- Email: [email protected]
Receptor Activator of NF-κB Ligand Promotes the Production of CCL17 from RANK+ M2 Macrophages
Accepted Article Preview: Published ahead of advance online publication www.jidonline.org
Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) Promotes the Production of CCL17 from RANK þ M2 Macrophages Taku Fujimura, Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba
Cite this article as: Taku Fujimura, Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba, Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) Promotes the Production of CCL17 from RANK þ M2 Macrophages, Journal of Investigative Dermatology accepted article preview 8 June 2015; doi: 10.1038/jid.2015.209. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 8 June 2015
© 2015 The Society for Investigative Dermatology
Receptor activator of nuclear factor kappa-B ligand (RANKL) promotes the production of CCL17 from RANK+ M2 macrophages. Taku Fujimura1)2), Yumi Kambayashi1)2), Sadanori Furudate1), Masayuki Asano1), Aya Kakizaki1), Setsuya Aiba1)
1)
Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai,
Japan 2)
These authors equally contributed to this work.
Short title: RANKL promotes the production of CCL17 from M2 macrophages.
Corresponding author: Taku Fujimura Department of Dermatology, Tohoku University Graduate School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Tel: +81 (22) 717-7271; Fax: +81 (22) 717-7361
1
© 2015 The Society for Investigative Dermatology
E-mail: [email protected]
Abbreviations: Treg: regulatory T cells; TAMs: tumor-associated macrophages; TNF: tumor necrosis factor; DC: dendritic cells; ultraviolet : UV; iNOS: inducible nitric oxide synthase; IL-4: interleukin-4, arginase 1: Arg1, magnetic-activated cell sorting: MACS, RANK: receptor of activator nuclear factor kappa-B, LC: Langerhans cell
Funding statement: None Disclosures: None declared Word count: 946 words Key words: RANKL/RANK, tumor-associated M2 macrophages, CCL17, extramammary Paget’s disease Conflicts of interest: The authors have no conflicting financial interests to declare.
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© 2015 The Society for Investigative Dermatology
TO THE EDITOR RANKL and RANK were originally identified as a cytokine and its receptor, respectively, that regulate the function of DCs (Anderson et al., 1997; Wong et al., 1997). RANK/RANKL also have a role in osteoclast differentiation (Yasuda et al., 1998). RANKL signaling promotes the survival of conventional DCs and ensures T-cell priming and activation, thereby enhancing the acquired immune response. On the other hand, in skin, RANKL-treated DCs maintain the number of CD4+CD25+ Tregs to suppress the immune response against self-antigens, food, and commensal flora. In addition, UV-irradiation upregulates the expression of RANKL in keratinocytes, which stimulates RANK on LCs leading to UV-induced cutaneous immunosuppression (Loser et al., 2006). In the accompanying report (Kambayashi et al. 2015) we showed that, in extramammary Paget’s disease (EMPD), 60% of the RANK+ cells are CD163+ macrophages, and a substantial amount of soluble RANKL (sRANKL) is released the lesional skin of EMPD. We therefore hypothesized that this RANKL might have an effect on CD163+RANK+ M2 macrophages. To explore the potential effects of sRANKL on M2 macrophages, we generated
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© 2015 The Society for Investigative Dermatology
monocyte-derived M2 macrophages (Fujimura et al., 2013; Martinez et al., 2006). This study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan (2013-1-521), and all healthy donors and patients gave written informed consent. Flow cytometric analysis indicated that all cells expressed CD163 and Arg1 (Fig. 1a, b), suggesting that they were M2 macrophages, while only a minor population very weakly expressed RANK (Fig. 1c). When these macrophages were further cultured with IL-4, however, most of them strongly expressed RANK (Fig. 1c). We stimulated these induced RANK+ M2 macrophages with sRANKL and then examined their mRNA expression of chemokines, which are known to be expressed by M2 macrophages (Hao et al., 2012; Mantovani et al., 2002). Stimulation with sRANKL augmented the expression of CCL11, CCL17, and CCL24 mRNA in a dose-dependent manner, while the expression of CCL18, CCL26, and Arg1 mRNA was not significantly affected (Fig. 1d). To confirm that the observed increased mRNA expression leads to protein expression, we used ELISA to examine chemokine protein production by M2 macrophages (Fig. 1e). Consistent with the mRNA expression, the production of CCL17 protein was
