAD-CHO (SantaCruz Biotechnology, Santa Cruz, CA), an IL-1b-converting enzyme/ caspase-1 inhibitor, or 20 mM Z-FA-FMK (R&D systems, Minneapolis, MN), a cathepsin-B inhibitor for 2 hours in a serum-free medium. We added human ACTH (1011 MB108 M) to the cells, which was purchased from Sigma.
Treatment of cells with Anti-MC1R and Anti-MC2R antibody HaCaT cells were pretreated with anti-MC1R or anti-MC2R antibodies (SantaCruz Biotechnology) for 1 hour in serum-free medium. The cells were washed with serum-free medium and then ACTH (1 nM) was added to the cells and incubated for 24 hours.
Reverse transcription-PCR After treatment, total RNA was extracted from HaCaT cells using the EasyBlue reagent (iNtRON, Inc., Sungnam, Korea). The reverse transcription was performed using a power cDNA synthesis kit (iNtRON, Inc.). IL-18 primers were: forward, 50 -AGGAATAAA GATGGCTGCTGAAC-30 ; reverse, 50 -GCTCACCACAACCTCTACC TCC-30 . The PCR amplification process consisted of 32 cycles of 951C for 1 minute; 601C for 1 minute; and 721C for 45 seconds. Human b-actin primers were: forward, 50 -GGCCATCTCTTGCTC GAAGT-30 ; reverse, 50 -GCCCAGAGCAAG AGAGGCAT-30 . The PCR amplification process consisted of 30 cycles of 941C for 30 seconds; 561C for 30 seconds; and 721C for 1 minute. Reaction products subjected to electrophoresis on 2.0% agarose gel were visualized with ethidium bromide. Signal strengths were quantified using a densitometric program (Quantity One 1-D Analysis software, Bio-Rad Laboratories, Hercules, CA). After normalizing versus bactin intensity, the increase or decrease in percentage was determined for each gene. Each experiment was repeated at least three times.
IL-18 ELISA The concentration of IL-18 in the culture supernatant was measured by a commercially available ELISA (MBL Medical & Biological Laboratories Co., Ltd, Nagoya, Japan). After stimulation, cell culture supernatants were collected, concentrated ( 30) by a microcon (Millipore, Bedford, MA), and then added to coated wells. After adding diluted biotinylated anti-IL-18 antibody to the wells, they were incubated for 3 hours at room temperature. After washing for four times with phosphate-buffered saline-Tween-20 (pH 7.4), streptavidin-horseradish peroxidase conjugated anti-human IL-18 antibody was added, which was incubated for an hour. The wells were washed four times with PBS-Tween-20, and substrate solution was added, which was incubated for an hour. Relative absorbance was measured at 450 nm, and the IL-18 concentration was calculated using a standard curve. Each supernatant was analyzed in triplicate.
Measurement of caspase-1 activity The caspase-1 activity assay was performed with an enzyme activity assay kit (Caspase-1 Colorimetric Assay kit, R&D systems) according to the manufacturer’s instructions. After stimulation, the cell pellets were lysed in a cell lysis buffer. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL). To 50 ml of cell lysate (200 mg protein), 50 ml of 2 reaction buffer (containing DTT 10 mmol/l) and 5 ml of the 4 mmol/l caspase-1 colorimetric substrate (WEHD-pNA) were added, and the mixture was incubated at 371C for 2 hours. The samples were read at 405 nm with a spectrophotometer.
Immunoprecipitation and Western blot analysis Whole-cell lysates were prepared by extracting proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM freshly added phenylmethylsulfonyl fluoride. To detect the phosphorylated form of p38 MAPK, the lysates were then immunoprecipitated with 2 mg of anti-phosphotyrosine antibody and 25 ml of protein-A agarose for 3 hours at 41C. After washing three times (20 minutes at 41C) with lysis buffer, the proteins bound to the beads were solubilized in elution buffer consisting of 1 M Tris (pH 6.8) and 0.1% SDS. The protein concentration was measured using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories). Equal amounts of protein were resolved by 10% SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with anti-p38 MAPK antibody. Blots were processed using horseradish peroxidaseconjugated secondary antibodies and enhanced chemiluminescence (ECL Detection Kit, Amersham Biosciences, Piscataway, NJ). To detect MC1R, MC2R, and the phosphorylation level of ERK and p38 MAPK, ACTH-treated and untreated cells were lysed with lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 1.0% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride in PBS (pH 7.4). Whole-cell lysates were directly resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) nonfat milk proteins. Blocked membranes were then incubated with primary antibodies (MC1R, MC2R rabbit polyclonal IgG antibodies at the final concentration of 1:50, Santa Cruz Biotechnology; phospho-ERK, total ERK or p38 rabbit polyclonal antibodies at the final concentration of 1:1,000, Cell Signaling, Beverly, MA) overnight at 41C, washed three times (5 minutes each) with PBS containing 0.1% Tween-20, and incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 hour at room temperature. The bands were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences). The band intensities were quantified using the Bio-Rad imaging system, and the quantity of the phosphorylated proteins was expressed as ratio of the phosphorylated over total protein in each case.
