Aya Kakizaki

The possible interaction between periostin expressed by cancer stroma and tumor‐associated macrophages in developing mycosis fungoides

Mycosis fungoides (MF) starts as an indolent disease, progresses from a patch stage to confluent plaques and ultimately develops skin tumors. Tumor‐associated macrophages (TAMs) play roles in maintaining the tumor microenvironment in MF. The purpose of this study was to elucidate the involvement of TAMs in the lesional skin of different stages of MF. First, we immunohistologically examined the percentage of CD163+ macrophages and CD206+ cells, as well as the levels of periostin and IL‐4 in cancer stroma. The percentage of CD206+ cells increased in parallel with tumor progression, while there was no significant difference in the percentage of CD163+ cells. Periostin was prominent in the stromal area at the patch and plaque stages but decreased at the tumor stage. In contrast, IL‐4 was prominently stained at both plaque and tumor stages. To further elucidate the molecular mechanisms of the effects of these stromal factors on TAMs, we examined their effects on mRNA expression in monocyte‐derived macrophages in vitro. Based on microarray analysis and gene ontology, we examined a series of chemokines and MMPs whose expression was strongly connected with periostin stimulation. The DNA microarray results were verified in M2 macrophages using real‐time PCR. We further examined the mRNA expression of these chemokines and MMPs in the presence of periostin and IL‐4 to simulate the advanced stages of MF and validated their protein expression by ELISA. Our present report suggests possible roles of periostin on TAMs in establishing the tumor microenvironment in MF.

The Possible Interaction between Receptor Activator of Nuclear Factor Kappa-B Ligand Expressed by Extramammary Paget Cells and its Ligand on Dermal Macrophages

TO THE EDITOR A mammary gland is a specific type of apocrine gland located in the subcutaneous fat of the breast (Ackerman et al., 2007). The interaction between receptor of activator nuclear factor kappa-B (RANK) and its ligand RANKL is the main mediator of progesterone-induced proliferation of mammary epithelial cells, and the activation of this pathway promotes mammary tumorigenesis (Bhatia et al., 2005; Gonzalez-Suarez, 2011). Extramammary Paget’s disease (EMPD) is an uncommon adenocarcinoma of apocrine origin. It usually affects older patients and is highly metastatic to other organs, including bone (Shiomi et al., 2013). On the basis of these findings, we hypothesized that the interaction between RANK and RANKL has a role in the tumorigenesis of EMPD. To test this hypothesis, we collected archival formalin-fixed paraffin-embedded skin specimens and cryosections from EMPD patients (Supplementary Table S1 and S2 online) for immunohistochemical and immunofluorescence analysis. This study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan (20131-521), and all patients gave written informed consent. The antibodies and antigen retrieval methods used are described in Supplementary Table S3 online. For laser capture microdissection, three samples of EMPD tissue and surrounding normal skin tissue were collected at the time of resection. For the preparation of samples for tissue culture, three samples of EMPD tissue and surrounding normal skin tissue were obtained and cultured in complete medium for 7 days. Full details are available in the Supplementary Methods online. Figure 1 shows that Paget cells strongly expressed RANKL (Figure 1a–c and Supplementary Table S1 online) and the matrix metalloprotease (MMP)-7, which cleaves RANKL to release a soluble form (sRANKL), (Figure 1d–f and Supplementary Table S1 online) on their surface (Lynch et al., 2005). Notably, keratinocytes of either lesional or normal epidermis did not express RANKL or MMP-7 (Figure 1a and d, and Supplementary Figure S1A online), whereas there were several RANKL-expressing stromal cells scattered in the lesional dermis (Figure 1c). In addition, apocrine glands in the lesional skin of EMPD expressed RANKL and MMP-7 (Supplementary Figure S1B and S1C online), whereas apocrine glands in normal skin did not express MMP-7 (Supplementary Figure S1D online). To analyze the mRNA expression of RANKL and MMP-7 by EMPD, we performed quantitative real-time reverse transcriptase–PCR using mRNA recovered from the laser-captured epidermis from the lesional skin containing Paget cells and that from the uninvolved skin of three cases of EMPD (Figure 1g and h and Supplementary Figure S1E online). The expression of RANKL mRNA (Figure 1g) and MMP-7 mRNA (Figure 1h) was significantly more abundant in the lesional epidermis of EMPD, suggesting the expression of RANKL and MMP-7 mRNA by Paget cells. As MMP-7 expression by Paget cells suggested that they might release sRANKL into the extracellular space of the lesional skin of EMPD, we cultured the lesional and non-lesional skin of EMPD and measured the concentration of sRANKL in the culture supernatants using ELISA. The results clearly demonstrated that the culture supernatants of the lesional skin contained significantly more sRANKL compared with those of the non-lesional skin (Figure 1i). These results indicate that the extracellular space surrounding EMPD tissue is rich in sRANKL. To clarify the significance of RANKL expression by Paget cells, we immunohistochemically stained cryosections of EMPD tissue for RANK. RANK was not expressed by Paget cells or surrounding keratinocytes but was instead expressed on scattered dermal cells (Figure 2a), as well as on epidermal dendritic cells (DCs; Figure 2a insert). In addition, RANK mRNA expression in the lesional epidermis of EMPD was lower than that in the non-lesional epidermis, which supported the immunohistochemical demonstration of the lack of RANK expression by Paget cells (Supplementary Figure S1F online). Although it is well known that epidermal Langerhans cell (LC)s express RANK (Loser et al., 2006), the origin of the RANK cells scattered in the dermis has not yet been clarified. When we immunohistochemically stained the lesional skin of EMPD with several antibodies against macrophages and DCs such as anti-CD163 Ab (Figure 2b), anti-CD1a Ab, anti-CD205 Ab, or anti-CD208 Ab (data not shown), the distribution LETTER TO THE EDITOR