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significantly augmented by sRANKL stimulation in a dose-dependent manner, while the expression of CCL18 protein was not significantly altered (Fig. 1e). The production of CCL24 protein was also augmented by sRANKL stimulation in a dose-dependent manner, although the increases were not statistically significant. In contrast to its mRNA expression, we could not find any significant effects of sRANKL on the production of IL-10 protein, even at the highest sRANKL concentration (Fig. 1e). CCL11 protein was not detected using ELISA (data not shown). Although the M2 macrophages stimulated by sRANKL significantly augmented production of CCL17, it was unclear whether the culture supernatants of the sRANKL-stimulated M2 macrophages contained T-cell chemotactic factors other than CCL17. We therefore examined the chemotactic activity of these culture supernatants for T cells in the presence or absence of anti-CCR4 Ab. The chemotactic activity of the culture supernatant of M2 macrophages for CD4+ T cells significantly increased with sRANKL stimulation in a dose-dependent manner, in parallel with the increased production of CCL17 (Fig. 2a). This T-cell chemotactic activity in the culture supernatant of sRANKL-stimulated M2 macrophages was significantly attenuated by
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the anti-CCR4 Ab (Fig. 2b). These findings suggest that RANK+ M2 macrophages stimulated with sRANKL preferentially recruit T cells via CCL17 stimulation of CCR4.
Since this in vitro study demonstrated that RANKL-stimulated M2 macrophages produced CCL17, we next examined whether CD163+ macrophages in the lesional skin of EMPD expressed CCL17. When we conducted double immunofluorescence staining of EMPD cryosections with a combination of antibodies against CD163 and CCL17, most of the CD163+ M2 macrophages expressed CCL17 (Fig. 2c). Notably, about half of these CD163+ M2 macrophages were previously shown to coexpress RANK (Kambayashi et al. 2015). Furthermore, when we compared the mRNA expression of CCL17 and T helper cell subset–specific transcription factors between the non-lesional and lesional skin of EMPD, the expression of CCL17, GATA3 and Foxp3 mRNA, but not that of T-bet or RORt mRNA, was significantly increased in the lesional skin (Fig. 2e). Consistent with the observed mRNA expression of the lesional skin, double immunofluorescence staining for CD163 and Foxp3 demonstrated that Foxp3+ cells surrounded CD163+ M2 macrophages as we previously reported (Fujimura et al., 2013;
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© 2015 The Society for Investigative Dermatology
Fujimura et al., 2012). (Fig. 2d). In addition, these previous papers suggested that Foxp3+ cells in the lesional skin of EMPD express CD3, CD4, CD25, and CD39, suggesting that these Foxp3+ cells are Tregs. These findings suggested that M2 macrophages in EMPD might attract Foxp3+ Tregs by chemotaxis via CCL17. These results suggest that M2 macrophages stimulated by RANKL/RANK signaling might recruit Tregs into the tumor microenvironment of EMPD, which thereby induces an immunosuppressive microenvironment in EMPD.
Gordon and Martinez classified the development of monocytes into mature and fully activated macrophages into three successive stages (Gordon and Martinez, 2010). During the third phase of activation, macrophages reach a mature functional phenotype in response to microbial and opsonic stimuli such as antibody complexes. In the present study, macrophages induced by M-CSF and IL-4 did not produce a substantial amount of CCL17; however, when these macrophages were stimulated by sRANKL, they produced CCL17 in an sRANKL dose-dependent manner. These results suggest that sRANKL may be another stimulus during the third phase of macrophage activation.