Statistical analysis The statistical significance was performed using Student’s t-test. Mean differences were considered to be significant when Po0.05. The results are shown as the mean7SD. CONFLICT OF INTEREST The authors state no conflict of interest.
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HJ Park et al. IL-18 Expression in Human HaCaT Keratinocytes
ACKNOWLEDGMENTS This work was supported by the SRC/ERC program of MOST/KOSEF (No. R112005-017) and the Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund, KRF-2005-204E00062).
REFERENCES Bohm M, Luger TA, Tobin DJ, Garcia-Borron JC (2006) Melanocortin receptor ligands: new horizons for skin biology and clinical dermatology. J Invest Dermatol 126:1966–75 Chakraborty AK, Funasaka Y, Slominski A, Emak G, Hwang J, Pawelek JM et al. (1996) Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B. Biochim Biophys Acta 1313:130–8 Chrousos GP (1995) The hypothalamic–pituitary–adrenal axis and immunemediated inflammation. N Engl J Med 332:1351–62 Conti B, Sugama S, Kim Y, Tinti C, Kim H, Baker H et al. (2000) Modulation of IL-18 production in the adrenal cortex following acute ACTH or chronic corticosterone treatment. Neuroimmunomodulation 8:1–7 Dinarello CA (1999) IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J Allergy Clin Immunol 103:11–24 Dugue B, Leppanen EA, Teppo AM, Fyhrquist F, Grasbeck R (1993) Effects of psychological stress on plasma interleukins-1 beta and 6, C-reactive protein, tumor necrosis factor alpha, antidiuretic hormone and serum cortisol. J Clin Lab Invest 53:555–61 Ghayur T, Banerjee S, Banerjee MD, Herzog L, Herzog A, Quintral L et al. (1997) Caspase-1 processed IFN-g- inducing factor and regulates LPS-induced IFN-g production. Nature 386:619–23 Gu Y, Kida K, Tsutsui H, Ku G, Hsiao K, Fleming MA et al. (1997) Activation of interferon-g- inducing factor mediated by interleukin-1b converting enzyme. Science 275:206–9 Hoshino T, Wiltrout RH, Young HA (1999) IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response. J Immunol 162:5070–7 Luger TA, Scholzen T, Brzoska T, Becher E, Slominski A, Paus R (1998) Cutaneous immunomodulation and coordination of skin responses by alpha-melanocyte-stimulating hormone. Ann NY Acad Sci 840:381–94 Mee JB, Alam Y, Groves RW (2000) Human keratinocytes constitutively produce but do not process interleukin-18. Br J Dermatol 143:330–6 Naik SM, Cannon G, Burbach GJ, Singh SR, Swerlick RA, Wilcox JN et al. (1999) Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with proinflammatory mediators and dinitrochlorobenzene. J Invest Dermatol 113:766–72
Pavey S, Gabrielli B (2002) Alpha-melanocyte stimulating hormone potentiates p16/CDKN2A expression in human skin after ultraviolet irradiation. Cancer Res 62:875–80 Schotte P, Schauvliege R, Janssens S, Beyaert R (2001) The cathepsin B inhibitor z-FA.fmk inhibits cytokine production in macrophages stimulated by lipopolysaccharide. J Biol Chem 276:21153–7 Sekiyama A, Useda H, Kashiwamura S, Sekiyama R, Takeda M, Rokutan K et al. (2005) A stress-induced, superoxide-mediated caspase-1 activation pathway causes plasma IL-18 upregulation. Immunity 22:669–77 Slominski A, Ermak G, Hwang J, Chakraborty A, Mazurkiewicz JE, Mihm M (1995) Proopiomelanocortin, corticotropin releasing hormone and corticotrophin releasing hormone receptor genes are expressed in human skin. FEBS Lett 374:113–6 Slominski A, Mihm MC (1996) Potential mechanism of skin response to stress. Int J Dermatol 35:849–51 Slominski A, Paus R, Wortsman J (1993) On the potential role of proopiomelanocortin in skin physiology and pathology. Mol Cell Endocrinol 93:C1–6 Slominski A, Pisarchik A, Tobin DJ, Mazurkiewicz JE, Wortsman J (2004a) Differential expression of a cutaneous corticotropin-releasing hormone system. Endocrinology 145:941–50 Slominski A, Szczesniewski A, Wortsman J (2000a) Liquid chromatographymass spectrometry detection of corticotropin-releasing hormone and proopio-melanocortin-derived peptides in human skin. J Clin Endocrinol Metab 85:3582–8 Slominski A, Wortsman J, Luger T, Paus R, Solomon S (2000b) Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 80:979–1020 Slominski A, Wortsman J, Pisarchik A, Zbytek B, Linton EA, Mazurkiewicz JE et al. (2001) Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors. FASEB J 15:1678–93 Slominski A, Zbytek B, Semak I, Sweatman T, Wortsman J (2005b) CRH stimulates POMC activity and corticosterone production in dermal fibroblasts. J Neuroimmunol 162:97–102 Slominski A, Zbytek B, Szczesniewski A, Semak I, Kaminski J, Sweatman T et al. (2005a) CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH. Am J Physiol Endocrinol Metab 288:E701–6 Slominski A, Tobin DJ, Shibahara S, Wortsman J (2004b) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155–228 Sugama S, Kim Y, Baker H, Tinti C, Kim H, Joh TH et al. (2000) Tissue-specific expression of rat IL-18 gene and response to adrenocorticotropic hormone treatment. J Immunol 165:6287–92
