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Background: Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by excessive fibrosis. FcγRIIB is a low-affinity receptor for the Fc fragment of IgG. FcγRIIB is expressed on the surface of various leukocyte subsets and signals negative feedback pathways to down-regulate B-cell antigen receptor signaling., Objective: The aim of the present study was to investigate the role of FcγRIIB in the development of a murine bleomycin-induced scleroderma model., Methods: The experimental fibrosis model was generated by the intradermal injection of bleomycin into wild-type (WT) and FcγRIIB-deficient (FcγRIIB -/- ) mice. We histologically assessed skin and lung fibrosis as well as inflammatory cell infiltration. Cytokine and chemokine expression levels were measured with RT-PCR., Results: The severity of fibrosis in the skin and lung was significantly worse in FcγRIIB -/- mice than in WT mice. In the skin of bleomycin-treated mice, the numbers of CD8 + T cells, F4/80 + macrophages, MPO + neutrophils, NK1.1 + NK cells, and B220 + B cells were significantly higher in FcγRIIB -/- mice than in WT mice. The expression of TNF-α and IL-1β was significantly higher in FcγRIIB -/- mice than in WT mice as was the expression of ICAM-1, CXCL2, and CCL3 in the affected skin. An adoptive transfer of splenic leukocytes from FcγRIIB -/- mice into WT mice showed exacerbated skin and lung fibrosis compared to WT mice without an adoptive transfer., Conclusion: These results indicate that FcγRIIB plays an inhibitory role in skin and lung fibrosis. Moreover, modulating FcγRIIB signaling has potential as a therapeutic approach for SSc., Competing Interests: Conflict of Interest The authors have declared no conflicts of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by excessive fibrosis. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function., Objective: The aim of the present study was to investigate the roles of CD22 and CD72 in a murine scleroderma model., Methods: The experimental fibrosis model was generated by subcutaneous injection of bleomycin or hypochlorous acid (HOCL) into wild-type (WT), CD22-deficient (CD22 -/- ), CD72-deficient (CD72 -/- ) and CD22 and CD72 double-deficient (CD22 -/- /CD72 -/- ) mice. We histologically assessed skin fibrosis and inflammatory cell infiltration. Cytokine and chemokine expression levels were measured by real-time polymerase chain reaction., Results: The severity of fibrosis in the skin and lung was significantly less in CD22 -/- , CD72 -/- , and CD22 -/- /CD72 -/- mice than in WT mice in the bleomycin-induced model. In the skin of bleomycin-treated mice, the numbers of CD3 + T cells, CD8 + T cells, and F4/80 + macrophages were significantly lower in CD22 -/- , CD72 -/- , and CD22 -/- /CD72 -/- mice than in WT mice. The expression levels of mRNAs for IL-6, TNF-α, TGF-β, CTGF, IL-1β, IL-13, CXCL2, and ICAM-1 were significantly lower in CD22 -/- , CD72 -/- , and CD22 -/- /CD72 -/- mice than in WT mice. In the HOCL-induced model, both skin and lung fibrosis were ameliorated in CD22 -/- , CD72 -/- and CD22 -/- /CD72 -/- mice compared to WT mice., Conclusion: These results indicate that CD22 and CD72 likely play crucial roles in skin and lung fibrosis. Moreover, the inhibition of CD22 and CD72 function has potential as a therapeutic approach to SSc., Competing Interests: Declaration of Competing Interest The authors have declared no conflicts of interest., (Copyright © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Local type III hypersensitivity reactions are acute inflammatory events induced by immune complex (IC) deposition. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function., Objective: To elucidate the roles of CD22 and CD72 in the development of IgG-mediated type III hypersensitivity reactions., Method: The reverse Arthus reaction model in the skin was induced in mice lacking CD22 (CD22 -/- ), CD72 (CD72 -/- ), and both of them (CD22 -/- /CD72 -/- ). Edema at 4h and hemorrhage at 8h after IC challenge were evaluated. Inflammatory cell infiltration and cytokine and chemokine expression were also examined., Results: Edema and hemorrhage were significantly reduced in CD22 -/- /CD72 -/- mice compared with wild-type mice. The loss of both membrane proteins resulted in a greater decrease in edema at 4h, but not hemorrhage at 8h, than the loss of each protein alone. Infiltration of neutrophils, macrophages, and T cells, and the expression of TNF-α, IL-6, MIP-1α, and CCR5 mRNA were also diminished in the knockout mice compared to wild-type mice, and most significantly reduced in CD22 -/- /CD72 -/- mice. Regulatory T (Treg) cells in the spleen were significantly increased in all knockout mice at 4h. Significant differences in the severity of edema and hemorrhage between wild-type and knockout mice were lost when Treg cells were depleted in the knockout mice., Conclusion: These results demonstrate that CD22 and CD72 expression contribute to the development of the reverse Arthus reaction model and CD22 and CD72 might be therapeutic targets for human IC-mediated diseases., (Copyright © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