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Denosumab, a fully human monoclonal antibody for RANKL, is used clinically to treat metastatic bone tumors (Azim and Azim, 2013). Our present study suggests that RANKL targeting with denosumab can be used in conjunction with the therapeutic elimination of primary EMPD to prevent local immunosuppression and metastatic disease.
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© 2015 The Society for Investigative Dermatology
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Grant Support This study was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (23791249 and 25461682).
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© 2015 The Society for Investigative Dermatology
References Anderson DM, Maraskovsky E, Billingsley WL, et al. (1997) A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175-9. Azim H, Azim HA, Jr. (2013) Targeting RANKL in breast cancer: bone metastasis and beyond. Expert Rev Anticancer Ther 13:195-201. Fujimura T, Furudate S, Kambayashi Y, et al. (2013) Potential use of bisphosphonates in invasive extramammary Paget's disease: an immunohistochemical Investigation. Clin Dev Immunol 2013:164982. Fujimura T, Kambayashi Y, Hidaka T, et al. (2012) Comparison of Foxp3+ regulatory T cells and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. Acta Derm Venereol 92:625-8. Gordon S, Martinez FO (2010) Alternative activation of macrophages: mechanism and functions. Immunity 32:593-604. Hao NB, Lu MH, Fan YH, et al. (2012) Macrophages in tumor microenvironments and the progression of tumors. Clin Dev Immunol 2012:948098. Jones DH, Nakashima T, Sanchez OH, et al. (2006) Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440:692-6. Kambayashi Y, Fujimura T, Furudate S, et al. (2015) The possible interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) expressed by extramammary Pagent cells and its ligand on dermal macrophages. J Invest Dermatol, in press. Loser K, Mehling A, Loeser S, et al. (2006) Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells. Nat Med 12:1372-9. 10
© 2015 The Society for Investigative Dermatology
Mantovani A, Sozzani S, Locati M, et al. (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23:549-55. Wong BR, Josien R, Lee SY, et al. (1997) TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 186:2075-80. Yasuda H, Shima N, Nakagawa N, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 95:3597-602.
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FIGURE LEGENDS Fig. 1. sRANKL induces the expression of CCL17 mRNA and protein by M2 macrophages. M2 macrophages were induced from PBMCs from healthy donors, as described in Materials and Methods. The expression of CD163 and Arg1 on CD14+ monocytes (a), and M-CSF–induced macrophages (b), and the expression of RANK on cultured M-CSF-induced macrophages with or without IL-4 (c) was analyzed using flow cytometry. Relative mRNA expression levels of CCL11, CCL17, CCL18, CCL24, CCL26, IL-10, Arg1 and iNOS by non-stimulated (NS) or sRANKL-stimulated M2 macrophages were measured using quantitative RT-PCR and were calculated using the ΔΔCt method (d). sRANKL induction of the secretion of CCL17, CCL18, CCL24 and IL-10 proteins from M2 macrophages was measured using ELISA (e). Data from each donor were obtained from triplicate assays, and the mean SD was calculated. * p<0.05, ** p<0.01 by Student’s t-test; n.s., not significant.
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Fig 2. M2 macrophages recruit CCR4-expressing cells in a tumor microenvironment. The chemoattractant activity of the culture supernatant of M2 macrophages for CD4+ T cells without (a) or with (b) an anti-CCR4 Ab was evaluated by using a Neuro Probe AP48 chamber, as described in Materials and Methods. CCL-17 was used as a positive control. Immunofluorescence staining of the lesional skin of EMPD for CD163 and CCL17 (c) and for CD163 and Foxp3 (d). Scale bar, 50 m. The expression of CCL17, T-bet, Foxp3, GATA3, and RORt mRNA in the lesional skin of EMPD was analyzed by quantitative RT-PCR using the ΔΔCt method (e). The mean SEM of data from three cases of EMPD is presented. * p<0.05 by Student’s t-test; n.s., not significant.