Nakanishi K, Yoshimoto T, Txutsui H (2001) Interleukin-18 regulates both Th1 and Th2 responses. Ann Rev Immunol 19:423–74
Tringali G, Pozzoli G, Vairano M, Mores N, Preziosi P, Navarra P (2005) Interleukin-18 displays effects opposite to those of interleukin-1 in the regulation of neuroendocrine stess axis. J Neuroimmunol 160:61–7
Navarra P, Tsagarakis S, Faria MS, Rees LH, Besser GM, Grossman A (1991) Interleukin-1b and -6 stimulate the release of corticotrophin releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway. Endocrinology 128:37–44
Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K et al. (1996) Cloning of the cDNA for human IFN g- inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 156:4274–9
Ohta Y, Hamada Y, Katsuoka K (2001) Expression of IL-18 in psoriasis. Arch Dermatol Res 293:334–42
Wakamatsu K, Graham A, Cook D, Thody AJ (1997) Characterization of ACTH peptides in human skin and their activation of the melanocortin-1 receptor. Pigment Cell Res 10:288–97
Okamura H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T et al. (1995) Cloning of a new cytokine that induces IFN-g production by T cells. Nature 378:88–91 Park HJ, Kim HJ, Lee JY, Cho BK, Kang SJ, Kang H et al. (2005) Corticotropinreleasing hormone (CRH) downregulates interleukin-18 expression in human HaCaT keratinocytes by activation of p38 mitogen-activated protein kinase (MAPK) pathway. J Invest Dermatol 124:751–5
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Wang S, Miura M, Jung YK, Zhu H, Li E, Yuan J (1998) Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92:501–9 Yoshimoto T, Mizutani H, Tsutsui H, Noben-Trauth N, Yamanaka K, Tanaka M et al. (2000) IL-18 induction of IgE: dependence on CD4+ T cells IL-4 and STAT6. Nat Immunol 1:132–7
Adrenocorticotropin Hormone Stimulates Interleukin-18 Expression in Human HaCaT Keratinocytes Hyun Jeong Park1,5, Hee Jung Kim1,5, Jun Young Lee1, Baik Kee Cho1, Richard L. Gallo2,3 and Dae Ho Cho4 The hypothalamic–pituitary–adrenal (HPA) axis is activated by stress. This involves the production of corticotropin releasing hormone (CRH) with the subsequent release of proopiomelanocortin (POMC) peptides, of which adrenocorticotropin hormone (ACTH) is most important. Although the skin has the capacity to produce CRH and POMC peptides, the immunomodulatory roles of ACTH in skin are yet unknown. IL-18 has been known to affect cells involved in the inflammatory response. In this study, we aimed to identify the regulatory effect of ACTH on IL-18 expression of skin keratinocytes. Exposure of HaCaT cells to ACTH stimulated formation of IL-18 mRNA transcript and its protein products in a dose-dependent manner. Furthermore, we suggest that ACTH-induced IL-18 production is via the caspase-1 activation pathway, as IL-18 production induced by ACTH could be suppressed by caspase-1 inhibitor, and ACTH could increase caspase-1 activity. The effect of ACTH on IL-18 production was blocked by specific inhibitors of p38 kinase (SB203580) or extracellular signalregulated kinase (ERK) (PD98059). In addition, ACTH-induced rapid phosphorylation of p38 kinase and ERK, and ACTH signaling occurred via melanocortin receptor 1 (MC1R) and receptor 2 (MC2R). These results suggest that ACTH stimulates IL-18 expression in human keratinocytes, which provides an insight into the interaction between ACTH and inflammatory mediators. Journal of Investigative Dermatology (2007) 127, 1210–1216. doi:10.1038/sj.jid.5700703; published online 18 January 2007
INTRODUCTION As an adaptive response to systemic stress, the hypothalamic– pituitary–adrenal (HPA) axis is activated (Chrousos, 1995). The process starts with the hypothalamic release of corticotropin-releasing hormone (CRH) which, in turn stimulates production of adrenocorticotropic hormone (ACTH) and other proopiomelanocortin (POMC) peptides via CRH receptor type 1 (CRH-R1). ACTH, a 39-amino-acid peptide, regulates glucocorticoid production through its effect on the adrenal cortex. Steroids are powerful anti-inflammatory agents that counteract stressors and buffer tissue damage 1
Department of Dermatology, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 2Department of Dermatology, University of California, San Diego, California, USA; 3VA San Diego Health Care System, San Diego, California, USA and 4Department of Life Science, Sookmyung Women’s University, Seoul, Korea
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These authors are co-first authors.