In tumor immunity, the participation of IL-10-producing regulatory B cells (Bregs), which play an important role in suppressing immune responses, is unclear. In this study, we demonstrated an increase in B16F10 melanoma growth and a decrease in the proportion of IFN-γ- and TNF-α-secreting tumor-infiltrating CD8 + T cells in B cell-specific PTEN-deficient mice in which Bregs were expanded. The number of tumor-infiltrating Bregs significantly increased in B cell-specific PTEN-deficient mice. More than 50% of tumor-infiltrating B cells consisted of Bregs, predominantly CD19 + CD5 + CD43 + B1a Bregs, in both B cell-specific PTEN-deficient and control mice. Adoptive B1a B cell transfer, which includes >30% of Bregs, increased melanoma growth, whereas non-B1a B cell transfer, which includes <2% of Bregs, exhibited no effect. In addition, adoptive transfer of B1a B cells from wild-type mice, but not IL-10 -/- mice, exacerbated B16F10 melanoma growth. The current study indicates that B1a Bregs negatively regulate anti-melanoma immunity by producing IL-10 and reducing T helper 1 type cytokine production in tumor-infiltrating CD8 + T cells. Therefore, B1a Bregs can be a potentially novel target for immunotherapy of melanomas., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Systemic sclerosis is a systemic autoimmune and connective tissue disorder associated with the human leukocyte antigen locus. However, the functional relationship between human leukocyte antigen gene(s) and disease development remains unknown. To elucidate major histocompatibility complex-linked systemic sclerosis genetics, we performed genotyping of major histocompatibility complex-borne microsatellites and HLA-DPB1 alleles using DNA obtained from 318 anti-topoisomerase I antibody-positive patients and 561 healthy controls, all of Japanese descent. Those results revealed two major histocompatibility complex haplotypes associated with systemic sclerosis. Exome sequencing and targeted analysis of these risk haplotypes identified rs17847931 in RXRB as a susceptibility variant (P = 1.3 × 10 -15 ; odds ratio [OR] = 9.4) with amino acid substitution p.V95A on the risk haplotype harboring HLA-DPB1∗13:01. No identical variant in the other haplotype including DPB1*09:01 was observed, though that haplotype also showed a significant association (P = 8.5 × 10 -22 ; OR = 4.3) with systemic sclerosis. Furthermore, the number of risk factors was shown to be a predominant factor, as individuals with two factors had elevated risk (P = 6.7 × 10 -13 ; OR = 30.2). We concluded that RXRB may be involved in antifibrotic activity in skin and chromatin remodeling., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Bone marrow transplantation (BMT) of B10.D2 mice into sublethally irradiated BALB/c mice across minor histocompatibility loci is a well-established animal model for human sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) and systemic sclerosis (SSc). The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammation and cytokine production. Furthermore, the activation of p38 MAPK plays an important role in collagen production in SSc. We investigated the effects of p38 MAPK inhibitor, VX-702, on Scl-cGVHD mice. VX-702 was orally administered to Scl-cGVHD mice from day 7 to 35 after BMT. We compared skin fibrosis of Scl-cGVHD mice between the VX-702-treated group and control group. Allogeneic BMT increased the phosphorylation of p38 MAPK in the skin. The administration of VX-702 attenuated the skin fibrosis of Scl-cGVHD compared to the control mice. Immunohistochemical staining showed that VX-702 suppressed the infiltration of CD4 + T cells, CD8 + T cells, and CD11b + cells into the dermis of Scl-cGVHD mice compared to the control mice. VX-702 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines, such as IL-6 and IL-13, in the skin of Scl-cGVHD mice. In addition, VX-702 directly inhibited collagen production from fibroblasts in vitro. VX-702 was shown to be a promising candidate for use in treating patients with Scl-cGVHD and SSc., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Autoantibodies against transcriptional intermediary factor 1 (TIF1) and Mi-2 are selectively detected in patients with dermatomyositis (DM). To measure these antibodies readily, the development of reliable ELISA systems has been needed., Objective: This study aimed to establish enzyme-linked immunosorbent assays (ELISAs) for anti-TIF1γ and anti-Mi-2β antibodies (Abs) and to assess their utility., Methods: Serum samples were obtained from 104 patients with classic DM, 68 with clinically amyopathic DM (CADM) and 70 with polymyositis, who were followed up at 8 medical centers across Japan. Serum samples from 190 patients with other connective tissue diseases (CTDs) and 123 healthy individuals were also assessed. Serum antibody levels were examined by ELISAs coated with full-length TIF1γ or Mi-2β proteins produced by a baculovirus expression system. To assess the cross-reactivity, partial-length Mi-2β proteins with or without mutations were produced and examined for reactivity., Results: When compared with immunoprecipitation assay, anti-TIF1γ Ab ELISA showed 100% sensitivity and 100% specificity, while anti-Mi-2β Ab ELISA showed 100% sensitivity and 99.6% specificity. Anti-TIF1γ Ab was positive in 30 (28.8%) with classic DM and 4 (5.9%) with CADM, whereas 14 (13.5%) with classic DM, but none with CADM, were positive for anti-Mi-2β Ab. Of 30 anti-TIF1γ Ab-positive DM patients, 23 (67.6%) had malignancy. Anti-Mi-2β Ab-positive serum samples exhibited modest cross-reactivity with the TIF1γ protein due to the homologous amino acid sequence containing cysteines in their plant homeodomains., Conclusion: The current study demonstrates the utility of newly established ELISAs for anti-TIF1γ and anti-Mi-2β Abs, which can serve as easier detection systems for routine testing., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

B cells are known to be critical mediators of tumor immunity; however, the mechanisms through which they exert this function remain unclear. B-cell linker protein (BLNK) is an essential component of the B-cell antigen receptor signaling machinery and is required for B-cell development, as evidenced by BLNK-deficient (BLNK(-/-)) mice, in which the development and function of B cells are severely impaired. Herein, we evaluated the role of B cells in the development of tumor immunity against B16F10 melanoma using BLNK(-/-) mice. B16F10 melanoma grew more aggressively in BLNK(-/-) mice, resulting in a twofold increase in tumor volume compared with wild-type mice. As predicted, tumor-infiltrating B-cell numbers were decreased in BLNK(-/-) mice. Paradoxically, tumor-infiltrating T-cell numbers were decreased in BLNK(-/-) mice, although inguinal lymph node T-cell numbers were increased. Adoptive transfer of B cells from wild-type mice into BLNK(-/-) mice attenuated B16F10 melanoma growth, with increasing numbers of B and T cells infiltrating into tumors. In addition, percentages of interferon-γ- and tumor necrosis factor-α-producing tumor-infiltrating T cells were restored. Taken together, our study supports the concept that B cells enhance tumor immunity against B16F10 melanoma by promoting T-cell infiltration into tumors and cytokine production within the tumor microenvironment.

Background: Murine sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) is a model for human Scl-cGVHD and systemic sclerosis (SSc). Syk is expressed in most of hematopoietic cells, fibroblasts, and endothelial cells. Syk is a protein tyrosine kinase that has an important role in transmitting signals from a variety of cell surface receptors., Objective: This study aims to investigate the effect of R788 (fostamatinib sodium), an oral prodrug that is rapidly converted to a potent inhibitor of Syk, R406, on Scl-cGVHD., Methods: R788 was orally administered twice a day to allogeneic recipients from day 14 to day 42 after bone marrow transplantation (BMT). In vitro, proliferation of GVHD-derived CD4(+) T cells and CD11b(+) cells was analyzed by R406., Results: Allogeneic BMT increased Syk phosphorylation in T, B, and CD11b(+) cells. The administration of R788 attenuated severity and fibrosis of Scl-cGVHD. The elevated expressions of CXCR4 on T cells, B cells, and CD11b(+) cells were significantly down-regulated by R788 treatment. R788 reduced memory CD4(+) T cells (CD44(hi)CD62L(-)CD4(+)). R406 inhibited proliferation of GVHD CD4(+) T cells and CD11b(+) cells in vitro. In addition, R788 treatment, inhibited proliferation of CD11b(+) cells in Scl-cGVHD mice. R788 treatment also reduced skin mRNA expressions of MCP-1, MIP-1α, IFN-γ, IL-13, IL-17A, and TGF-β1, but not influenced RANTES, CXCL12, and TFN-α., Conclusion: Blockade of Syk suppressed migration factor of immune cells and antigen-specific memory CD4(+) T cells and proliferation and activation of GVHD CD4(+) T cells and CD11b(+) cells. The current studies suggested that Syk inhibitor is a potential candidate for use in treating patients with Scl-cGVHD and SSc., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent cause of morbidity and mortality of allogeneic hematopoietic stem-cell transplantation. Sclerodermatous cGVHD (Scl-cGVHD) is characterized by fibrosis and autoimmune features resembling those of systemic sclerosis (SSc). Transplantation of B10.D2 bone marrow and splenocytes into irradiated BALB/c mice is an established model of human Scl-cGVHD. To examine the role of B cells in Scl-cGVHD, CD19-deficient (CD19(-/-)) mice were used as donors or recipients. CD19(-/-) donors induced more severe Scl-cGVHD than wild-type donors, but use of CD19(-/-) recipients resulted in no significant differences compared with wild-type recipients. Moreover, CD19 deficiency on donor B cells resulted in the expansion of splenic interleukin (IL) -6-producing monocytes/macrophages, cytotoxic CD8(+) T cells, and Th1 cells during the early stage of disease and increased the infiltration of T cells, TGF-β-producing monocytes/macrophages, and Th2 cells into the skin in the later stage of Scl-cGVHD. IL-10-producing regulatory B cells (B10 cells) were not reconstituted by CD19(-/-) donor cells, and early adoptive transfer of B10 cells attenuated the augmented manifestations of CD19(-/-) donor-induced Scl-cGVHD. Therefore, donor-derived B10 cells have a suppressive role in Scl-cGVHD development, warranting future investigation of regulatory B-cell-based therapy for treatment of Scl-cGVHD and SSc.

