Sei-ichiro Motegi

Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions

A new langerin+ DC subset has recently been identified in murine dermis (langerin+ dDC), but the lineage and functional relationships between these cells and langerin+ epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin+ dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin+ dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin+ dDC, in contrast to LC, did not require TGFβ1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin+ dDC and LC revealed that langerin+ dDC were required for optimal production of β-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin+ dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.

Negative Regulation of Phagocytosis in Macrophages by the CD47-SHPS-1 System

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted FcγR-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of FcγRs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of FcγR-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, FcγR-mediated tyrosine phosphorylation of Syk, Cbl, or the γ subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the FcγR-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS‐1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP‐2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47‐Fc fusion protein or antibodies to SHPS‐1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS‐1. Overexpression of wild‐type SHPS‐1 promoted CHO cell migration, whereas expression of the SHPS‐1‐4F mutant, which lacks the phosphorylation sites required for SHP‐2 binding, had no effect. Antibodies to SHPS‐1 failed to inhibit migration of CHO cells expressing SHPS‐1‐4F. SHPS‐1 ligands induced the dephosphorylation of SHPS‐1 and dissociation of SHP‐2. Antibodies to SHPS‐1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS‐1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47–SHPS‐1 system and SHP‐2 might thus contribute to the inhibition of cell migration by cell–cell contact.

MFG-E8 Regulates Angiogenesis in Cutaneous Wound Healing

Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF-PDGFRβ signaling mediated by integrin-growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31(+) blood vessels, and MFG-E8 colocalized with PDGFRβ(+), αSMA(+), and NG2(+) pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.

Beneficial effect of botulinum toxin A on Raynaud's phenomenon in Japanese patients with systemic sclerosis: A prospective, case series study

Currently, there is no satisfactory treatment for Raynauds phenomenon (RP) in systemic sclerosis (SSc). Recently, it has been reported that botulinum toxin A (BTX‐A) injection was effective for the treatment of RP in SSc patients. The objective was to assess the efficacy and safety of BTX‐A on RP in Japanese SSc patients. In the prospective, case series study, 10 Japanese SSc patients with RP received 10 U of BTX‐A injections into the hand. The change in severity of RP, including the frequency of attacks/pain, color changes, duration time of RP and the severity of pain, was assessed by Raynauds score and pain visual analog scale (VAS) at each visit during 16 weeks. The recovery of skin temperature 20 min after cold water stimulation was examined by thermography at baseline and 4 weeks after injection. The number of digital ulcers (DU) and adverse effects were assessed at each visit. BTX‐A injection decreased Raynauds score and pain VAS from 2 weeks after injection, and the suppressive effect was continued until 16 weeks after injection. Skin temperature recovery after cold water stimulation at 4 weeks after injection was significantly enhanced compared with that before injection. All DU in five patients were healed within 12 weeks after injection. Neither systemic nor local adverse effects were observed in all cases. We conclude that BTX‐A injection significantly improved the activity of RP in SSc patients without any adverse events, suggesting that BTX‐A may have possible long‐term preventive and therapeutic potentials for RP in Japanese SSc patients.

Global DNA hypomethylation and hypoxia‐induced expression of the ten eleven translocation (TET) family, TET1, in scleroderma fibroblasts

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF‐1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5‐hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF‐1α‐independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.

The prevalence of Merkel cell polyomavirus in Japanese patients with Merkel cell carcinoma

BACKGROUND A novel polyomavirus, the Merkel cell polyomavirus (MCPyV) has been implicated in the pathogenesis of Merkel cell carcinoma (MCC); however, the prevalence of MCPyV in Japan has not been extensively investigated. OBJECTIVE To clarify the prevalence of MCPyV in Japanese patients with MCC. METHODS MCPyV DNA was examined by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples from 26 patients with MCC diagnosed in four medical centers in Japan. Immunohistochemistry was simultaneously performed using a monoclonal antibody against the viral large T (LT) antigen. FFPE samples from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) were also analyzed as controls. RESULTS Twenty-three out of 26 cases (88.5%) were positive for MCPyV DNA by PCR. The amplified products harbored 4 patterns of mutations. Phylogenetic analysis demonstrated that one of our strains was closely related to the other Japanese strains previously reported. The LT antigen was expressed in various degrees in 20 of 26 cases (76.9%) by immunohistochemistry. Histological type had little relation to CM2B4 positivity, whereas 3 of 5 trabecular-type tumors showed no staining. The immunoreactivity for CM2B4 did not correlate with the relative viral DNA load. In BCC and SCC, the LT antigen was immunohistochemically positive, but MCPyV DNA was not detected by PCR. The cells around some MCC and non-MCC tumors were stained with CM2B4 with a distribution similar to CD20- and CD45RO- (especially CD8-) positive lymphocytes. CONCLUSION MCPyV was highly positive in Japanese patients with MCC. It is of note that the positive rate differs depending upon the detection method.