Tumor-associated macrophages in skin: How to treat their heterogeneity and plasticity.

Immunosuppressive tumor-associated macrophages (TAMs) promote an immunosuppressive environment in the tumor-bearing host, together with regulatory T cells (Tregs). TAMs compose cancer stroma in skin cancers including melanomas and non-melanomas. The majority of tumor-associated macrophages (TAMs) are alternatively activated M2 macrophages that favor tumor development, and they comprise one of the main populations of inflammatory cells in skin cancers. On the other hand, TAMs could be modulated into M1-type macrophages that suppress tumor growth by stimulating and recruiting Th1 and effector cells in the tumor sites. Therefore, TAMs are a target for immunotherapy in various cancers. In this review, we discuss the definition and suppressive mechanisms of TAMs, as well as their biological activities in tumor-bearing hosts to assess potential therapeutic strategies.

Immunomodulatory effect of peritumorally administered interferon-beta on melanoma through tumor-associated macrophages.

An imbalance of immunosuppressive cells and cytotoxic cells plays an important role in the tumor-bearing host. Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Since interferon beta (IFN-β) has been clinically used for the treatment of malignant melanoma, we investigated the immunomodulatory effect of IFN-β during melanoma growth to elucidate the effects of IFN-β on the tumor microenvironment by using the B16F10 melanoma model. Peritumorally administered IFN-β significantly decreased the mRNA expression and production of Th2-related chemokines, which suppressed the recruitment of Tregs in B16F10 melanoma. Since the administration of IFN-β augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN-β for the treatment of B16F10 melanoma. Moreover, in parallel with the mouse model, in the human system, IFN-β decreased the production of Th2-related chemokines and augmented the production of Th1-related chemokines from monocyte-derived M2 macrophages. Since these immunomodulatory effects of IFN-β on macrophages were also observed in the lesional skin of human in-transit melanoma, our present data suggest one of the possible immunomodulatory effects of IFN-β and support the possibility of IFN-β in combination with anti-PD-1 Ab for the treatment of melanoma.