Background: Dermokine-β is abundant in stratified epithelia and in differentiating keratinocytes in culture. We have recently shown that treatment of keratinocytes with dermokine-β attenuates phosphorylation of extracellular signal-regulated kinase, however, the roles of dermokine-β in vivo remain unknown., Objective: Dermokine-β is overexpressed in marginal keratinocytes during wound healing. This study was conducted to investigate the roles of dermokine-β in the wound healing process., Methods: Recombinant human dermokine-β or its active peptide was topically applied to excisional wounds in mice and the relative wound area was calculated. Histological and chemokine expression analyses in wounds were also performed. The chemokine expression levels as well as the chemotactic activity of dermokine-β in cultured keratinocytes were determined., Results: Topical application of recombinant dermokine-β as well as its carboxy-terminal domain peptide inhibited mouse wound healing at an early phase, reduced infiltration of neutrophils and macrophages into the wounds, inhibited angiogenesis, and decreased the number of myofibroblasts in the wounds. Treatment with dermokine-β augmented IL-10 expression, but attenuated expression of transforming growth factor-β and tumor necrosis factor-α. In addition, application of dermokine-β to skin wounds reduced the expression of CXCL1 and CXCL5, both of which are chemoattractant for neutrophils into wounds. Both dermokine-β and its active peptide decreased the expression of CXCL1, CXCL6, and CXCL8 in cultured human keratinocytes. Treatment of human keratinocytes with dermokine-β inhibited neutrophil chemotaxis., Conclusion: These results suggest that dermokine-β delays early cutaneous wound healing in part by inhibiting expression of CXC chemokines containing the ERL-sequence motif., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM), and develop under the influence of certain cytokines. We previously established a mouse model of skin fibrosis induced by exogenous application of cytokines. We have revealed that both the number of macrophages and the levels of macrophage chemoattractant protein-1 (MCP-1) mRNA positively correlate with the extent of skin fibrosis. Macrophages can be divided into two subsets, the first expressing CCR2, and the second expressing CX3CR1., Objective: To elucidate the role of skin infiltrating macrophages based on CCR2 and CX3CR1 in this cytokine-induced murine fibrosis model., Methods: We examined the amounts of collagen deposited in granulation tissues, the numbers of macrophages and the levels of several mRNA in wild type (WT) mice, CCR2(-/-) mice, and CX3CR1(-/-) mice during injections of transforming growth factor-β (TGF-β) followed by injections of connective tissue growth factor (CTGF)., Results: TGF-β injection increased the expressions of MCP-1, fractalkine, CCR2 and CX3CR1 mRNA in WT mice. The overproduction of collagen induced by TGF-β was significantly reduced by CCR2 deficiency, while collagen contents induced by CTGF were restored to wild-type levels. In contrast, overproduction of collagen in CX3CR1-deficient mice decreased nearly 50% by both TGF-β and CTGF stimulations., Conclusion: The involvement of CCR2/MCP-1 interaction (CCR2-dependent loop) was during the TGF-β phase. In contrast, the fractalkine/CX3CR1 interaction contributes to the initiation of fibrosis by TGF-β and its maintenance by CTGF. Collectively, two subsets of macrophages both cooperatively and independently play important roles in the development of fibrosis., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Systemic sclerosis (SSc) is a systemic inflammatory and fibrotic disease characterized by activation of circulating T lymphocytes., Objective: To determine phenotypic abnormalities of γ/δ T cells and whether γ/δ T cells influence fibroblasts activation in SSc patients., Methods: Number and proportion of peripheral γ/δ T lymphocytes, and their expressions of cell surface molecules were evaluated by flow cytometry. Isolated γ/δ T cells were cocultured with fibroblast, and mRNA expressions of proalpha1(I) collagen and proalpha2(I) collagen (COL1A2) of fibroblasts were analyzed by real time RT-PCR. γ/δ T cell infiltrations in the skin were examined histopathologically., Results: No significant difference in number and proportion of γ/δ T cells was observed in SSc patients compared to healthy controls (HCs). Geometric mean fluorescence intensity (GMFI) of CD16 and CD69 on γ/δ T cells was significantly increased in patients with diffuse cutaneous SSc (dcSSc) compared to HCs. CD62L expression was significantly decreased in patients with dcSSc compared to HCs. The percentage of CD69 positive γ/δ T cells was significantly higher in patients with SSc than HCs. COL1A2 mRNA expression was significantly higher in fibroblasts cocultured with γ/δ T cells from SSc than that from HCs in cell contact independent manner. Additionally, γ/δ T cell infiltrations were observed in SSc patients' skin., Conclusion: Our results suggest that γ/δ T cells showed activated phenotype in SSc and suggest that SSc γ/δ T cells may play an important role on fibrotic process by upregulation of COL1A2 mRNA expression in fibroblasts., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Systemic sclerosis (scleroderma) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs. Serum IL-6 levels are reported to be elevated in human scleroderma and chronic graft-versus-host disease (cGVHD) patients. IL-6 blockade using anti-IL-6 receptor mAb (anti-IL-6R mAb) results in amelioration of the pathologic symptoms of some autoimmune diseases such as rheumatoid arthritis and juvenile idiopathic arthritis. In this study, we examined the effects of anti-IL-6R mAb on either prevention or treatment of murine sclerodermatous cGVHD (Scl-cGVHD). We found that serum IL-6 levels in Scl-cGVHD mice gradually increased after bone marrow transplantation. Administration of anti-IL-6R mAb attenuated the development of severe Scl-cGVHD and fibrosis and resulted in an increase in CD4(+)CD25(+)FoxP3(+) regulatory T cells. However, treatment of established Scl-cGVHD with anti-IL-6R mAb showed no effects on disease severity. The effects of anti-IL-6R mAb were mostly inhibited by anti-CD25 mAb. Together, our results indicate that IL-6 has an important role in the pathogenesis of Scl-cGVHD. IL-6 blockade may be an effective approach for preventing Scl-cGVHD and treating cGVHD and scleroderma in humans.