Essential roles of SHPS-1 in induction of contact hypersensitivity of skin.

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 and is abundant on the surface of CD11c(+) dendritic cells (DCs). We recently showed that SHPS-1 is essential for priming by DCs of CD4(+) T cells and for development of Th17 cell-mediated experimental autoimmunity. We have now further evaluated the importance of SHPS-1 and that of its ligand CD47 in contact hypersensitivity (CHS) to 2,4-dinitro-1-fluorobenzene (DNFB). Whereas the DNFB-induced CHS response was impaired in mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region, it was unaffected in CD47-deficient mice. Moreover, treatment of wild-type mice with mAbs to SHPS-1 that either block or do not block the binding of SHPS-1 to CD47 inhibited the CHS response. A mAb to CD47 had no such effect. The 2,4-dinitro-benzenesulfonic acid-induced proliferation of, and production of IFN-gamma or IL-17 by, T cells from DNFB-sensitized wild-type mice were inhibited by either mAb to SHPS-1 but not by that to CD47. In contrast, the blocking mAbs to SHPS-1, but not that to CD47, inhibited an allogeneic mixed leukocyte reaction. Both mAbs to SHPS-1, but not that to CD47, also inhibited the lipopolysaccharide- or polyinosinic-polycytidylic acid-induced production of TNF-alpha by DCs. These results suggest that SHPS-1 is essential for development of CHS, likely as a result of its positive regulation of the priming by DCs of CD4(+) T cells. However, such regulation by SHPS-1 does not appear to require its interaction with CD47.

MFG-E8 Drives Melanoma Growth by Stimulating Mesenchymal Stromal Cell–Induced Angiogenesis and M2 Polarization of Tumor-Associated Macrophages

Secretion of the powerful angiogenic factor MFG-E8 by pericytes can bypass the therapeutic effects of anti-VEGF therapy, but the mechanisms by which MFG-E8 acts are not fully understood. In this study, we investigated how this factor acts to promote the growth of melanomas that express it. We found that mouse bone marrow-derived mesenchymal stromal cells (MSC) expressed a substantial amount of MFG-E8. To assess its expression from this cell type, we implanted melanoma cells and MSC derived from wild type (WT) or MFG-E8 deficient [knockout (KO)] into mice and monitored tumor growth. Tumor growth and M2 macrophages were each attenuated in subjects coimplanted with KO-MSC compared with WT-MSC. In both xenograft tumors and clinical specimens of melanoma, we found that MFG-E8 expression was heightened near blood vessels where MSC could be found. Through in vitro assays, we confirmed that WT-MSC-conditioned medium was more potent at inducing M2 macrophage polarization, compared with KO-MSC-conditioned medium. VEGF and ET-1 expression in KO-MSC was significantly lower than in WT-MSC, correlating in vivo with reduced tumor growth and numbers of pericytes and M2 macrophages within tumors. Overall, our results suggested that MFG-E8 acts at two levels, by increasing VEGF and ET-1 expression in MSC and by enhancing M2 polarization of macrophages, to increase tumor angiogenesis. Cancer Res; 76(14); 4283-92. ©2016 AACR.

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the induction of langerhans cell maturation

Recently, we reported that Src homology 2 domain‐containing protein tyrosine phosphatase substrate 1 (SHPS‐1) plays an important role in the migration of Langerhans cells (LC). Here, we show that SHPS‐1 is involved in the maturation of LC. Immunofluorescence analysis on epidermal sheets for I‐A or CD86 revealed that LC maturation induced by 2,4‐dinitro‐1‐fluorobenzene (DNFB) or by TNF‐α was inhibited by pretreatment with an anti‐SHPS‐1 monoclonal antibody (mAb) or with CD47‐Fc fusion protein, a ligand for SHPS‐1. Further, FACS analysis demonstrated that I‐A+ LC that had emigrated from skin explants expressed CD80 or CD86, whereas CD47‐Fc protein reduced CD80high+ or CD86high+ cells. CD47‐Fc protein also reduced the up‐regulation of surface CD80 or CD86 by LC remaining in the skin explants. In SHPS‐1 mutant mice, we observed that the up‐regulation of surface CD86 and CCR7 by LC induced by DNFB as well as that of surface CD80 and CD86 by LC in skin explants was attenuated. Finally, contact hypersensitivity (CHS) response was suppressed in SHPS‐1 mutant mice and in wild‐type mice treated with an anti‐SHPS‐1 mAb. These observations indicate that SHPS‐1 plays an important role in the maturation of LC ex vivo and in vivo, and that SHPS‐1‐CD47 interaction may negatively regulate CHS.