Correspondence: Dr Hyun Jeong Park, Department of Dermatology, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. E-mail: [email protected] and Dae Ho Cho, Department of Life Science, Sookmyung Women’s University, Chungpa-Dong 2-Ka, Yongsan-Ku, Seoul 140-742, South Korea. E-mail: [email protected] Abbreviations: ACTH, adrenocorticotropin hormone; CRH, corticotropin releasing hormone; ERK, extracellular signal-regulated kinase; HPA, hypothalamic–pituitary–adrenal; MAPK, mitogen-activated protein kinase; MCIR, melanocortin receptor 1; PBS, phosphate-buffered saline; POMC, proopiomelanocortin Received 6 July 2006; revised 27 October 2006; accepted 7 November 2006; published online 18 January 2007
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(Chrousos, 1995). Recent evidence suggests that ACTH is produced outside the pituitary tissue, which includes the skin (Chakraborty et al., 1996; Wakamatsu et al., 1997; Slominski et al., 2000a). Slominski et al. have acknowledged the skin as a cutaneous equivalent of the central HPA (Slominski and Mihm, 1996; Slominski et al., 2000b, 2001, 2004a). Although similar to its systemic equivalent, the cutaneous HPA axis is responsive to local stressors (solar, thermal, chemical, biological, etc.) and resultantly activates the neuronal, endocrine, and immune systems in the skin. A variety of cytokines (e.g., IL-1b, IL-6, and TNF-a) are upregulated by stress, suggesting their role in mediating stress responses (Dugue et al., 1993). However, the molecular mechanism of stress-induced cytokine in impairing host defenses is not well understood. IL-18 was originally discovered as IFN-g-inducing factor (Okamura et al., 1995; Ushio et al., 1996). It is normally expressed in its precursor form (24 kDa), but can be activated (18 kDa) by caspase-1 (IL-1b-converting enzyme) (Ghayur et al., 1997; Gu et al., 1997). IL-18 performs multiple biological activities, which include Fas (CD95, APO-1) ligand induction, enhancement of T-cell cytolytic activity, and production of T helper type 2 (Th2) cytokines (Hoshino et al., 1999; Yoshimoto et al., 2000; Nakanishi et al., 2001). IL-18 is expressed in a wide range of cells, including Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, and adrenal cortex cells. It plays an important role in the Th1 response to toxic shock and shares functional similarities with IL-12 (Dinarello, 1999). As a & 2007 The Society for Investigative Dermatology
HJ Park et al. IL-18 Expression in Human HaCaT Keratinocytes
proinflammatory cytokine, overproduction of IL-18 induces severe inflammatory disorders. Thus, it is important to know how IL-18 expression is regulated in inflammatory response and in maintaining the balance of immune system function. Recently, IL-18 by keratinocytes was reported to play an important role in response to cutaneous inflammation (Mee et al., 2000). In this study, we investigated the effect of ACTH on IL-18 expression of human HaCaT keratinocytes, and also examined its regulatory mechanism. RESULTS Effect of ACTH on IL-18 production in HaCaT cells
To measure the effect of ACTH on IL-18 expression in HaCaT cells, we treated the cells with the fixed concentrations of ACTH, and then assessed IL-18 mRNA and its protein expression by reverse transcriptase-PCR and ELISA, respectively. IL-18 mRNA expression was increased in a dosedependent manner by ACTH, with the peak response at 109 M (Figure 1a). IL-18 assessed by ELISA was also increased in a dose-dependent manner (Figure 1b). As shown
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Because caspase-1 is required for cleaving pro-IL-18 into its mature form, its involvement in IL-18 production was examined by pretreating HaCaT cells for 2 hours with the caspase-1-specific inhibitor Ac-YVAD-CHO before stimulation with ACTH. We observed Ac-YVAD-CHO to suppress the production of IL-18 (Figure 2a). Next, we investigated the molecular mechanism underlying ACTH-induced IL-18 production by performing caspase-1 colorimetric assay. Caspase-1 activity in HaCaT cells were elevated 10-fold by ACTH (Figure 2b). The results indicate that ACTH is involved in IL-18 production of HaCaT cells probably via a caspase-1 activation pathway. In a previous report, caspase-1 was activated by caspase-11 (Wang et al., 1998). It has also been reported that caspase-11 can be activated by cathepsins B (Schotte et al., 2001). To understand the involvement of caspase-11 in the ACTH-induced caspase-1 activation, we examined it by using Z-FA-FMK, an inhibitor of cathepsins B. In our observation, Z-FA-FMK suppressed the production of IL-18 induced by ACTH, indicating that caspase-11 is also involved in the ACTH-induced IL-18 production in HaCaT cells (Figure 2c). Effect of mitogen-activated protein kinase inhibitors on IL-18 expression
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Figure 1. Effects of ACTH on the mRNA expression and production of IL-18 in HaCaT keratinocytes. (a) HaCaT cells were harvested after ACTH treatment for 6 hours. Total RNA was extracted, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to b-actin by densitometry. (b) Cells were treated with indicated concentration of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control.