Background: The pathogenic role of IgE has been implicated in a variety of allergic and inflammatory diseases. We have previously established an IgE-mediated cutaneous reverse passive Arthus model in which eosinophil infiltration is a prominent feature. This uniquely provides a model of type III hypersensitivity in which Fc classes of Ig that forms immune complex differentially determine the disease manifestation., Objective: To investigate the mechanisms of how mast cells and basophils regulate this IgE-mediated Arthus reaction., Methods: IgE-mediated cutaneous reverse passive Arthus reaction was induced in wild-type C57BL/6 or WBB6F1-+/+ mice and mast-cell-deficient WBB6F1-W/W(v) mice by intradermal injection of IgE anti-trinitrophenyl antibodies followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. Basophils were depleted in vivo using anti-CD200R3 monoclonal antibody prior to the IC challenge., Results: Hemorrhage and infiltration of eosinophils, neutrophils, and basophils were significantly reduced but were not completely abrogated in WBB6F1-W/W(v) mice compared with those in wild-type WBB6F1-+/+ mice. Wild-type C57BL/6 mice treated by basophil-depleting mAb also showed significantly decreased hemorrhage and inflammatory cell infiltration, especially that of eosinophils, compared with control mice. Furthermore, basophil depletion in WBB6F1-W/W(v) mice led to nearly complete inhibition of eosinophil recruitment. By contrast, basophil depletion did not further decrease neutrophil infiltration in WBB6F1-W/W(v) mice., Conclusion: While mast cells play a central role, basophils also have an important function, especially for eosinophil recruitment, in IgE-mediated cutaneous reverse passive Arthus reaction., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Leukocytic infiltration into malignant melanoma lesions is tightly regulated by chemokines. To assess the role of the CC chemokines monocyte chemotactic protein-1 (MCP-1/chemokine ligand 2) and macrophage inflammatory protein-1α (MIP-1α/chemokine ligand 3) in this process, s.c. primary and metastatic B16 F10 melanoma tumor growth levels were examined in mice lacking MCP-1 or MIP-1α. Primary s.c. B16 F10 melanoma growth was augmented by loss of MCP-1 or MIP-1α. Similarly, lung metastasis was enhanced by the deficiency of MCP-1 or MIP-1α. Enhanced tumor outgrowth was associated with decreased percentages of infiltrating CD4(+) T cells, CD8(+) T cells, and natural killer cells. In the absence of MCP-1 or MIP-1α, melanoma outgrowth was correlated with reduced local expression of interferon-γ, IL-6, tumor necrosis factor-α, and transforming growth factor-β. Among these cytokines, reduced expression levels of interferon-γ and tumor necrosis factor-α on leukocytes from the spleen were associated with the development of lung metastasis in chemokine-deficient mice. The local s.c. administration of these four cytokines significantly augmented another chemokine's expression and suppressed primary melanoma growth in mice deficient for MCP-1 or MIP-1α. The s.c. injection of MCP-1 or MIP-1α significantly inhibited the primary tumor growth in wild-type mice. These results indicate that host-derived MCP-1 and MIP-1α regulate protective anti-tumor immunity to B16 F10 melanoma by promoting lymphocyte infiltration into the tumor and subsequent cytokine production., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal pulmonary fibrotic disease and useful biomarkers are required to diagnose and predict disease activity. CCN2 (connective tissue growth factor; CTGF) has been reported as one of the key profibrotic factors associated with transforming growth factor-β (TGF-β), and its assay has potential as a non-invasive measure in various fibrotic diseases. Recently, we developed a new subtraction method for determination of plasma CCN2 levels. We examined the utility of plasma CCN2 levels as a surrogate marker in IPF., Methods: Plasma CCN2 levels were calculated in 33 patients with IPF, 14 patients with non-IPF idiopathic interstitial pneumonias (IIPs) and 101 healthy volunteers by sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies for two distinct epitopes of human CCN2. We evaluated the utility of plasma CCN2 levels by comparison with clinical parameters., Results: Plasma CCN2 levels were significantly higher in patients with IPF than in those with non-IPF IIPs and healthy volunteers. Importantly, plasma CCN2 levels showed significantly negative correlation with 6-month change of forced vital capacity (FVC) in patients with IPF., Conclusions: Plasma CCN2 is a potential biomarker for IPF., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Skin wound healing is mediated by inflammatory cell infiltration of the wound site. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor-ligand pair. Although the ICOS-ICOSL pathway participates in adaptive immunity, its roles in skin wound healing, which is mediated by innate immune responses, remain unknown. To clarify these roles, repair of excisional wounds was examined in ICOS(-/-) mice, ICOSL(-/-) mice, and ICOS(-/-)ICOSL(-/-) mice. Each mutant strain showed similar, dramatic delays in wound healing, especially at early times. Knockout mice showed suppressed keratinocyte migration, angiogenesis, and granulation tissue formation, and diminished T-cell, macrophage, and neutrophil infiltration. The loss of ICOS and/or ICOSL resulted in marked suppression of cytokine expression in wounds, especially the Th2 cytokines interleukin (IL)-4, IL-6, and IL-10. T-cell transfer experiments and T-cell depletion therapy further clarified the important roles of ICOS expressed on T cells and its interaction with ICOSL. Application of IL-6, but not IL-4, to the wounds significantly increased the onset of early wound healing in mutant mice. Thus, our results indicate that ICOS-ICOSL costimulatory signaling has critical roles during wound healing, most likely by inducing IL-6 production., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Prominent eosinophil infiltration is a characteristic of some forms of vasculitis, such as Churg-Strauss syndrome, also known as allergic granulomatous vasculitis. In the current study, we established a mouse model of cutaneous eosinophilic vasculitis by the cutaneous reverse passive Arthus reaction using IgE injection instead of IgG. Wild-type C57BL/6 mice were injected with IgE anti-trinitrophenyl antibodies, followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. IgE-mediated immune complex challenge induced substantial hemorrhage with marked infiltration of eosinophils in which neutrophils, mast cells, and macrophages were also mixed. This finding contrasted remarkably with the neutrophil-dominant infiltration pattern in IgG-mediated immune complex challenge. In the lesion, the expression level of monocyte chemotactic protein-3 was increased, and anti-monocyte chemotactic protein-3 treatment resulted in a significant but incomplete blockade of eosinophil recruitment. Furthermore, mice lacking E-selectin, P-selectin, L-selectin, or intercellular adhesion molecule-1, as well as wild-type mice that received anti-vascular cell adhesion molecule-1-blocking antibodies were assessed for the IgE-mediated Arthus reaction. After 24 hours, the loss of P-selectin resulted in a significant reduction in eosinophil accumulation compared with both wild-type mice and other mouse mutants. Collectively, the Fc class of immunoglobulins, which forms these immune complexes, critically determines the disease manifestation of vasculitis. The IgE-mediated cutaneous reverse passive Arthus reaction may serve as an experimental model for cutaneous eosinophilic infiltration in vasculitis as well as in other diseases.

