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• W2072462906 abstract "Stimulation with tumor necrosis factor (TNF)α and interferon (IFN)γ synergistically induced thymus- and activation-regulated chemokine (TARC)/CCL17 production from HaCaT keratinocytes (KC). Inhibitors for nuclear factor kappa B (NFκB), parthenolide, and Bay 11-7085, and an inhibitor of p38, SB202190, inhibited TNFα- and IFNγ-induced production of CCL17 by HaCaT KC. Surprisingly, an inhibitor of epidermal growth factor receptor tyrosine kinase, PD153035, enhanced the production of CCL17 in HaCaT KC. Roxithromycin (RXM), a 14-membered ring macrolide, suppressed CCL17 production by HaCaT KC induced by IFNγ and TNFα. RXM partially suppressed p38 phosphorylation and NFκB-driven luciferase activity induced by TNFα and IFNγ. Degradation of inhibitor of nuclear factor kappa B (IκB) α upon stimulation with IFNγ and TNFα was not affected by the addition of RXM. Through elucidating the mechanism of CCL17 production, our study indicates that RXM suppresses the production through the inhibition of p38 and NFκB, independent of the inhibition of IκB degradation. Stimulation with tumor necrosis factor (TNF)α and interferon (IFN)γ synergistically induced thymus- and activation-regulated chemokine (TARC)/CCL17 production from HaCaT keratinocytes (KC). Inhibitors for nuclear factor kappa B (NFκB), parthenolide, and Bay 11-7085, and an inhibitor of p38, SB202190, inhibited TNFα- and IFNγ-induced production of CCL17 by HaCaT KC. Surprisingly, an inhibitor of epidermal growth factor receptor tyrosine kinase, PD153035, enhanced the production of CCL17 in HaCaT KC. Roxithromycin (RXM), a 14-membered ring macrolide, suppressed CCL17 production by HaCaT KC induced by IFNγ and TNFα. RXM partially suppressed p38 phosphorylation and NFκB-driven luciferase activity induced by TNFα and IFNγ. Degradation of inhibitor of nuclear factor kappa B (IκB) α upon stimulation with IFNγ and TNFα was not affected by the addition of RXM. Through elucidating the mechanism of CCL17 production, our study indicates that RXM suppresses the production through the inhibition of p38 and NFκB, independent of the inhibition of IκB degradation. epidermal growth factor receptor interferon inhibitor of nuclear factor kappa B keratinocyte mitogen-activated protein kinase nuclear factor kappa B roxithromycin thymus- and activation-regulated chemokine tumor necrosis factor Thymus- and activation-regulated chemokine (TARC)/CCL17 is a member of the CC chemokine group that is constitutively expressed in the thymus and is produced by monocyte-derived dendritic cells (Imai et al., 1996Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. Molecular cloning of a novel T cell-derived CC chemokine expressed in thymus by signal sequence trap using Epstein–Barr virus vector.J Biol Chem. 1996; 271: 21514-21521Crossref PubMed Scopus (272) Google Scholar; Zlotnik and Yoshie, 2000Zlotnik A. Yoshie O. Chemokines: A new classification system and their role in immunity.Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3144) Google Scholar) and endothelial cells (Campbell et al., 1999Campbell J.J. Haraldsen G. Pan J. et al.The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (719) Google Scholar). It is a ligand for CC chemokine receptor (CCR) 4 and CCR8 and serves in the recruitment and migration of cells expressing these receptors (Bernardini et al., 1998Bernardini G. Hedrick J. Sozzani S. et al.Identification of the CC chemokine TARC and macrophage inflammatory protein-1b as novel functional ligands for the CCR8 receptor.Eur J Immunol. 1998; 28: 582-588Crossref PubMed Scopus (99) Google Scholar; Sallusto et al., 1998Sallusto F. Lenig D. Mackay C.R. Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes.J Exp Med. 1998; 187: 875-883Crossref PubMed Scopus (1335) Google Scholar). It is considered a Th2-type chemokine because CCR4-expressing T cells mainly produce interleukin (IL)-4. We focused on TARC/CCL17 production from keratinocytes (KC), because TARC/CCL17 has been proven to be essential in skin inflammation (Reiss et al., 2001Reiss Y. Proudfoot A.E. Power C.A. Campbell J.J. Butcher E.C. CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.J Exp Med. 2001; 194: 1541-1547Crossref PubMed Scopus (434) Google Scholar), its existence in the epidermis has been shown (Kakinuma et al., 2001Kakinuma T. Nakamura K. Wakugawa M. et al.Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is closely related with disease activity.J Allergy Clin Immunol. 2001; 107: 535-541Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), and the serum concentration of TARC/CCL17 is elevated in several inflammatory skin diseases such as atopic dermatitis, and mycosis fungoides (Kakinuma et al., 2001Kakinuma T. Nakamura K. Wakugawa M. et al.Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is closely related with disease activity.J Allergy Clin Immunol. 2001; 107: 535-541Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar,Kakinuma et al., 2003Kakinuma T. Sugaya M. Nakamura K. Kaneko F. Wakugawa M. Matsushima K. Tamaki K. Thymus and activation-regulated chemokine (TARC/CCL17) in mycosis fungoides: Serum TARC levels reflect the disease activity of mycosis fungoides.J Am Acad Dermatol. 2003; 48: 23-30Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), which suggested to us that TARC/CCL17 produced from KC could be the key molecule in attracting inflammatory lymphocytes to the skin. SinceKudoh et al., 1987Kudoh S. Uetake T. Hagiwara K. Hirayama M. Hus L.J. Kimura H. Sugiyama Y. Clinical effect of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis.Nippon Kyobu Shikkan Gakkai Zasshi. 1987; 25: 632-642PubMed Google Scholar,Kudoh et al., 1998Kudoh S. Azuma A. Yamamoto M. Izumi T. Ando M. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin.Am J Respir Crit Care Med. 1998; 157: 1829-1832Crossref PubMed Scopus (533) Google Scholar) reported their effectiveness in the treatment of diffuse panbronchiolitis (DPB), macrolide antibiotics have been recognized for their anti-inflammatory effects. Macrolides suppress the activity of neutrophils (Oishi et al., 1994Oishi K. Sonoda F. Kobayashi S. Iwagaki A. Nagatake T. Matsushima K. Matsumoto K. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway disease.Infect Immunity. 1994; 62: 4145-4152PubMed Google Scholar; Matsuyama et al., 1997Matsuyama T. Hidaka K. Furuno T. Hara N. Neutrophil-induced endothelial cell damage: Inhibition by a 14-membered ring macrolide through the action of nitric oxide.Int Arch Allergy Immunol. 1997; 114: 111-115Crossref PubMed Scopus (24) Google Scholar), production of IL-6 and IL-8 in bronchial epithelial cells (Takizawa et al., 1995Takizawa H. Desaki M. Ohtoshi T. et al.Erythromycin suppresses interleukin-6 expression by human bronchial epithelial cells; a potential mechanism of its actiinflammatory action.Biochem Biophys Res Commun. 1995; 210: 781-786Crossref PubMed Scopus (122) Google Scholar,Takizawa et al., 1997Takizawa H. Desaki M. Ohtoshi T. et al.Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells.Am J Respir Crit Care Med. 1997; 156: 266-271Crossref PubMed Scopus (217) Google Scholar), and the function of macrophages (Sugiyama et al., 1999Sugiyama Y. Yanagisawa K. Tominaga S.I. Kitamura S. Effects of long-term administration of erythromycin on cytokine production in rat alveolar macrophages.Eur Respir J. 1999; 14: 1113-1116Crossref PubMed Scopus (30) Google Scholar). They also inhibit the production of inflammatory cytokines by nasal epithelial cells (Fujita et al., 2000Fujita K. Shimizu T. Majima Y. Sakakura Y. Effects of macrolides on interleukin-8 secretion from human nasal epithelial cells.Eur Arch Otorhinolaryngol. 2000; 257: 199-204Crossref PubMed Scopus (35) Google Scholar). Their clinical effects in patients with dermatological diseases, such as psoriasis (Komine and Tamaki, 2000Komine M. Tamaki K. An open trial of oral macrolide treatment for psoriasis vulgaris.J Dermatol. 2000; 27: 508-512Crossref PubMed Scopus (41) Google Scholar) and prurigo pigmentosa (Yazawa et al., 2001Yazawa N. Ihn H. Yamane K. Etoh T. Tamaki K. The successful treatment of prurigo pigmentosa with macrolide antibiotics.Dermatol. 2001; 202: 67-69Crossref PubMed Scopus (46) Google Scholar), were also reported. Recently, macrolides have been reported to be effective against bronchial asthma (Shoji et al., 1999Shoji T. Yoshida S. Sakamoto H. Hasegawa H. Nakagawa H. Amayasu H. Anti-inflammatory effect of roxithromycin in patients with aspirin-intolerant asthma.Clin Exp Allergy. 1999; 29: 950-956Crossref PubMed Scopus (70) Google Scholar), acting on eosinophils (Cui et al., 2001Cui C.H. Honda K. Saito N. et al.Effect of roxithromycin on eotaxin-primed reactive oxygen species from eosinophils.Int Arch Allergy Immunol. 2001; 125: 38-41Crossref PubMed Scopus (9) Google Scholar) and lymphocytes (Noma and Ogawa, 2003Noma T. Ogawa N. Roxithromycin enhances lymphocyte apoptosis in Dermatophagoides-sensitive childhood asthma.J Allergy Clin Immunol. 2003; 111: 646-647Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar), (Noma et al., 2001Noma T. Aoki K. Hayashi M. Yoshizawa I. Kawano Y. Effect of roxithromycin on T lymphocyte proliferation and cytokine production elicited by mite antigen.Int Immunopharmacol. 