The mitogen-activated protein kinases (MAPKs) control a wide variety of cellular events, which include very complex cellular programs such as cell differentiation, proliferation and apoptosis to processes involved in immune responses. To identify its involvement in ACTH-induced IL-18 expression, HaCaT cells were treated with 10 mM of p38 MAPK inhibitor (SB203580) or extracellular signal-regulated kinase (ERK) inhibitor (PD98059) for 2 hours before adding ACTH (1 nM). We then assessed the expression of IL-18 mRNA transcripts and its products. As shown in Figure 3, both SB203580 and PD98059 had an inhibitory effect over ACTH on IL-18 mRNA expression and its production. To further identify the role of MAP kinase signaling pathways in ACTH-induced IL-18 expression, HaCaT cells were treated with ACTH, and the phosphorylation status of p38 kinase and ERK were analyzed by immunoprecipitation and Western blotting using commercially available antibodies specific for the active forms of the two kinases. ACTH induced transient phosphorylation of both p38 MAP kinase and ERK. The maximal activation of p38 kinase and ERK by ACTH was observed after 5 minutes (Figure 4a and b). These data demonstrate that ACTH stimulates IL-18 production through p38 kinase and ERK signaling pathways. Melanocortin receptor 1 and MC2R expression in HaCaT cells
To study whether melanocortin receptor 1 (MC1R) and/or MC2R are involved in the mediating ACTH-induced IL-18 production in HaCaT cells, the cells were treated with ACTH and then MC1R and MC2R were analyzed by Western blotting. www.jidonline.org 1211
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Figure 2. Caspase-1 is essential for the ACTH-induced IL-18 production in HaCaT keratinocytes. (a) HaCaT cells were pretreated with 40 mM of the caspase-1 inhibitor Ac-YVAD-CHO for 1 hour, and then challenged with 1 nM ACTH. The IL-18 concentration in the culture supernatants was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer’s instruction. The data normalized to the negative control are shown as fold increases in caspase activity. (c) Effects of Z-FA-fmk on ATCH-induced IL-18 production. HaCaT cells were pretreated with 20 mM of Z-FA-FMK for 2 hours, and then stimulated with 1 nM ACTH. The IL-18 concentration in the supernatant was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
We determined that MC1R and MC2R were both expressed on HaCaT cells (Figure 5a). Next, we used anti-MC1R and antiMC2R antibody to examine whether they could neutralize the ACTH-induced IL-18 production. Anti-MC2R antibody was more effective and almost reduced it to the control level (Figure 5b). These results suggest that the induction of IL-18 by ACTH may be mainly mediated by MC2R. Effect of ACTH on IL-18 production in human primary keratinocytes
To see whether ACTH regulates IL-18 production in primary human epidermal keratinocytes, we assessed IL-18 production by ELISA. As shown in Figure 6a, IL-18 production was significantly stimulated by ACTH treatment in human primary keratinocytes. Next, we observed that caspase-1 activity in primary human epidermal keratinocytes was stimulated by ACTH (Figure 6b). Ac-YVAD-CHO, a caspase-1 inhibitor, effectively suppresses IL-18 production by ACTH in primary human epidermal keratinocytes, as shown in the result from human keratinocyte cell line HaCaT (Figure 6a). In the end, we showed that SB203580 and PD98059 could suppress the ACTH-induced IL-18 production, suggesting that p38 kinase and ERK were both involved in the signaling pathway in primary human epidermal keratinocytes (Figure 6c). DISCUSSION In this study, we investigated the regulatory effect of ACTH on expression of IL-18 in HaCaT and normal keratinocytes. 1212 Journal of Investigative Dermatology (2007), Volume 127
Our results show that ACTH stimulates human keratinocytes to secrete IL-18 through MC1R and MC2R. Furthermore, ACTH activates the MAPK p38 and ERK pathways that are further required for IL-18 expression. In response to systemic stress, CRH stimulates the pituitary CRH-R, which leads to production and secretion of POMCderived peptides, including melanocyte-stimulating hormone (MSH) and ACTH (Chrousos, 1995). Recent evidence suggests that CRH stimulation of corticosteroids production in melanocytes and dermal fibroblasts is mediated by ACTH (Slominski et al., 2005a, b). CRH, CRH-R, and POMC genes are expressed in human skin as well as in cultured keratinocytes and melanocytes (Slominski et al., 1993, 1995, 2001, 2004a, b). Slominski et al. (2000b, 2001) proposed that an equivalent of HPA axis, composed of the CRH-CRHR-POMC loop, is conserved in the skin that may be activated in a skin stress response system. Recently, MSH and ACTH act as regulators of skin pigmentation and inhibitors of the skin immune systems (Luger et al., 1998; Slominski et al., 2004a, b). In this study, ACTH induced IL-18 production, which is known as an inflammatory cytokine. However, it is also known that the closely related POMC peptide alphaMSH exhibits anti-inflammatory effects through MC1R in keratinocytes (Pavey and Gabrielli, 2002). To date, five melanocortin receptors are known, and they have a variety of important biological functions (Bohm et al., 2006). To understand the difference between ACTH and alpha-MSH, we detected the expression of MC receptors in HaCaT cells.