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-lambda. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P=0.0018, odds ratio=2.67). A SNP in the 5' flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P=0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P=0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.

The formation of pressure ulcers is dependent on multiple factors including ischemia-reperfusion (IR). This study assessed the mechanism of a previously reported murine model of cutaneous IR injury. Three cycles of IR (days 1-3) by external application of two magnetic plates were performed to induce pressure ulcer formation. Increased infiltration of neutrophils and macrophages, and augmented expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS), were observed during IR cycles. In this model, monocyte chemoattractant protein-1 (MCP-1) was remarkably increased at day 1 in the skin followed by inflammatory cell infiltration. Therefore, IR cycles were performed in MCP-1-deficient (MCP-1(-/-)) mice to evaluate the role of this chemokine in pressure ulcer development. MCP-1(-/-) mice showed reduced macrophage infiltration and expression of tumor-necrosis factor-alpha (TNF)-alpha and iNOS during IR cycles leading to attenuated apoptosis and skin injury. Importantly, MCP-1 played a role in apoptosis and injury via inducing iNOS during the reperfusion rather than the ischemic period. These findings indicate that MCP-1 may be a critical factor for macrophage recruitment and subsequent skin inflammation and injury during IR cycles. We propose that this is a useful model for investigating the mechanism of pressure ulcer formation using various transgenic mice.

Netherton syndrome (NS) is a congenital ichthyosiform dermatosis caused by serine protease inhibitor Kazal-type 5 (SPINK5) mutations. Tissue kallikreins (KLKs) and lymphoepithelial Kazal-type-related inhibitor (LEKTI) (SPINK5 product) may contribute to the balance of serine proteases/inhibitors in skin and influence skin barrier function and desquamation. SPINK5 mutations, causing NS, lead to truncated LEKTI; each NS patient possesses LEKTI of a different length, depending on the location of mutations. This study aims to elucidate genotype/phenotype correlations in Japanese NS patients and to characterize the functions of each LEKTI domain. Since we were unable to demonstrate truncated proteins in tissue from patients with NS, we used recombinant protein to test the hypothesis that the length of LEKTI correlated with protease inhibitory activity. Genotype/phenotype correlations were observed with cutaneous severity, growth retardation, skin infection, stratum corneum (SC) protease activities, and KLK levels in the SC. Predominant inhibition by LEKTI domains against overall SC protease activities was trypsin-like (Phe-Ser-Arg-) activity by LEKTI domains 6-12, plasmin- and trypsin-like (Pro-Phe-Arg-) activities by domains 12-15, chymotrypsin-like activity by all domains, and furin-like activity by none. KLK levels were significantly elevated in the SC and serum of NS patients. These data link LEKTI domain deficiency and clinical manifestations in NS patients and pinpoints to possibilities for targeted therapeutic interventions.

The tight-skin (TSK/+) mouse, a genetic model for systemic sclerosis (SSc), develops cutaneous fibrosis and autoimmunity. Although immunological abnormalities have been demonstrated in TSK/+ mice, the roles of B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell survival factor, have not been investigated. Serum BAFF levels in TSK/+ mice were examined by ELISA. Newborn TSK/+ mice were treated with BAFF antagonist, and then skin fibrosis of 8-week-old mice was assessed. Serum BAFF levels were significantly elevated in TSK/+ mice. Remarkably, BAFF antagonist inhibited the development of skin fibrosis, hyper-gamma-globulinemia, and the autoantibody production in TSK/+ mice. The skin from TSK/+ mice showed upregulated expressions of fibrogenic cytokines, such as IL-6 and IL-10, while BAFF antagonist significantly suppressed them. Reciprocally, BAFF antagonist augmented antifibrogenic cytokines, such as IFN-gamma, in the skin of TSK/+ mice. Furthermore, TSK/+ B cells with BAFF stimulation had a significantly enhanced ability to produce IL-6. The results suggest that BAFF/BAFF receptor system is critical for the development of skin fibrosis in TSK/+ mice and could be a potent therapeutical target.

Chronic contact hypersensitivity (CH) models induced by repeated hapten exposure exhibit chronic dermatitis and immunological abnormalities resembling atopic dermatitis. To assess the contribution of endothelial selectins (P- and E-selectins) to cutaneous chronic inflammation, chronic CH responses were assessed in mice lacking P- or E-selectin. Elicitation with oxazolone on the ears of P-selectin(-/-) mice 7 days after the sensitization induced a typical delayed-type hypersensitivity response similar to that found in wild-type mice. By contrast, a significant increase in ear swelling was observed in E-selectin(-/-) mice 36 to 48 hours after first elicitation. E-selectin(-/-) mice showed augmented P-selectin up-regulation, and administration of anti-P-selectin monoclonal antibody significantly inhibited the enhanced ear response, suggesting that the enhanced ear-swelling response in E-selectin(-/-) mice resulted from compensatory increase in P-selectin expression. In the late phase of chronic CH, acceleration of ear swelling was significantly reduced in both E- and P-selectin(-/-) mice relative to wild-type littermates. Thus, the loss of P- or E-selectin suppressed inflammatory responses during the chronic phase of the chronic models, whereas early-phase inflammatory responses were exacerbated by E-selectin blockade. Collectively, P- and E-selectins cooperatively regulate CH response, although their roles may be different depending on the phase of the reaction.