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© 2015 The Society for Investigative Dermatology
© 2015 The Society for Investigative Dermatology
© 2015 The Society for Investigative Dermatology
Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) Promotes the Production of CCL17 from RANK þ M2 Macrophages Taku Fujimura, Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba
Cite this article as: Taku Fujimura, Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba, Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) Promotes the Production of CCL17 from RANK þ M2 Macrophages, Journal of Investigative Dermatology accepted article preview 8 June 2015; doi: 10.1038/jid.2015.209. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 8 June 2015
© 2015 The Society for Investigative Dermatology
Receptor activator of nuclear factor kappa-B ligand (RANKL) promotes the production of CCL17 from RANK+ M2 macrophages. Taku Fujimura1)2), Yumi Kambayashi1)2), Sadanori Furudate1), Masayuki Asano1), Aya Kakizaki1), Setsuya Aiba1)
1)
Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai,
Japan 2)
These authors equally contributed to this work.
Short title: RANKL promotes the production of CCL17 from M2 macrophages.
Corresponding author: Taku Fujimura Department of Dermatology, Tohoku University Graduate School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Tel: +81 (22) 717-7271; Fax: +81 (22) 717-7361
1
© 2015 The Society for Investigative Dermatology
E-mail: [email protected]
Abbreviations: Treg: regulatory T cells; TAMs: tumor-associated macrophages; TNF: tumor necrosis factor; DC: dendritic cells; ultraviolet : UV; iNOS: inducible nitric oxide synthase; IL-4: interleukin-4, arginase 1: Arg1, magnetic-activated cell sorting: MACS, RANK: receptor of activator nuclear factor kappa-B, LC: Langerhans cell
Funding statement: None Disclosures: None declared Word count: 946 words Key words: RANKL/RANK, tumor-associated M2 macrophages, CCL17, extramammary Paget’s disease Conflicts of interest: The authors have no conflicting financial interests to declare.
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© 2015 The Society for Investigative Dermatology
TO THE EDITOR RANKL and RANK were originally identified as a cytokine and its receptor, respectively, that regulate the function of DCs (Anderson et al., 1997; Wong et al., 1997). RANK/RANKL also have a role in osteoclast differentiation (Yasuda et al., 1998). RANKL signaling promotes the survival of conventional DCs and ensures T-cell priming and activation, thereby enhancing the acquired immune response. On the other hand, in skin, RANKL-treated DCs maintain the number of CD4+CD25+ Tregs to suppress the immune response against self-antigens, food, and commensal flora. In addition, UV-irradiation upregulates the expression of RANKL in keratinocytes, which stimulates RANK on LCs leading to UV-induced cutaneous immunosuppression (Loser et al., 2006). In the accompanying report (Kambayashi et al. 2015) we showed that, in extramammary Paget’s disease (EMPD), 60% of the RANK+ cells are CD163+ macrophages, and a substantial amount of soluble RANKL (sRANKL) is released the lesional skin of EMPD. We therefore hypothesized that this RANKL might have an effect on CD163+RANK+ M2 macrophages. To explore the potential effects of sRANKL on M2 macrophages, we generated
3
© 2015 The Society for Investigative Dermatology
monocyte-derived M2 macrophages (Fujimura et al., 2013; Martinez et al., 2006). This study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan (2013-1-521), and all healthy donors and patients gave written informed consent. Flow cytometric analysis indicated that all cells expressed CD163 and Arg1 (Fig. 1a, b), suggesting that they were M2 macrophages, while only a minor population very weakly expressed RANK (Fig. 1c). When these macrophages were further cultured with IL-4, however, most of them strongly expressed RANK (Fig. 1c). We stimulated these induced RANK+ M2 macrophages with sRANKL and then examined their mRNA expression of chemokines, which are known to be expressed by M2 macrophages (Hao et al., 2012; Mantovani et al., 2002). Stimulation with sRANKL augmented the expression of CCL11, CCL17, and CCL24 mRNA in a dose-dependent manner, while the expression of CCL18, CCL26, and Arg1 mRNA was not significantly affected (Fig. 1d). To confirm that the observed increased mRNA expression leads to protein expression, we used ELISA to examine chemokine protein production by M2 macrophages (Fig. 1e). Consistent with the mRNA expression, the production of CCL17 protein was