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Figure 3. Effects of the p38 MAPK inhibitor SB203580, or the ERK inhibitor PD98059 on ACTH-induced IL-18 expression in HaCaT cells. HaCaT cells were pretreated with SB203580 (10 mM) or PD98059 (10 mM) for 2 hours before addition of 1 nM of ACTH. (a) Total RNA was extracted at 6 hours following ACTH treatment, and cDNA was synthesized for reverse transcriptase-PCR. The histogram shows the IL-18 mRNA expression level relative to b-actin by densitometry. (b) After cells were harvested for 24 hours, IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
Figure 5. Involvement of MC1R and MC2R in ACTH-induced IL-18 production. (a) HaCaT cells were treated with 1 nM of ACTH. After that, cells were harvested for 24 hours. The expression level of MC1R and MC2R were analyzed by Western blot. (b) Cells were pretreated with anti-MC1R (2 mg/mL) or anti-MC2R antibody (2 mg/mL) for 1 hour and then incubated with 1 nM of ACTH for 24 hours. The supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
Figure 5 showed that ACTH signals through MC1R and MC2R. This might be an explanation for the differential effects of ACTH and alpha-MSH in inflammation as MC1R and MC2R have different signal pathways (Bohm et al., 2006). IL-18 has multiple and complicated functions as it participates in innate immunity and in Th1- and Th2mediated responses. During inflammation, the activation of the stress system, through induction of a Th2 shift, protects the organism from systemic ‘‘overshooting’’ with Th1/proinflammatory cytokines. Interestingly, it has been reported that the expression of IL-18 is markedly increased in suprabasal keratinocytes in psoriatic lesions (Naik et al., 1999; Ohta et al., 2001), raising the possibility that IL-18 expression is upregulated in differentiated keratinocytes. Therefore, ACTH induced IL-18 expression by keratinocytes may provide new insights into the mechanism underlying the pathogenesis of skin disorders.
In a previous report, cytokines IL-1b and IL-6 were able to stimulate CRH secretion from the hypothalamus (Navarra et al., 1991), and IL-18 was originally identified as a member of the IL-1 family (Dinarello, 1999) and expected to be able to stimulate CRH production. Interestingly, recent evidence documented that IL-18 inhibited basal and stimulated CRH secretion from isolated rat hypothalami, and reduced CRH gene expression (Tringali et al., 2005). We have recently reported that CRH inhibits IL-18 expression in HaCaT cells, suggesting that IL-18 and CRH act antagonistically to each other (Park et al., 2005). Conti et al. (2000) have shown that the production of IL-18 in the adrenal cortex is stimulated by ACTH and cannot be inhibited by the direct action of corticosterone. In this study, we also showed that IL-18 mRNA expression and its production were significantly stimulated by ACTH in a dose-dependent manner in HaCaT keratinocytes (Figure 1). The above findings suggest that IL-18 may play an www.jidonline.org 1213
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Figure 6. Effects of ACTH on the production of IL-18 in primary human epidermal keratinocytes. (a) Primary human epidermal keratinocytes were pretreated with or without Ac-YVAD-CHO (40 mM) for 2 hours before addition of 1 nM of ACTH for 24 hours. Cell supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. (b) Cells were stimulated with 1 nM ACTH for 6 hours. Caspase-1 enzymatic activity was assayed with a caspase-1 colorimetric assay kit according to the manufacturer’s instruction. The data, normalized to the negative control, are shown as fold increases in caspase activity. (c) Cells were treated with or without SB203580 (10 mM) or PD98059 (10 mM) for 2 hours before addition of 1 nM of ACTH for 24 hours. Supernatants were collected and then IL-18 concentration was measured by ELISA, each performed in triplicate. Data are mean7SD. *Po0.05 versus control, yPo0.05 versus ACTH treated group.