Background: Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro., Methods: Expression of 12 of the KLKs (KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities., Results: We here describe for the first time expression of numerous KLKs (KLKs 5-8, 10-14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4-6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro. Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro. Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation., Conclusion: We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

Systemic sclerosis (scleroderma) is an autoimmune disease characterized by excessive extracellular matrix deposition in the skin. A direct role for B lymphocytes in disease development or progression has remained controversial, although autoantibody production is a feature of this disease. To address this issue, skin sclerosis and autoimmunity were assessed in tight-skin mice, a genetic model of human systemic sclerosis, after circulating and tissue B-cell depletion using an anti-mouse CD20 monoclonal antibody before (day 3 after birth) and after disease development (day 56). CD20 monoclonal antibody treatment (10 to 20 microg) depleted the majority (85 to 99%) of circulating and tissue B cells in newborn and adult tight-skin mice by days 56 and 112, respectively. B-cell depletion in newborn tight-skin mice significantly suppressed (approximately 43%) the development of skin fibrosis, autoantibody production, and hypergammaglobulinemia. B-cell depletion also restored a more normal balance between Th1 and Th2 cytokine mRNA expression in the skin. By contrast, B-cell depletion did not affect skin fibrosis, hypergammaglobulinemia, and autoantibody levels in adult mice with established disease. Thereby, B-cell depletion during disease onset suppressed skin fibrosis, indicating that B cells contribute to the initiation of systemic sclerosis pathogenesis in tight-skin mice but are not required for disease maintenance.

The development of bleomycin-induced lung injury, which is a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. Therefore, bleomycin-induced lung fibrosis was examined in E-selectin-/- mice, P-selectin-/- mice, and E-selectin-/- mice treated with anti-P-selectin monoclonal antibody (mAb) in comparison of wild-type mice. E-selectin-/- mice treated with anti-P-selectin mAb exhibited augmented lung fibrosis histologically, increased lung collagen deposition, and increased mortality compared to wild-type mice. Furthermore, lung interferon-gamma mRNA expression decreased in E-selectin-/- mice treated with anti-P-selectin mAb relative to wild-type mice, while tumor necrosis factor-alpha and interleukin-6 mRNA expression increased in these mice. Similar changes were observed in E-selectin-/- mice, albeit to a lesser extent than those treated with anti-P-selectin mAb. Remarkably, flow cytometric analysis revealed that the frequency of interferon-gamma-producing natural killer T (NKT) cells in the bronchoalveolar lavage was decreased in E-selectin-/- mice and E-selectin-/- mice treated with anti-P-selectin mAb compared with wild-type mice. Moreover, the majority of NKT cells expressed high levels of CXCR3, suggesting that NKT cell infiltration is also dependent on CXCR3 expression. These results suggest that E- and P-selectins synergistically inhibit lung fibrosis by promoting the recruitment of NKT cells.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nerve system that is considered a T helper type 1 (Th1)-mediated autoimmune disease. EAE currently serves as an experimental animal model for multiple sclerosis in human. Cytokines, such as interferon-gamma and interleukin-10, play a key role in the development and remission of EAE. Recent studies have also shown a role for B cells in the pathogenesis of EAE. Therefore, we examined the role of CD19, a B cell-specific surface molecule that defines signaling thresholds critical for B-cell responses and autoimmunity, on the development of EAE. Following immunization with myelin oligodendrocyte glycoprotein (MOG) peptide, CD19-deficient (CD19(-/-)) mice exhibited higher clinical and pathological severity scores of EAE than wild-type mice. The increased severity of EAE in CD19(-/-) mice was associated with polarized Th1 cytokines in the inflamed central nerve system but not with anti-MOG antibodies in the serum. MOG-primed CD19(-/-) B cells produced high levels of interferon-gamma, and transfer of MOG-primed CD19(-/-) B cells to wild-type mice worsened the disease. Thus, CD19 modulates the Th1/Th2 cytokine balance in B cells and plays a critical role as a suppressive molecule in the development of EAE.

Human tissue kallikreins are a family of 15 trypsin or chymotrypsin-like secreted serine proteases (hK1-hK15). hK5, hK6, hK7, hK8, and hK13 have been identified in the stratum corneum (SC), stratum granulosum, and skin appendages. It has been reported that hK5 and hK7 degrade desmosomes/corneodesmosomes, suggesting that kallikreins are responsible for desquamation. We report the quantification of hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 in the SC by ELISA and their variation among age groups. The total SC trypsin and chymotrypsin-like activities were also measured. The amount of hK7, hK8, and hK11 (ng per mg dry weight) were high, and varied from 6 to 14, hK5 (2.0-4.0) was present at intermediate levels, and hK10 (0.65-1.0), hK14 (0.1-0.3), hK6 (0.1-0.3), and hK13 (0.02-0.1) were present at lower levels. hK6 and hK14 were significantly lower in females between 20 and 59 y. hK5, hK7, hK10, hK11, and hK14 were not significantly different across the age groups. hK8 was lowest at extremes of age (highest at 30-39 y), hK6 was lower at >30 y, and hK13 was lower at >20 y. Overall trypsin-like activity did not differ across age groups but was higher in subjects <11 y. Overall chymotrypsin-like activity was not related to age. In conclusion, we found multiple kallikreins in the SC and suggest that these enzymes may be responsible for desquamation through an enzymatic cascade pathway.

Natural killer (NK) cells are innate immune effectors that produce various immunoregulatory cytokines. Recent studies have shown that NK cells are involved in the initiation of autoimmunity. In this study, we determined abnormalities of NK cells in systemic sclerosis (SSc), an autoimmune connective tissue disease, by assessing the frequency and absolute number, activation marker expression, cytokine production, and killing activity. The frequency and absolute number of NK cells increased in diffuse cutaneous SSc (dcSSc), whereas they were normal in limited cutaneous SSc (lcSSc). NK cells from both dcSSc and lcSSc patients exhibited activated phenotypes characterized by up-regulated CD16 and CD69 expression and downregulated CD62L expression. Interferon (IFN)-gamma production by non-stimulated NK cells from both dcSSc and lcSSc patients was increased compared to the normal control, whereas on stimulation, a reduced amount of IFN-gamma was produced. Interleukin (IL)-5 and IL-10 production by non-stimulated NK cells and IL-6 production by stimulated NK cells were augmented in dcSSc patients, but not in lcSSc patients. Despite the augmented cytokine production by non-stimulated NK cells, natural cytotoxicity activity and granzyme B secretion was reduced in NK cells from dcSSc and lcSSc patients. These results suggested that altered NK cell function contributes to immunological abnormalities in SSc.

Systemic sclerosis (SSc) is characterized by autoimmunity and tissue fibrosis. There is a close association between specific autoantibodies and clinical features in patients with SSc. A number of studies have demonstrated that various cytokines, such as transforming growth factor-beta, modulate the synthesis of extracellular matrix by fibroblasts. However, it is not clear as to how autoimmunity and tissue fibrosis interact with each other. Recent studies have revealed that B cells play a critical role in various systemic autoimmune disorders. CD19 is a central regulator of B cell signaling threshold, and B cells from SSc patients exhibit an increased expression of CD19 that induces SSc-specific autoantibody production in transgenic mice. Furthermore, SSc patients have intrinsic B cell abnormalities characterized by decreased but activated memory B cells, which is possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a model of SSc, show augmented CD19 signaling and chronic B cell activation. Remarkably, CD19 loss results in inhibition of chronic B cell hyper-reactivity and elimination of autoantibody production, which is associated with improvement in skin fibrosis and a parallel decrease in IL-6 production by B cells. Therefore, augmented cytokine production by B cells is a potential candidate for the induction of skin sclerosis. Alternatively, B cells may influence tissue fibrosis by regulating T cell activation and cytokine production through their antigen-presenting and co-stimulatory abilities. Thus, altered B cell function may result in tissue fibrosis, as well as autoimmunity, in SSc.