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significantly augmented by sRANKL stimulation in a dose-dependent manner, while the expression of CCL18 protein was not significantly altered (Fig. 1e). The production of CCL24 protein was also augmented by sRANKL stimulation in a dose-dependent manner, although the increases were not statistically significant. In contrast to its mRNA expression, we could not find any significant effects of sRANKL on the production of IL-10 protein, even at the highest sRANKL concentration (Fig. 1e). CCL11 protein was not detected using ELISA (data not shown). Although the M2 macrophages stimulated by sRANKL significantly augmented production of CCL17, it was unclear whether the culture supernatants of the sRANKL-stimulated M2 macrophages contained T-cell chemotactic factors other than CCL17. We therefore examined the chemotactic activity of these culture supernatants for T cells in the presence or absence of anti-CCR4 Ab. The chemotactic activity of the culture supernatant of M2 macrophages for CD4+ T cells significantly increased with sRANKL stimulation in a dose-dependent manner, in parallel with the increased production of CCL17 (Fig. 2a). This T-cell chemotactic activity in the culture supernatant of sRANKL-stimulated M2 macrophages was significantly attenuated by
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© 2015 The Society for Investigative Dermatology
the anti-CCR4 Ab (Fig. 2b). These findings suggest that RANK+ M2 macrophages stimulated with sRANKL preferentially recruit T cells via CCL17 stimulation of CCR4.
Since this in vitro study demonstrated that RANKL-stimulated M2 macrophages produced CCL17, we next examined whether CD163+ macrophages in the lesional skin of EMPD expressed CCL17. When we conducted double immunofluorescence staining of EMPD cryosections with a combination of antibodies against CD163 and CCL17, most of the CD163+ M2 macrophages expressed CCL17 (Fig. 2c). Notably, about half of these CD163+ M2 macrophages were previously shown to coexpress RANK (Kambayashi et al. 2015). Furthermore, when we compared the mRNA expression of CCL17 and T helper cell subset–specific transcription factors between the non-lesional and lesional skin of EMPD, the expression of CCL17, GATA3 and Foxp3 mRNA, but not that of T-bet or RORt mRNA, was significantly increased in the lesional skin (Fig. 2e). Consistent with the observed mRNA expression of the lesional skin, double immunofluorescence staining for CD163 and Foxp3 demonstrated that Foxp3+ cells surrounded CD163+ M2 macrophages as we previously reported (Fujimura et al., 2013;
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© 2015 The Society for Investigative Dermatology
Fujimura et al., 2012). (Fig. 2d). In addition, these previous papers suggested that Foxp3+ cells in the lesional skin of EMPD express CD3, CD4, CD25, and CD39, suggesting that these Foxp3+ cells are Tregs. These findings suggested that M2 macrophages in EMPD might attract Foxp3+ Tregs by chemotaxis via CCL17. These results suggest that M2 macrophages stimulated by RANKL/RANK signaling might recruit Tregs into the tumor microenvironment of EMPD, which thereby induces an immunosuppressive microenvironment in EMPD.
Gordon and Martinez classified the development of monocytes into mature and fully activated macrophages into three successive stages (Gordon and Martinez, 2010). During the third phase of activation, macrophages reach a mature functional phenotype in response to microbial and opsonic stimuli such as antibody complexes. In the present study, macrophages induced by M-CSF and IL-4 did not produce a substantial amount of CCL17; however, when these macrophages were stimulated by sRANKL, they produced CCL17 in an sRANKL dose-dependent manner. These results suggest that sRANKL may be another stimulus during the third phase of macrophage activation.