important role on the negative feedback loop, CRH upregulating POMC and, subsequently, ACTH leads to the induction of IL-18, and then the downregulation of CRH. Sugama et al. (2000) have reported differential IL-18 promoter usage in the adrenal gland and immune cells with adrenal gland-specific expression of IL-18 mRNA by ACTH, suggesting that IL-18 may be induced in the adrenal gland during stress. Besides, Sekiyama et al. (2005) demonstrated that ACTH can enhance IL-18 production through superoxide-mediated caspase-1 activation and p38 MAPK kinase. In these studies, animal models were used to show the systematic effects of ACTH by injecting ACTH into the animals. In this study, the aim was to test the direct effect of ACTH in keratinocytes and to compare the difference between the adrenal glands and keratinocytes. The results showed for the first time that ACTH can directly regulate immune functions of keratinocytes, and that the mechanism was the same as that of the adrenal gland. These findings suggest that caspase-1 activation and p38 MAPK kinase pathways are commonly involved in ACTH-induced IL-18 production. In conclusion, we demonstrated that ACTH stimulates IL18 expression by phosphorylation of p38 and ERK MAPK signaling pathway. Therefore, our findings may provide an insight into cellular interactions between ACTH and inflammatory mediators. Because IL-18 is involved in both Th1 and Th2 functions, the ability of ACTH to induce the production of inflammatory mediators by keratinocytes provides a new mechanism for the involvement of ACTH in innate and 1214 Journal of Investigative Dermatology (2007), Volume 127
adaptive immunity and its role in the pathogenesis of skin disorders. MATERIALS AND METHODS Cell culture The human keratinocyte cell line HaCaT was cultured in RPMI 1640 (Gibco-BRL, Gaithersburg, MD) medium supplemented with 2mM L-glutamine, antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL). This cell line was used for experiments while in their log phase of growth. Primary cultures of normal human epidermal keratinocytes were prepared from foreskin of three children who underwent circumcision. The ethical committee of the Catholic University of Korea approved the study, and all patients provided an informed consent. Our experiments adhered to the Declaration of Helsinki Principles. Skin specimens were cut into small pieces and incubated overnight in dispase II (2.4 U/ml; Roche Applied Science, Mannheim, Germany) at 41C. The epidermis was detached from the dermis with fine forceps and incubated in Hank’s solution with 0.25% trypsin (Gibco-BRL) for 20 minutes at 371C. Trypsin activity was stopped by adding FBS. The cell suspension passed through a 40 mm sterile gauze and the keratinocytes obtained were washed twice. Freshly isolated keratinocytes were cultured in keratinocyte basal medium (BioWhittaker, Heidelberg, Germany) with full supplements (0.1 ng/ml human epidermal growth factor, 0.5 mg/ml hydrocortisone, 5 mg/ml insulin, 7.5 mg/ml bovine pituitary extract, 50 mg/ml gentamicin, 50 ng/ml amphotericin-B, and 0.15 mM calcium) according to the manufacturer’s instruction. All cells were incubated in a humidified
HJ Park et al. IL-18 Expression in Human HaCaT Keratinocytes
atmosphere containing 5% CO2 at 371C and were used at the passage two to five.
Cell treatment with inhibitors HaCaT cells were seeded at a density of 1 106 cells/100-mm dishes. After 48 hours, the cells were washed with a serum-free medium and then placed at least 16 hours before experiments in a media without FBS. The cells were pretreated with 10 mM SB203580 (Sigma, St Louis, MO), a p38 MAPK inhibitor, 10 mM PD98059 (Sigma), an ERK inhibitor, 40 mM Ac-YVAD-CHO (SantaCruz Biotechnology, Santa Cruz, CA), an IL-1b-converting enzyme/ caspase-1 inhibitor, or 20 mM Z-FA-FMK (R&D systems, Minneapolis, MN), a cathepsin-B inhibitor for 2 hours in a serum-free medium. We added human ACTH (1011 MB108 M) to the cells, which was purchased from Sigma.
Treatment of cells with Anti-MC1R and Anti-MC2R antibody HaCaT cells were pretreated with anti-MC1R or anti-MC2R antibodies (SantaCruz Biotechnology) for 1 hour in serum-free medium. The cells were washed with serum-free medium and then ACTH (1 nM) was added to the cells and incubated for 24 hours.