2001; 1: 201-210Crossref PubMed Scopus (22) Google Scholar). Roxithromycin (RXM) is one of the newly synthesized 14-membered ring macrolide antibiotics that is effective in treating acne vulgaris and bacterial infections (Bryskier, 1998Bryskier A. Roxithromycin: Review of its antimicrobial activity.J Antimicrob Chemother. 1998; 41: 1-21Crossref PubMed Scopus (51) Google Scholar). It is also effective in treating DPB, and chronic sinusitis, probably due to its anti-inflammatory effects (Scaglione and Rossoni, 1998Scaglione F. Rossoni G. Comparative anti-inflammatory effects of roxithromycin, azithromycin and clarithromycin.J Antimicrob Chemother. 1998; 41: 47-50Crossref PubMed Scopus (122) Google Scholar). In this study, we first investigated the signaling pathways by which tumor necrosis factor (TNF)α and interferon (IFN)γ stimulate HaCaT KC to produce CCL17, and then tested the ability of RXM to suppress CCL17 production by HaCaT KC. Furthermore, we investigated the inhibitory effect of RXM on various signaling molecules in order to elucidate the mechanism of the suppression of CCL17 production. Either TNFα or IFNγ alone slightly induced TARC/CCL17 production from HaCaT KC, and the addition of both of these cytokines synergistically induced TARC/CCL17 production (Figure 1). Normal human KC in a monolayer culture did not produce TARC/CCL17 in a similar way, as previously reported (Tsuda et al., 2004Tsuda T. Tohyama M. Yamasaki K. et al.Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro.J Dermatol Sci. 2004; 31: 37-42Abstract Full Text Full Text PDF Scopus (35) Google Scholar) (data not shown). We incubated HaCaT KC with the inhibitors of NFκB, p38 mitogen-activated protein kinase (MAPK), ERK MAPK, or EGFR tyrosine kinase for 1 h before stimulation with TNFα and IFNγ. Supernatants were collected and subjected to ELISA. Parthenolide and Bay 11-7085, inhibitors of NFκB activation, and SB202190, the inhibitor of p38 MAPK, strongly inhibited the induction of CCL17 production by TNFα and IFNγ, but PD98059, the inhibitor of ERK MAPK, did not show a significant suppression, which suggests that the induction of CCL17 production by TNFα and IFNγ is dependent on NFκB and p38 MAPK, but not on ERK MAPK. Unexpectedly, the addition of PD153035, the inhibitor of EGFR tyrosine kinase, caused a significant increase in CCL17 production in HaCaT KC, suggesting that the phosphorylation of EGFR has an inhibitory effect on the production of CCL17 by HaCaT KC (Figure 2a). These results indicate that either NFκB or p38 is indispensable for TNFα and IFNγ-induced CCL17 production, whereas ERK is not, and that the phosphorylation of EGFR induced by TNFα and IFNγ inhibits CCL17 production. We tried western blotting utilizing the antibody against the phosphorylated tyrosine to observe the tyrosine phosphorylation state of the whole EGFR molecule. Stimulation with TNFα and IFNγ time dependently enhanced the intensity of the EGFR bands that appeared at the molecular weight of 170 kDa, and PD153035 completely abolished these bands (data not shown). To investigate the role of STAT1 in the induction of TARC/CCL17 by TNFα and IFNγ, we infected HaCaT KC with an adenovirus vector encoding the STAT1 dominant-negative gene. The blocking of the STAT1 pathway by the STAT1 dominant-negative genes (AxCAdnSTAT1) did not cause any inhibition of TNFα and IFNγ-induced TARC/CCL17 production compared with the induction in cells transferred with STAT1 wild-type genes (AxCAwtSTAT1) (Figure 2b). The induction of TARC/CCL17 was weaker when compared with non-transfected cells, probably because of the transfection procedure. This result indicates that STAT1 activation is not essential in the induction of TARC/CCL17 production by TNFα and IFNγ. Supernatants from cultured HaCaT KC stimulated with TNFα and IFNγ with or without RXM treatment were subjected to ELISA. RXM suppressed the induction of CCL17 production at concentrations of 10−4 and 10−5 M. The effect was concentration dependent, with almost 80% suppression at 10−4 M, and 60% suppression at 10−5 M, but was not significant at 10−6 M (Figure 3) or lower concentrations. We assumed that RXM suppresses NFκB transcriptional activation since NFκB was shown to be involved in the induction of CCL17 production by TNFα and IFNγ. We transfected HaCaT KC with an NFκB-driven luciferase construct together with a cytomegalovirus (CMV) renilla luciferase construct. The relative luciferase activity was calculated with CMV renilla luciferase activity as a control. Additions of TNFα and IFNγ caused an almost 9-fold increase in NFκB activity. At 10−5 M, RXM significantly suppressed TNFα- and IFNγ-induced NFκB activity in HaCaT KC (Figure 4a). These results suggest that the stimulation of HaCaT KC with TNFα and IFNγ leads to NFκB activation, which was essential for CCL17 production. The fact that RXM partially inhibited NFκB activation by TNFα and IFNγ indicated that the suppression of CCL17 by RXM was partially dependent on its NFκB suppression. We performed a similar experiment in normal human KC in order to confirm the suppression of NFκB activation by RXM, because HaCaT KC have been reported to have abnormal NFκB signaling (Chaturvedi et al., 2001Chaturvedi V. Qin J.Z. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. Abnormal NF-kappaB signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes.J Dermatol Sci. 2001; 26: 67-78Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). RXM also inhibited the induction of NFκB-driven luciferase activity by TNFα and IFNγ in normal human KC (data not shown). Inhibitor of nuclear factor kappa B (IκB) is an inhibitory protein that binds to NFκB. Upon stimulation, IκB is phosphorylated and subsequently degraded by the ubiquitin–proteasome pathway. Western blotting with the IκBα antibody revealed that IκBα degradation occurred upon stimulation with TNFα and IFNγ in a time-dependent manner, which was restored after 1 h. This time course of degradation was not affected by the addition of RXM (Figure 4b). IκB degradation usually leads to NFκB activation. The fact that RXM did not alter the course of IκB degradation suggests that RXM suppresses NFκB activation independent of IκB degradation, probably at the point after IκB degradation. To investigate whether the phosphorylation of signaling molecules, including p38, is affected by the addition of RXM, western blotting was performed using monoclonal antibodies against phosphorylated forms of these signaling molecules. RXM partially suppressed the phosphorylation of p38 MAPK induced by TNFα and IFNγ (Figures 5a and b), which showed significant suppression at the 15 and 30 min time points. Phosphorylation of ERK was also slightly suppressed (Figures 5c and d), which showed significant suppression only at the 5-min time point. But, it did not affect the phosphorylation of c-Jun-N-terminal kinase (JNK) or STAT1 (Fig S1, Fig S2). p38 is crucial for the CCL17 production induced by TNFα and IFNγ as shown in Figure 1, which means that p38 could also be the target of RXM. Download .pdf (.01 MB) Help with pdf files Figure S1TNFα and IFNγ induced phosphorylation of the EGF receptor, which was completely abolished with the addition of PD153035. Download .pdf (.02 MB) Help with pdf files Figure S2Phosphorylations OF JNK and STAT1 were not attenuated by the addition of RXM. We confirmed the suppressive effect of RXM on the phosphorylation of p38 in normal human KC. Normal human KC were incubated in keratinocyte-serum free medium (keratinocyte-SFM) without epidermal growth factor (EGF) or bovine pituitary extract (BPE), with or without RXM for 24 h, and then stimulated with TNFα and IFNγ for indicated time periods. The phosphorylation of p38 was suppressed when pre-incubated with RXM; phosphorylations of ERK, JNK, and STAT1 were not affected by the addition of RXM in normal human KC (data not shown). Our result clearly showed that RXM suppressed CCL17 production in HaCaT KC, which is a strong evidence for its action against Th2-type reactions. These findings support its clinical effectiveness against Th2-type inflammations, such as asthma and eosinophilic pustular folliculitis. 1Suzuki S: Personal communication. TNFα has been reported to cause the activation of various signaling molecules including NFκB (Duh et al., 1989Duh E.J. Maury W.J. Folks T.M. Fauci A.S. Rabson A.B. Tumor necrosis factor alpha activates human immunodeficiency viorus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.Proc Natl Acad Sci USA. 1989; 86: 5974-5978Crossref PubMed Scopus (613) Google Scholar), ERK, JNK, and p38 MAP kinases (Kyriaskis et al., 1994Kyriaskis J.M. Banerjee P. Nikolakaki E. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. The stress-activated protein kinase subfamily of c-Jun kinases.Nature. 1994; 369: 156-160Crossref PubMed Scopus (2386) Google Scholar), and EGFR (Donato et al., 1989Donato N.J. Gallick G.E. Steck P.A. Rpsenblum M.G. Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity.J Biol Chem. 1989; 264: 20474-20481Abstract Full Text PDF PubMed Google Scholar) in many cell types. In HaCaT KCs, TNFα also activates signaling molecules such as ERK, JNK, and p38 MAP kinases (Holvoet et al., 2003Holvoet S. Vincent C. Schmitt D. Serres M. The inhibition of MAPK pathway is correlated with down-regulation of MMP-9 secretion induced by TNF-alpha in human keratinocytes.Exp Cell Res. 2003; 290: 108-119Crossref PubMed Scopus (56) Google Scholar), and NFκB (Tomic-Canic et al., 1998Tomic-Canic M. Komine M. Freedberg I.M. Blumenberg M. Epidermal signal transduction and transcription factor activation in activated keratinocytes.J Dermatol Sci. 1998; 17: 167-181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). IFNγ activates STAT, ERK, and p38 MAP kinases, but not JNK in several cell types (Goh et al., 1999Goh K.C. Haque S.J. Williams B.R. p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons.EMBO J. 1999; 118: 5601-5608Crossref Scopus (321) Google Scholar), (Nguyen et al., 2000Nguyen V.A.T. Chen J. Hong F. Ishac E.J.N. Gao B. Interferons activate the p42/44 mitogen-activated protein kinase and JAK-STAT signaling pathways in hepatocytes: Differential regulation by acute ethanol via protein kinase C-dependent mechanism.Biochem J. 2000; 349: 427-434Crossref PubMed Scopus (65) Google Scholar). In our experiment, co-stimulation with TNFα and IFNγ of HaCaT KC caused the synergistic upregulation of TARC. It also caused the phosphorylations of ERK, p38 and JNK MAP kinases, the EGFR and STAT1, and degradation of IκB in a time-dependent manner. TNFα alone caused the phosphorylations of ERK, JNK, p38 MAP kinases, and the EGFR, and degradation of IκB, whereas IFNγ alone caused only STAT1 phosphorylation and very weak phosphorylation of p38 within 1 h, among the above molecules investigated (data not shown). The mechanism of synergy is unclear; however,Fujii-Maeda et al., 2004Fujii-Maeda S. Kajiwara K. Ikizawa K. et al.Reciprocal regulation of thymus and activation-regulated chemokine/macrophage-derived chemokine production by interleukin (IL)-4/IL-13 and interferon-gamma in HaCaT keratinocytes is mediated by alternations in E-cadherin distribution.J Invest Dermatol. 2004; 122: 20-28Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar recently reported that E-cadherin is essential in the production of TARC/CCL17 in HaCaT KC, whose expression is upregulated by IFNγ and it could be involved in synergistic induction by TNFα and IFNγ. TNFα is also capable of inducing the IFNγ receptor, thereby inducing the synergistic activation of indoleamine deoxygenase by TNFα and IFNγ (Robinson et al., 2003Robinson C.M. Shirey K.A. Carlin J.M. Synergistic transcriptional activation of indoleamine dioxygenase by IFN-gamma and tumor necrosis factor-alpha.J Interferon Cytokine Res. 2003; 223: 413-421Crossref Scopus (96) Google Scholar). The interaction of signaling molecules, such as STAT1 and NFκB, is also a possible mechanism of synergy, as reported in the transcriptional activation of CXCL9 (Hiroi and Ohmori, 2003Hiroi M. Ohmori Y. The transcriptional coactivator CREB-binding protein cooperates with STAT1 and NF-kappa B for synergistic transcriptional activation of the CXC ligand 9/monokine induced by interferon-gamma gene.J Biol Chem. 2003; 278: 651-660Crossref PubMed Scopus (79) Google Scholar); however, dominant negative STAT1-transfected cells showed a response similar to wild-type STAT1-transfected cells, suggesting that STAT1 activation is not essential in the induction of TARC/CCL17 production. Interferon regulatory factors (IRF) are the transcription factors involved in IFNγ signaling, and have been reported to interact with NFκB, which causes the synergistic induction of inducible nitric oxide synthase (Saura et al., 1999Saura M. Zaragoza C. Bao C. McMillan A. Lowenstein C.J. Interaction of interferon regulatory factor-1 and nuclear factor kappaB during activation of inducible nitric oxide synthase transcription.J Mol Biol. 1999; 289: 459-471Crossref PubMed Scopus (151) Google Scholar) and regulated on activation, normal T-cell expressed and secreted (RANTES) (Genin et al., 2000Genin P. Algarte M. Roof P. Lin R. Hiscott J. Regulation of RANTES chemokine gene expression requires cooperativity between NF-kappa B and IFN-regulatory factor transcription factors.J Immunol. 2000; 164: 5352-5361Crossref PubMed Scopus (181) Google Scholar). IRF-1, the main IRF family member in IFNγ signaling, however, is induced through STAT1 signaling, which should be STAT1 dependent. Recent investigation revealed that a number of genes are regulated by IFNγ in a STAT1-independent manner. Among them are several chemokines such as macrophage-inflammatory protein-1α and monocyte chemoattractant protein 1 (MCP-1) (Gil et al., 2001Gil M.P. Bohn E. O'Guin A.K. et al.Biologic consequences of Stat1-independent IFN signaling.Proc Natl Acad Sci USA. 2001; 98: 6680-6685Crossref PubMed Scopus (285) Google Scholar). Several transcription factors such as EGR-1, C/EBPβ, and c-jun are induced in STAT-1 null cells (Ramana et al., 2001Ramana C.V. Gil M.P. Han Y. Ransohoff R.M. Schreiber R.D. Stark G.R. Stat1-independent regulation of gene expression in response to IFN-gamma.Proc Natl Acad Sci USA. 2001; 98: 6674-6679Crossref PubMed Scopus (204) Google Scholar). Production of TARC by TNFα and IFNγ could be induced through STAT1-independent pathways, the mechanism of which needs further investigation. Recently,Banno et al., 2003Banno T. Adachi M. Mukkamala L. Blumenberg M. Unique keratinocyte-specific effects of interferon-gamma that protect skin from viruses, identified using transcriptional profiling.Antivir Ther. 2003; 8: 541-554PubMed Google Scholar,Banno et al., 2004Banno T. Gazel A. Blumenberg M. Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.J Biol Chem. 2004; 279: 32633-32642Crossref PubMed Scopus (221) Google Scholar reported on a wide spectrum of genes regulated by TNFα or IFNγ utilizing a DNA microarray system. TNFα and IFNγ are pro-inflammatory cytokines involved in innate immunity and inflammation, and the synergistic effect of these cytokines would be a substantial driving force of immunity and inflammation in a biological context. The fact that TARC/CCL17 is only slightly induced by TNFα or IFNγ alone, and strongly induced when they are applied at the same time, suggests that TARC would have a biological meaning, especially when inflammation reaches the level at which both TNFα and IFNγ are abundant, and, at such a level, TARC may have the role of directing the inflammation in a Th2-balanced direction. The EGFR tyrosine phosphorylation inhibitor enhanced the production of CCL17 stimulated by TNFα and IFNγ. A similar phenomenon has been reported with other chemokines such as CCL2 (MCP-1), CCL5 (RANTES), and CXCL10 (interferon-γ-induced protein of 10 kDa, IP-10) in normal human KC (Mascia et al., 2003Mascia F. Mariani V. Girolomoni G. Pastore S. Blockade of the EGF receptor induces a deranged chemokine expression in keratinocytes leading to enhanced skin inflammation.Am J Pathol. 2003; 163: 303-312Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). EGFR is known to activate various signaling cascades such as ERK, PI3K, and STATs (STAT1, 3, 5). The finding that the EGFR inhibitor enhanced the production of CCL17, whereas the ERK inhibitor had no effect means that the activated EGFR exerts its signal through signaling molecules other than ERK to inhibit CCL17 production. The mechanism of CCL17 production enhancement by the EGFR inhibitor needs further investigation. Our experiment showed that the mechanisms of CCL17 production in HaCaT and normal human KC are dependent on NFκB activation. This is consistent with a previous report, in which the NFκB inhibitor, MG-132, and mutant IκBα introduced by retroviral vectors inhibited the production of CCL17 in A549 cells, a bronchial epithelial cell line (Berin et al., 2001Berin M.C. Eckmann L. Broide D.H. Kagnoff M.F. Regulated production of the T helper 2-type T-cell chemoattractant TARC by human bronchial epithelial cells in vitro and in human lung xenografts.Am J Respir Cell Mol Biol. 2001; 24: 382-389Crossref PubMed Scopus (107) Google Scholar). Our results also indicate that CCL17 production in HaCaT and normal human KC is dependent on p38 MAPK, but not on ERK MAPK, which is also activated in HaCaT KC stimulated by TNFα and IFNγ. A recent study reported on the involvement of p38 and NFκB in CCL17 production in B cells (Nakayama et al., 2004Nakayama T. Hieshima K. Nagakubo D. Sato E. Nakayama M. Kawa K. Yoshie O. Selective induction of Th2-attracting chemokine CCL17 and CCL22 in human B cells by latent membrane protein 1 of Epstein–Barr virus.J Virol. 2004; 78: 1665-1674Crossref PubMed Scopus (130) Google Scholar), which suggests similar signaling pathways of CCL17 production in B cells and HaCaT KC. Increasing evidence suggests an important role for p38 in adhesion-induced differentiation and signal transduction in epithelial cells. E-cadherin forms adherens junctions in epithelial tissues, providing tight cell–cell contacts. E-cadherin engagement is essential in the activation of p38 in intestinal epithelial cells, which plays a crucial role in intestinal epithelial cell differentiation (Laprise et al., 2002Laprise P. Chailler P. Houde M. Beaulieu J.F. Boucher M.J. Rivard N. Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.J Biol Chem. 2002; 277: 8226-8234Crossref PubMed Scopus (135) Google Scholar). P38 is also involved in the hypotonic stress-induced expression of E-cadherin in HaCaT KC (Kippenberger et al., 2005Kippenberger S. Loitsch S. Guschel M. Muller J. Kaufmann R. Bernd A. Hypotonic stress induces E-cadherin expression in cultured human keratinocytes.FEBS Lett. 2005; 579: 207-214Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Taking into consideration the fact that E-cadherin is essential in the spontaneous production of TARC/CCL17 (Fujii-Maeda et al., 2004Fujii-Maeda S. Kajiwara K. Ikizawa K. et al.Reciprocal regulation of thymus and activation-regulated chemokine/macrophage-derived chemokine production by interleukin (IL)-4/IL-13 and interferon-gamma in HaCaT keratinocytes is mediated by alternations in E-cadherin distribution.J Invest Dermatol. 