Background: Although abnormalities of various chemokines are detected in systemic sclerosis (SSc), there are few findings concerning Th1 or Th2 chemoattractants., Objective: To determine whether serum levels of chemokines preferentially chemotactic for Th1 cells (IP-10 and MIG) and predominantly chemotactic for Th2 cells (TARC and MDC) are elevated and whether they correlate with clinical features in patients with SSc., Methods: Serum samples from patients with diffuse cutaneous SSc (dSSc; n = 34), limited cutaneous SSc (lSSc; n = 30), dermatomyositis (DM; n = 15), systemic lupus erythematosus (SLE; n = 22), and normal controls (n = 30) were examined by sandwich ELISA., Results: Serum TARC levels were significantly elevated in dSSc patients (P < 0.0002) and lSSc patients (P < 0.0001) compared with normal controls. Similarly, serum MDC levels were significantly increased in patients with dSSc (P < 0.02) or lSSc (P < 0.05) relative to normal controls. In addition, serum IP-10 was detected significantly more frequently in patients with dSSc (44%), lSSc (30%), or DM (53%) than normal controls (0%) and patients with SLE (0%). Furthermore, elevated TARC levels correlated with the presence of pitting scars and anti-topoisomerase I antibody, increased titers of anti-topoisomerase I and antinuclear antibody, and decreased glomerular filtration rate. Increased MDC levels were associated with pitting scars and younger ages at onset., Conclusion: These results suggest that both Th2 chemoattractants, TARC and MDC, and a Th1 chemoattractant IP-10 play a role in the development of SSc., (Copyright 2004 Japanese Society for Investigative Dermatology)

Background: Increased serum IgE levels are occasionally found in patients with severe bullous pemphigoid (BP). CD23, a low affinity Fc receptor for IgE, is mainly expressed on mature B lymphocytes. Studies have suggested that serum levels of soluble CD23 (sCD23) correlate with serum IgE levels and disease severity in BP., Objective: The purpose of our study is to examine whether the expression of CD23 is elevated in BP and whether this expression correlates with serum IgE levels and disease severity., Methods: We measured CD23 expression on B cells from patients with active BP, pemphigus vulgaris, pemphigus foliaceus, and atopic dermatitis (AD), as well as healthy control subjects, using a flow cytometer. Serum levels of IgE and sCD23 were also measured., Results: The expression of CD23 was significantly higher in BP patients compared with healthy control subjects (P < 0.05), whereas the levels were normal in the other bullous diseases. CD23 expression tended to be higher in severe BP compared with moderate BP, and the levels in severe BP were comparable to the levels in AD. Furthermore, CD23 expression correlated positively with serum IgE levels (P < 0.002), and the IgE levels were significantly higher in severe BP than in moderate BP (P < 0.01 ). CD23 expression in BP did not correlate with sCD23 levels., Conclusions: These results suggest that the up-regulated surface CD23 on B cells may be involved in IgE synthesis and inflammatory events in BP., (Copyright 2004 Japanese Society for Investigative Dermatology)

Background: Chemokines and their receptors are important elements for the selective attraction and activation of various subsets of leukocytes. Expression of CXCR3 ligands, such as monokine induced by IFN-gamma (Mig) leads to preferential Th1 recruitment, whereas CCR4 ligands, thymus and activation regulated chemokine (TARC) or macrophage derived chemokine (MDC), mediate preferential Th2 recruitment. Although atopic dermatitis (AD) has been shown to be a Th2-type disease, recent studies have revealed that Th1-type cytokines, such as IFN-gamma, especially in chronic skin lesions, play important roles in pathogenesis of AD., Objective: The purpose of this study was to investigate serum levels of Th2 chemokines TARC and MDC and a Th1 chemokine Mig in the same samples from patients with AD and their clinical correlation., Methods: Serum chemokine levels in patients with AD (n = 55), contact dermatitis (CD; n = 15), and normal controls (n = 30) were examined by ELISA., Results: Serum levels of TARC and MDC in AD patients and CD patients were significantly higher than those found in normal controls. Serum levels of these chemokines were similar for AD patients and CD patients. Furthermore, these levels correlated positively with disease severity, total IgE levels, and peripheral eosinophilia in AD patients. Serum Mig levels in AD patients and CD patients were significantly higher than those in control subjects. However, serum Mig levels were significantly elevated in CD patients relative to AD patients. Furthermore, serum Mig levels correlated positively with levels of both TARC and MDC in AD patients., Conclusion: These results suggest that both Th2 and Th1 chemokines may play roles in the development of AD., (Copyright 2004 Japanese Society for Investigative Dermatology)

Abnormal production of reactive oxygen species (ROS) induces tissue damage and superoxide dismutase (SOD) that converts superoxide radicals to hydrogen peroxide functions as defense against ROS. Cu/Zn SOD administration has been shown to be effective for various fibrotic conditions by inhibiting the fibrogenic effects of ROS. We hypothesized that autoimmune background in localized scleroderma induced anti-Cu/Zn SOD autoantibodies that inhibited SOD activity and thereby contributed to fibrosis by increasing ROS. ELISA using human purified Cu/Zn SOD revealed that IgG or IgM anti-Cu/Zn SOD Ab was detected in the serum of 89% of localized scleroderma patients, especially 100% of patients with generalized morphea, the severest form of localized scleroderma, but was positive only in the serum of less than 15% of patients with other autoimmune disorders, including systemic sclerosis, systemic lupus erythematosus, dermatomyositis, and autoimmune bullous disorders. The immunoblotting analysis confirmed the presence of IgG anti-Cu/Zn SOD Ab in sera from localized scleroderma patients. Remarkably, anti-Cu/Zn SOD autoantibody could inhibit Cu/Zn SOD enzymatic activity. Collectively, these results indicate that anti-Cu/Zn SOD Ab is a novel, major autoantibody in localized scleroderma, and also suggest that the autoantibody may play a role in the development of fibrosis by directly inhibiting SOD activity.