Comparison of CD163+ CD206+ M2 Macrophages in the Lesional Skin of Bullous Pemphigoid and Pemphigus Vulgaris: The Possible Pathogenesis of Bullous Pemphigoid

Background: M2 macrophages play a critical role in the induction of T helper 2 (Th2) polarization. Methods: To investigate the contribution of M2 macrophages to the pathogenesis of bullous pemphigoid (BP) and pemphigus vulgaris (PV), we employed immunohistochemical staining for CD163 and CD206, as well as pSTAT1, pSTAT6, interleukin (IL)-4, IL-13, CCL17, CCL18 and Foxp3 in the lesional skin of 10 cases of BP and PV. Results: The numbers of CD163+ CD206+ M2 macrophages were higher in BP than in PV. Moreover, pSTAT6+ cells, CCL17+ cells, CCL18+ cells and Foxp3+ regulatory T cells were prominent only in the lesional skin of BP. To further investigate the function of M2 macrophages, we examined the mRNA expression and production of Th2-related chemokines from M2 macrophages in vitro, which showed a significant increase in the mRNA expression and production of CCL18 when stimulated by IL-4 or IL-13. Conclusion: Our study sheds light onto one of the possible immunological mechanisms of BP and PV.

Tumor-associated M2 macrophages in mycosis fungoides acquire immunomodulatory function by interferon alpha and interferon gamma

BACKGROUND Tumor-associated M2 macrophages (TAMs) produce chemokines that affect the formation of cutaneous T-cell lymphoma (CTCL) by stromal factors. Since IFNs are an effective treatment for advanced-stage mycosis fungoides (MF), we hypothesized that IFNs might modulate M2 macrophages. OBJECTIVE To prove our hypothesis, we stimulated monocyte-derived M2 macrophages with IFN-α2a or IFN-γ and examined the mRNA expression of chemokines. METHODS By using a microarray, we selected a series of chemokines and MMPs that were strongly connected with the IL-4 stimulation. Then, we investigated the effects of IFN-α2a and IFN-γ on these chemokines. RESULTS IFN-α2a and IFN-γ decreased the expression and production of CCL17 and CCL18 and increased those of CXCL10 and CXCL11. Moreover, the subcutaneous administration of IFN-α2a increased the CXCL11-producing cells in the lesional skin of patients with advanced MF. CONCLUSION Our data suggest one possible mechanism of the therapeutic effects of IFNs through TAMs for the treatment of advanced-stage MF.

Immunomodulatory Effect of Bisphosphonate Risedronate Sodium on CD163 + Arginase 1 + M2 Macrophages: The Development of a Possible Supportive Therapy for Angiosarcoma

An imbalance of immunosuppressive cells and cytotoxic cells plays an important role in inhibiting the antitumor immune response of the tumor-bearing host. We previously reported the profiles of tumor infiltrating leukocytes in cutaneous angiosarcoma (AS) and suggested that a combination of docetaxel (DTX) with bisphosphonate risedronate sodium (RS) might be effective for MMP9-expressing AS by targeting immunosuppressive cells such as M2 macrophages. To further confirm the effect of this combination therapy, in this report we investigated the immunomodulatory effect of DTX and RS on CD163+ arginase 1 (Arg1)+ M2 macrophages in vitro. Interestingly, our present study demonstrated that DTX in combination with RS significantly upregulated the mRNA expression of CXCL10 on M2 macrophages and significantly decreased the mRNA expression of CCL17 and Arg1. Moreover, the production of CXCL10 and CXCL11 from M2 macrophages was significantly increased by DTX with RS though there was no effect of DTX with RS on the production of CCL5 and CCL17. Furthermore, DTX with RS significantly decreased the production of CCL18, which was previously reported to correlate with the severity and prognosis in cancer patients. Our present report suggests one of the possible mechanisms of DTX with RS in the supportive therapy for angiosarcoma.

Contact immunotherapy enhances the therapeutic effects of nivolumab in treating in‐transit melanoma: Two cases reports

Because nivolumab significantly prolongs survival in patients with metastatic melanoma, enhancing its antitumor immune response is of great interest to dermato‐oncologists. In this report, we describe two cases of metastatic melanoma successfully treated with nivolumab in combination with contact immunotherapy, using contact sensitizing agents, such as squaric acid dibutylester and diphencyprone. In addition, immunohistochemical staining supported one of the possible mechanisms of this combination therapy. Our present cases suggested a possible therapy for metastatic melanoma using nivolumab in combination with contact immunotherapy.