2004; 122: 20-28Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and that its expression is induced by IFNγ, p38 may be involved in E-cadherin expression induced by IFNγ or in signaling pathways evoked by E-cadherin engagement. Our results also showed that RXM partially inhibited TNFα- and IFNγ-induced NFκB activation in HaCaT KC, which can explain the inhibitory effect of RXM on CCL17 production in our system. It has been reported that RXM inhibited NFκB activation induced by reactive oxygen intermediates (Abeyama et al., 2003Abeyama K. Kawahara K. Iino S. et al.Antibiotic cyclic AMP signaling by “primed” leukocytes confers anti-inflammatory cytoprotection.J Leukoc Biol. 2003; 74: 908-915Crossref PubMed Scopus (14) Google Scholar) or by Propionibacterium acnes (Chen et al., 2002Chen Q. Koga T. Uchi H. et al.Propionibacterium acnes-induced IL-8 production may be mediated by NF-kappaB activation in human monocytes.J Dermatol Sci. 2002; 29: 97-103Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The inhibition by RXM of NFκB activity induced by TNFα and IFNγ was demonstrated in our experiment. Erythromycin has been reported to suppress cycloxygenase-2 synthesis in rheumatoid synovial cells by inhibiting p38 activation (Fumimori et al., 2004Fumimori T. Honda S. Migita K. et al.Erythromycin suppresses the expression of cycloxygenase-2 in rheumatoid synovial cells.J Rheumatol. 2004; 31: 436-441PubMed Google Scholar). Clarithromycin suppressed mucin production by inhibiting ERK phosphorylation in the lungs in a murine model of DPB (Kaneko et al., 2003Kaneko Y. Yanagihara K. Seki M. et al.Clarithromycin inhibits overproduction of muc5ac core protein in murine model of diffuse panbronchiolitis.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L847-L853Crossref PubMed Scopus (53) Google Scholar). Evidence of RXM inhibiting any of the MAP kinases, however, has not been demonstrated. This report demonstrates that RXM inhibits the phosphorylation of p38 induced by TNFα and IFNγ in KC. This study, by demonstrating the signaling pathways triggered by TNFα and IFNγ in KC, and the mechanism of RXM action, shed new light on the development of skin inflammation, and also suggests some new therapeutic approaches to inflammatory skin diseases. Anti-phospho-ERK1/2, anti-phospho-JNK, anti-phospho-p38, anti-phospho-EGFR tyr845, anti-phosphoEGFR tyr992, anti-phosphoEGFR tyr1045, and anti-phospho-EGFR tyr1068 were purchased from Cell Signaling (Beverly, Massachusetts). Rabbit polyclonal anti-human IκBα antibody, monoclonal mouse anti-ERK2, anti-JNK, anti-p38, anti-phospho-STAT1, anti-STAT1α, anti-rabbit IgG horseradish peroxidase (HRP) conjugate, and anti-mouse IgG HRP conjugate were from Santa Cruz (Santa Cruz, California). Recombinant human TNFα and recombinant human IFNγ were from R & D systems (Minneapolis, Minnesota). Several signal transduction inhibitors were added 1 h before stimulation with TNFα and IFNγ. PD98059 was purchased from Alexis Biochemicals (San Diego, California), PD153035, Parthenolide, Bay 11-7085, and SB202190 were purchased from Calbiochem (San Diego, California). HaCaT KC were a generous gift from Dr Kuroki (Showa University, Tokyo, Japan) with the permission of Dr Fusenig (Institute Fur Zell- und Tumourbiologie, Deutsches Kresforschungszentrum, Heidelberg, Germany). They were grown routinely in Eagle's minimum essential medium (MEM, SIGMA, St. Louis, Missouri) supplemented with 10% fetal calf serum (FCS) in a humidified CO2 incubator. Cells of the 30th to 50th passage were used for experiments. Normal neonatal foreskin human KC were purchased from Clonetics (San Diego, California), and cultured in keratinocyte-SFM supplemented with BPE and EGF, from Invitrogen (Carlsbad, California). HaCaT KC were trypsinized into six-well plates. When they reached subconfluency, the medium was changed to MEM without FCS. After incubation in MEM without FCS for 24 h, RXM (provided by Eisai, Tokyo, Japan) at a concentration of 10−4 to 10−8 M was added prior to the addition of with 10 ng per mL TNFα and 100 U per mL IFNγ. Supernatants were harvested after 24 h and subjected to ELISA utilizing 96-well plates coated with a murine monoclonal antibody against human CCL17 (TECHNE, Minneapolis, Minnesota). ELISA was