Tissue kallikreins are a group of serine proteases that are found in many organs and biologic fluids. Tissue kallikrein genes (KLKs) are found on chromosome 19q13.3-4 as a gene cluster encoding 15 different serine proteases. In skin, two tissue kallikrein proteins, hK5 and hK7, are expressed in the stratum corneum and are known to be involved in desquamation of corneocytes. The possible involvement of other kallikrein proteins has not been clarified, however, nor has the significance of each member in the serine protease activity of skin been delineated. In the study described here, we examined expression and localization of KLK mRNA in normal human skin by means of RT-PCR and in situ hybridization. Quantitative RT-PCR analysis showed abundant expression of KLK1 and KLK11 mRNA, moderate expression of KLK4, KLK5, KLK6, KLK7, and KLK13 mRNA, and low expression of KLK8 mRNA in normal human skin. For KLK4, KLK8, and KLK13 mRNA, splice variants were identified to be their major mRNA species. Two variants for KLK13 mRNA were novel. The amount of the serine protease inhibitor Kazal-type 5 (SPINK5) mRNA was comparable to KLK1 and KLK11 mRNA. In situ hybridization revealed intense expression of all KLK mRNA studied except KLK12 mRNA in the stratum granulosum of normal epidermis, where SPINK5 mRNA coexisted. Excluding KLK13 mRNA, they are also expressed in hair sheath, eccrine sweat glands, and sebaceous glands. Coexpression of various KLK and SPINK5 mRNA suggests that their proteins are the candidates to balance and maintain serine protease activities in both the skin and appendages.

Immune complex-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. To assess the relative contribution of adhesion molecules, including selectins and ICAM-1, in this pathogenetic process, the cutaneous passive Arthus reaction was examined in mice lacking E-selectin, P-selectin, or both L-selectin and ICAM-1 with anti-P- or E-selectin mAbs. Edema and hemorrhage were significantly reduced in P-selectin(-/-) mice compared with wild-type mice while they were not inhibited in E-selectin(-/-) mice. Combined E- and P-selectin blockade resulted in more significant reduction relative to L-selectin/ICAM-1(-/-) as well as P-selectin(-/-) mice. Remarkably, both E- and P-selectin blockade in L-selectin/ICAM-1(-/-) mice completely abrogated edema and hemorrhage. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells that expressed significant levels of P-selectin glycoprotein ligand-1. Similarly reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated partly with the decreased production of tumor necrosis factor-alpha and interleukin-6. The results of this study indicate that both endothelial selectins contribute predominantly to the Arthus reaction by regulating mast cell and neutrophil infiltration and that the full development of the Arthus reaction is mediated cooperatively by all selectins and ICAM-1.

Systemic sclerosis is characterized by fibrosis and systemic autoimmunity; however, roles of autoantibodies in the development of fibrosis remain unknown in systemic sclerosis. The net accumulation of extracellular matrix is dependent on the balance between the synthesis and degradation of extracellular matrix components, the latter process regulated by matrix metalloproteinases. Matrix metalloproteinase-1 (interstitial collagenase-1) can initiate degradation of collagen types I-III that are major extracellular matrix constituents in affected skin of systemic sclerosis. In this study, we tested the hypothesis that systemic autoimmunity in systemic sclerosis induced anti-matrix metalloproteinase-1 autoantibodies that inhibited matrix metallo-proteinase-1 activity, resulting in collagen accumulation. Enzyme-linked immunosorbent assay using human recombinant matrix metalloproteinase-1 revealed that IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly elevated in sera from patients with systemic sclerosis, but not patients with active systemic lupus erythematosus or dermatomyositis, relative to normal controls. IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly higher in patients with diffuse cutaneous systemic sclerosis than those found in patients with limited cutaneous systemic sclerosis. Furthermore, IgG anti-matrix metalloproteinase-1 antibody levels significantly correlated with the extent of fibrosis in the skin, lung, and renal blood vessels. The presence of IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients was confirmed by immunoblotting analysis. Remarkably, IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients inhibited matrix metalloproteinase-1 collagenase activity. Collectively, the results of this study suggest that anti-matrix metalloproteinase-1 autoantibody contributes to the development of fibrosis by inhibiting matrix metalloproteinase-1 collagenase activity and reducing the extracellular matrix turnover and suggest that the presence of anti-matrix metalloproteinase-1 autoantibody in systemic sclerosis is the link between systemic autoimmunity and fibrosis.

The development of bleomycin-induced lung injury, a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. At present, the identity and role of the adhesion molecules involved in the fibrotic process are unknown. Therefore, bleomycin-induced pulmonary fibrosis was examined in mice lacking L-selectin (L-selectin(-/-)) expression, intercellular adhesion molecule-1 (ICAM-1) expression, or both. After 16 days of intratracheal bleomycin challenge, collagen deposition was inhibited in both L-selectin(-/-) and ICAM-1(-/-) mice when compared with wild-type littermates. Interestingly, collagen deposition was virtually eliminated in L-selectin/ICAM-1(-/-) mice relative to either the L-selectin(-/-) or ICAM-1(-/-) mice. Decreased pulmonary fibrosis was associated with reduced accumulation of leukocytes, including neutrophils and lymphocytes. Decreased mRNA expression of proinflammatory cytokines and transforming growth factor (TGF)-beta1 paralleled the inhibition of collagen deposition. The present study indicates that L-selectin and ICAM-1 play a critical role in pulmonary fibrosis by mediating the accumulation of leukocytes, which regulate the production of proinflammatory cytokines and TGF-beta1. This suggests that these adhesion molecules are potential therapeutic targets for inhibiting human pulmonary fibrosis.

Netherton syndrome is a congenital ichthyosis associated with erythroderma, hair shaft defects, and atopic features. The mutations of the secretory serine protease inhibitor Kazal-type 5 gene have been identified in Netherton syndrome patients; however, the actual physiologic substrates of the serine protease inhibitor Kazal-type 5 proprotein are unknown, and how the genetic defects cause characteristic skin phenotype remains uncertain. Here, we describe the serine protease inhibitor Kazal-type 5 gene mutations, including two novel non-sense mutations, and genotype-phenotype correlation in three Netherton syndrome patients in two unrelated Japanese families. Furthermore, based on the reappraisal of the structure of the serine protease inhibitor Kazal-type 5 proprotein, demonstration of the presence of carboxypeptidase in normal keratinocytes, and the observation of mRNA localization of the serine protease inhibitor Kazal-type 5 transcripts in the uppermost epidermis as well as pilosebaceous units, we propose a hypothetical model of proteolytic processing of the serine protease inhibitor Kazal-type 5 proprotein in the epidermis and inhibitory regulation of corneocyte desquamation by a set of serine protease inhibitor Kazal-type 5-derived peptides. This hypothesis is supported by the marked increase of trypsin-like hydrolytic activity demonstrated in stratum corneum samples from our Netherton syndrome patients. The findings in this study suggest that the defective inhibitory regulation of desquamation due to the serine protease inhibitor Kazal-type 5 gene mutations may cause over-desquamation of corneocytes in Netherton syndrome, leading to severe skin permeability barrier dysfunction.