Isolated adrenocorticotropic hormone deficiency possibly caused by nivolumab in a metastatic melanoma patient.

Dear Editor, Although nivolumab significantly prolongs survival in patients with metastatic melanoma, severe adverse events sometimes occur. Therefore, to predict adverse events from nivolumab is of great interest. This study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan (20151-731). A 68-year-old Japanese man visited us with multiple nodules on his left foot. He had been treated for acral lentiginous melanoma and had undergone excision of the tumors 3 years prior. Physical examination revealed multiple, skincolored, dome-shaped nodules on his left lower extremities (Fig. 1a). A biopsy specimen revealed dense infiltration of spindle-shaped atypical cells with pigmentation in dermis (Fig. 1b). We diagnosed this patient as having in-transit melanoma, and administrated nivolumab at 2 mg/kg every 3 weeks. Six months after the administration of nivolumab, follow-up magnetic resonance imaging (MRI) revealed multiple brain metastases. Then, we employed a CyberKnife (Accuray, Sunnyvale, CA, USA) (14 Gy in five fractions). One month after finishing the irradiation, the serum thyroid-stimulating hormone level was 7.07 lIU/mL (normal range, 0.50–5.00), though the serum free T3 and T4 were within normal ranges. Because we suspected hypopituitarism, we performed corticotropin-releasing hormone load test, which revealed decreased levels of the serum adrenocorticotropic hormone (ACTH; <1.0 ng/mL) and cortisol (<0.8 lg/dL). Thyrotropin-releasing hormone load test, growth hormone-releasing peptide-2 load test, prolactin test and luteinizing hormone–releasing hormone load test were within normal range. MRI revealed a normally shaped pituitary. Because decreased level of ACTH had been observed in a clinical course of treatment by nivolumab, we diagnosed this patient with isolated ACTH deficiency possibly caused by nivolumab. To further analyze the mechanisms resembling hypophysitis, we retrospectively analyzed the serum soluble (s) CD163, CXCL5 and tumor necrosis factor (TNF)-a. Compared with the serum from seven cases of nivolumab-treated patients without adverse events, our present case showed strikingly upregulated serum levels of sCD163 (Fig. 1c) and CXCL5 (Fig. 1d) 6 weeks after the administration of nivolumab. The serum TNF-a was not detected in all cases. Because nivolumab significantly prolongs survival in melanoma patients, the number of patients administrated nivolumab is increasing. While several reagents were reported to

A possible interaction between periostin and CD163+ skin-resident macrophages in pemphigus vulgaris and bullous pemphigoid

Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are autoimmune blistering diseases, and substantial numbers of CD163+ tissue‐associated macrophages (TAMs) are detected in both diseases. PV and BP possess different subsets of helper T cells, suggesting that the cytokine profiles of PV and BP might be different. The purpose of this study was to investigate the microenvironment of lesional skin and serum of PV and BP patients, focusing on the immunomodulatory factors related to TAMs, such as periostin (POSTN), chemokines, cytokines and matrix metalloproteinases (MMPs). We first performed immunohistological staining of POSTN in PV and BP lesions. POSTN was prominent in the superficial dermis in both PV and BP lesions. Next, to validate the activation of CD163+ TAMs in PV and BP patients, we examined the serum levels of soluble (s)CD163. The serum sCD163 levels in PV and BP patients are significantly higher than in healthy controls. To further elucidate the molecular mechanisms of the effects of POSTN on CD163+ TAMs in PV and BP, we examined chemokines, MMPs and cytokines selected by DNA microarray database. The serum CXCL5 levels from PV patients are significantly higher than those in BP patients and healthy controls. The IL‐36γ expression on infiltrating macrophages was prominent only in the lesional skin of PV, while the MMP12 deposition was detected in both PV and BP lesions. Our results shed light on the novel pathogenesis of PV through CD163+ TAMs.