Reverse transcription-PCR After treatment, total RNA was extracted from HaCaT cells using the EasyBlue reagent (iNtRON, Inc., Sungnam, Korea). The reverse transcription was performed using a power cDNA synthesis kit (iNtRON, Inc.). IL-18 primers were: forward, 50 -AGGAATAAA GATGGCTGCTGAAC-30 ; reverse, 50 -GCTCACCACAACCTCTACC TCC-30 . The PCR amplification process consisted of 32 cycles of 951C for 1 minute; 601C for 1 minute; and 721C for 45 seconds. Human b-actin primers were: forward, 50 -GGCCATCTCTTGCTC GAAGT-30 ; reverse, 50 -GCCCAGAGCAAG AGAGGCAT-30 . The PCR amplification process consisted of 30 cycles of 941C for 30 seconds; 561C for 30 seconds; and 721C for 1 minute. Reaction products subjected to electrophoresis on 2.0% agarose gel were visualized with ethidium bromide. Signal strengths were quantified using a densitometric program (Quantity One 1-D Analysis software, Bio-Rad Laboratories, Hercules, CA). After normalizing versus bactin intensity, the increase or decrease in percentage was determined for each gene. Each experiment was repeated at least three times.
IL-18 ELISA The concentration of IL-18 in the culture supernatant was measured by a commercially available ELISA (MBL Medical & Biological Laboratories Co., Ltd, Nagoya, Japan). After stimulation, cell culture supernatants were collected, concentrated ( 30) by a microcon (Millipore, Bedford, MA), and then added to coated wells. After adding diluted biotinylated anti-IL-18 antibody to the wells, they were incubated for 3 hours at room temperature. After washing for four times with phosphate-buffered saline-Tween-20 (pH 7.4), streptavidin-horseradish peroxidase conjugated anti-human IL-18 antibody was added, which was incubated for an hour. The wells were washed four times with PBS-Tween-20, and substrate solution was added, which was incubated for an hour. Relative absorbance was measured at 450 nm, and the IL-18 concentration was calculated using a standard curve. Each supernatant was analyzed in triplicate.
Measurement of caspase-1 activity The caspase-1 activity assay was performed with an enzyme activity assay kit (Caspase-1 Colorimetric Assay kit, R&D systems) according to the manufacturer’s instructions. After stimulation, the cell pellets were lysed in a cell lysis buffer. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL). To 50 ml of cell lysate (200 mg protein), 50 ml of 2 reaction buffer (containing DTT 10 mmol/l) and 5 ml of the 4 mmol/l caspase-1 colorimetric substrate (WEHD-pNA) were added, and the mixture was incubated at 371C for 2 hours. The samples were read at 405 nm with a spectrophotometer.
Immunoprecipitation and Western blot analysis Whole-cell lysates were prepared by extracting proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM freshly added phenylmethylsulfonyl fluoride. To detect the phosphorylated form of p38 MAPK, the lysates were then immunoprecipitated with 2 mg of anti-phosphotyrosine antibody and 25 ml of protein-A agarose for 3 hours at 41C. After washing three times (20 minutes at 41C) with lysis buffer, the proteins bound to the beads were solubilized in elution buffer consisting of 1 M Tris (pH 6.8) and 0.1% SDS. The protein concentration was measured using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories). Equal amounts of protein were resolved by 10% SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with anti-p38 MAPK antibody. Blots were processed using horseradish peroxidaseconjugated secondary antibodies and enhanced chemiluminescence (ECL Detection Kit, Amersham Biosciences, Piscataway, NJ). To detect MC1R, MC2R, and the phosphorylation level of ERK and p38 MAPK, ACTH-treated and untreated cells were lysed with lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 1.0% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride in PBS (pH 7.4). Whole-cell lysates were directly resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) nonfat milk proteins. Blocked membranes were then incubated with primary antibodies (MC1R, MC2R rabbit polyclonal IgG antibodies at the final concentration of 1:50, Santa Cruz Biotechnology; phospho-ERK, total ERK or p38 rabbit polyclonal antibodies at the final concentration of 1:1,000, Cell Signaling, Beverly, MA) overnight at 41C, washed three times (5 minutes each) with PBS containing 0.1% Tween-20, and incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 hour at room temperature. The bands were visualized by an enhanced chemiluminescence detection system (Amersham Biosciences). The band intensities were quantified using the Bio-Rad imaging system, and the quantity of the phosphorylated proteins was expressed as ratio of the phosphorylated over total protein in each case.
Statistical analysis The statistical significance was performed using Student’s t-test. Mean differences were considered to be significant when Po0.05. The results are shown as the mean7SD. CONFLICT OF INTEREST The authors state no conflict of interest.
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ACKNOWLEDGMENTS This work was supported by the SRC/ERC program of MOST/KOSEF (No. R112005-017) and the Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund, KRF-2005-204E00062).
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