performed according to the manufacturer's directions. Briefly, samples and standards were applied to antibody-coated 96-well plates, incubated for 2 h, washed, incubated with secondary conjugated antibodies for another 2 h, washed again, and incubated with substrate for 30 min before the reaction was terminated. The optical density of each well was determined using a microplate reader (Model 550, Bio-Rad, Hercules, California) set to 450 nm. Adenovirus vectors containing the genes for HA-tagged wild-type STAT1 (AxCAwtSTAT1) and HA-tagged dominant-negative STAT1 (AxCAdnSTAT1) (Nakajima et al., 1996Nakajima K. Yomanaka Y. Nakae K. et al.A central role for Stat-3 in IL-6-induced regulation of growth and differentiation in M1 kukemia cells.EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (512) Google Scholar), which contain a CAG promoter (chicken β-actin promoter withCMV enhancer), were prepared by homologous recombination in 293 cells as described previously (Hanakawa et al., 2000Hanakawa Y. Amagai M. Shirakata Y. Sayama K. Hashimoto K. Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes.J Cell Sci. 2000; 113: 1803-1811Crossref PubMed Google Scholar). DNA encoding wtSTAT1 and dnSTAT1 were kind gifts from Dr Nakajima of Osaka University. HaCaT KC were infected with AxCAwtSTAT1 or AxCAdnSTAT1, at a multiplicity of infection (MOI) of 10 in MEM containing 10% FBS. They were starved of serum for 24 h, and incubated with or without TNFα (10 ng per mL) and IFNγ (100 U per mL) for 24 h. Concentrations of TARC/CCL17 in supernatants were examined by ELISA. The concentration of TARC/CCL17 was normalized with the protein amount because of the anti-proliferative effect of the STAT1 wild-type vector. The luciferase construct containing four NFκB consensus sequences in tandem and the CMV–renilla luciferase construct were purchased from Promega (Madison, Wisconsin). HaCaT KCs were trypsinized into 100 mm dishes, and cultured in MEM with 10% FCS until subconfluent. The NFκB–luciferase (NFκB–luc) construct together with the CMV–renilla construct were co-transfected using Fugene6 from Roche Diagnostics (Tokyo, Japan) as recommended by the manufacturer. Six hours after transfection, cells were trypsinized into six-well plates to remove the toxic effect of Fugene6, and incubated overnight before being stimulated with TNFα and IFNγ. RXM (20 μg per mL) was added 2 h before treatment with TNFα (10 ng per mL) and IFNγ (100 U per mL), and the cells were harvested after 16 h of incubation. The cells were disrupted with lysis buffer provided by the manufacturer, and then subjected to a dual-luciferase assay (Promega) with a luminometer (Luminescencer-PSN, AB-2200, ATTO, Tokyo, Japan) as described by the manufacturer. HaCaT KC or normal human KC pre-incubated overnight with or without RXM (20 μg per mL) were stimulated with TNFα (10 ng per mL) and IFNγ (100 U per mL), and disrupted in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate) with 2 mM phenyl methyl sulfonyl fluoride (PMSF) (Boehringer Mannheim, Germany), 1 mg per mL leupeptin (Sigma-Aldrich, St. Louis, Missouri), and 1 mM sodium orthovanadate (Na3VO4) from Sigma-Aldrich. The concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (Pierce, Rockford, Illinois). The samples were boiled in sample buffer (50 mM Tris (pH 7.4)/0.14% sodium dodecylsulfate (SDS)/1% β-mercaptoethanol (vol/vol)), and separated by 12.5% SDS-polyacrylamide gel electrophoresis (10 μg of protein per lane). After transfer to an Immobilon-P transfer membrane (Millipore, Billerica, Massachusetts), the membrane was incubated in blocking buffer (5% bovine serum albumin in 25 mM Tris/0.02% KCl/0.8% NaCl (pH 7.4) tris-buffered saline (TBS)) for 1 h at 4°C, followed by an appropriate primary antibody overnight at 4°C. The membrane was washed and incubated with a secondary antibody for 1 h, and the bands were visualized using a chemiluminescence method (Phototope-HRP Western Blot Detection Kit, New England BioLabs, Beverly, Massachusetts). The following supplementary material is available for this article online. Figure S1 TNFα and IFNγ induced phosphorylation of the EGF receptor, which was completely abolished with the addition of PD153035. Figure S2 Phosphorylations of JNK and STAT1 were not attenuated by the addition of RXM." @default.
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• W2072462906 title "Mechanism of Thymus- and Activation-Regulated Chemokine (TARC)/CCL17 Production and its Modulation by Roxithromycin" @default.
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• W2072462906 doi "https://doi.org/10.1111/j.0022-202x.2005.23840.x" @default.
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