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© 2015 The Society for Investigative Dermatology
Denosumab, a fully human monoclonal antibody for RANKL, is used clinically to treat metastatic bone tumors (Azim and Azim, 2013). Our present study suggests that RANKL targeting with denosumab can be used in conjunction with the therapeutic elimination of primary EMPD to prevent local immunosuppression and metastatic disease.
8
© 2015 The Society for Investigative Dermatology
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
Grant Support This study was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (23791249 and 25461682).
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© 2015 The Society for Investigative Dermatology
References Anderson DM, Maraskovsky E, Billingsley WL, et al. (1997) A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175-9. Azim H, Azim HA, Jr. (2013) Targeting RANKL in breast cancer: bone metastasis and beyond. Expert Rev Anticancer Ther 13:195-201. Fujimura T, Furudate S, Kambayashi Y, et al. (2013) Potential use of bisphosphonates in invasive extramammary Paget's disease: an immunohistochemical Investigation. Clin Dev Immunol 2013:164982. Fujimura T, Kambayashi Y, Hidaka T, et al. (2012) Comparison of Foxp3+ regulatory T cells and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. Acta Derm Venereol 92:625-8. Gordon S, Martinez FO (2010) Alternative activation of macrophages: mechanism and functions. Immunity 32:593-604. Hao NB, Lu MH, Fan YH, et al. (2012) Macrophages in tumor microenvironments and the progression of tumors. Clin Dev Immunol 2012:948098. Jones DH, Nakashima T, Sanchez OH, et al. (2006) Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440:692-6. Kambayashi Y, Fujimura T, Furudate S, et al. (2015) The possible interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) expressed by extramammary Pagent cells and its ligand on dermal macrophages. J Invest Dermatol, in press. Loser K, Mehling A, Loeser S, et al. (2006) Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells. Nat Med 12:1372-9. 10
© 2015 The Society for Investigative Dermatology
Mantovani A, Sozzani S, Locati M, et al. (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23:549-55. Wong BR, Josien R, Lee SY, et al. (1997) TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 186:2075-80. Yasuda H, Shima N, Nakagawa N, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 95:3597-602.
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FIGURE LEGENDS Fig. 1. sRANKL induces the expression of CCL17 mRNA and protein by M2 macrophages. M2 macrophages were induced from PBMCs from healthy donors, as described in Materials and Methods. The expression of CD163 and Arg1 on CD14+ monocytes (a), and M-CSF–induced macrophages (b), and the expression of RANK on cultured M-CSF-induced macrophages with or without IL-4 (c) was analyzed using flow cytometry. Relative mRNA expression levels of CCL11, CCL17, CCL18, CCL24, CCL26, IL-10, Arg1 and iNOS by non-stimulated (NS) or sRANKL-stimulated M2 macrophages were measured using quantitative RT-PCR and were calculated using the ΔΔCt method (d). sRANKL induction of the secretion of CCL17, CCL18, CCL24 and IL-10 proteins from M2 macrophages was measured using ELISA (e). Data from each donor were obtained from triplicate assays, and the mean SD was calculated. * p<0.05, ** p<0.01 by Student’s t-test; n.s., not significant.
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Fig 2. M2 macrophages recruit CCR4-expressing cells in a tumor microenvironment. The chemoattractant activity of the culture supernatant of M2 macrophages for CD4+ T cells without (a) or with (b) an anti-CCR4 Ab was evaluated by using a Neuro Probe AP48 chamber, as described in Materials and Methods. CCL-17 was used as a positive control. Immunofluorescence staining of the lesional skin of EMPD for CD163 and CCL17 (c) and for CD163 and Foxp3 (d). Scale bar, 50 m. The expression of CCL17, T-bet, Foxp3, GATA3, and RORt mRNA in the lesional skin of EMPD was analyzed by quantitative RT-PCR using the ΔΔCt method (e). The mean SEM of data from three cases of EMPD is presented. * p<0.05 by Student’s t-test; n.s., not significant.
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© 2015 The Society for Investigative Dermatology
© 2015 The Society for Investigative Dermatology