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A phase 2 trial of tocilizumab showed preliminary evidence of efficacy in systemic sclerosis. We assessed skin fibrosis and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in a phase 3 trial to investigate the safety and efficacy of tocilizumab, an anti-interleukin-6 receptor antibody, in the treatment of systemic sclerosis.

Systemic sclerosis is a connective tissue disorder characterized by microvascular damage and excessive fibrosis of the skin and internal organs. One hallmark of the immunological abnormalities in systemic sclerosis is the presence of anti-nuclear antibodies, which are detected in more than 90% of patients with systemic sclerosis. Anti-centromere antibodies, anti-DNA topoisomerase I antibodies, and anti-RNA polymerase III antibodies are the predominant anti-nuclear antibodies found in systemic sclerosis patients. Other systemic sclerosis–related anti-nuclear antibodies include those targeted against U3 ribonucleoprotein, Th/To, U11/U12 ribonucleoprotein, and eukaryotic initiation factor 2B. Anti-U1 ribonucleoprotein, anti-Ku antibodies, anti-PM–Scl, and anti-RuvBL1/2 antibodies are associated with systemic sclerosis overlap syndrome. Anti-human upstream binding factor, anti-Ro52/TRIM21, anti-B23, and anti-centriole antibodies do not have specificity to systemic sclerosis, but are sometimes detected in sera from patients with systemic sclerosis. Identification of each systemic sclerosis–related antibody is useful to diagnose and predict organ involvement, since the particular type of systemic sclerosis–related antibodies is often predictive of clinical features, severity, and prognosis. The clinical phenotypes are largely influenced by ethnicity. Currently, an immunoprecipitation assay is necessary to detect most systemic sclerosis–related antibodies; therefore, the establishment of an easy, reliable, and simple screening system is warranted.

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-DPB1alleles 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 RXRBas 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*09:01was 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 RXRBmay be involved in antifibrotic activity in skin and chromatin remodeling.

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.

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.

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.

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.

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.

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.

OBJECTIVE: To clarify the clinical significance of concentrations of serum Clara cell 16-kDa protein (CC16; previously denoted CC10) in the diagnosis and monitoring of pulmonary fibrosis (PF) in patients with systemic sclerosis (SSc); and to compare CC16 levels with levels of the current most reliable serum markers for PF, such as Krebs von den Lungen-6 (KL-6) antigen and surfactant protein-D (SP-D). METHODS: Serum levels of CC16, KL-6, and SP-D were determined by ELISA in 92 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy controls. In a retrospective longitudinal study, correlation of serum CC16 levels with the activity of PF was assessed in 16 SSc patients with PF. RESULTS: Although CC16 levels were higher in patients with SSc than in SLE patients or healthy controls, the difference was not significant. Increased serum CC16 levels were associated with involvement of PF, especially active PF, as well as KL-6 and SP-D. Receiver operating characteristic curve analysis revealed that the utility of CC16 is slightly inferior to KL-6, but was comparable with that of SP-D for detecting PF in patients with SSc. In the longitudinal study, serum levels of CC16, KL-6, and SP-D were significantly decreased in the inactive disease phase compared to the active disease phase. CONCLUSION: CC16 levels can be used as a potential serum biomarker for PF in addition to KL-6 and SP-D in patients with SSc.

OBJECTIVE To clarify the association of clinical and prognostic features with dermatomyositis (DM)-specific autoantibodies (Abs) in adult Japanese patients with DM. DESIGN Retrospective study. SETTING Kanazawa University Graduate School of Medical Science Department of Dermatology and collaborating medical centers. PATIENTS A total of 376 consecutive adult Japanese patients with DM who visited our hospital or collaborating medical centers between 2003 and 2008. MAIN OUTCOME MEASURES Clinical and laboratory characteristics of adult Japanese patients with DM and DM-specific Abs that include Abs against Mi-2, 155/140, and CADM-140. RESULTS In patients with DM, anti–Mi-2, anti–155/140, and anti–CADM-140 were detected in 9 (2%), 25 (7%), and 43 (11%), respectively. These DM-specific Abs were mutually exclusive and were detected in none of 34 patients with polymyositis, 326 with systemic sclerosis, and 97 with systemic lupus erythematosus. Anti–Mi-2 was associated with classical DM without interstitial lung disease or malignancy, whereas anti–155/140 was associated with malignancy. Patients with anti–CADM-140 frequently had clinically amyopathic DM and rapidly progressive interstitial lung disease. Cumulative survival rates were more favorable in patients with anti–Mi-2 compared with those with anti–155/140 or anti–CADM-140 (P < .01 for both comparisons). Nearly all deaths occurred within 1 year after diagnosis in patients with anti–CADM-140. CONCLUSION Dermatomyositis-specific Abs define clinically distinct subsets and are useful for predicting clinical outcomes in patients with DM.Arch Dermatol. 2011;147(4):391-398--

OBJECTIVE: To determine serum concentrations of soluble CXCL16 and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum CXCL16 levels from 89 patients with SSc were examined by ELISA. In a retrospective longitudinal study, 68 sera from 28 patients with SSc were analyzed (followup 1.3 to 7.3 yrs). RESULTS: Serum CXCL16 levels were elevated in patients with SSc compared with healthy controls (n = 42). Patients with diffuse cutaneous SSc (n = 52) had higher levels of CXCL16 than those with limited cutaneous SSc (n = 37). Serum CXCL16 levels correlated positively with the extent of skin sclerosis. In the longitudinal study, CXCL16 levels generally decreased on a parallel with the improvement in skin sclerosis. CONCLUSION: CXCL16 levels were increased in patients with SSc, and correlated with the extent of skin sclerosis, suggesting that CXCL16 may have a role in the development of skin fibrosis in SSc. Blockade of CXCL16 interaction might be a potential therapeutic target in patients with SSc.

OBJECTIVE: To determine serum levels of tumor necrosis factor-related weak inducer of apoptosis (TWEAK) and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum TWEAK levels from 70 patients with SSc were examined by ELISA. In a retrospective longitudinal study, sera from 23 patients with SSc were analyzed (followup 0.8–7.2 yrs). RESULTS: Serum TWEAK levels were elevated in patients with SSc (n = 70) compared with healthy controls (n = 31) and patients with systemic lupus erythematosus (n = 22). Among patients with SSc, there were no differences in serum TWEAK levels between limited cutaneous SSc and diffuse cutaneous SSc. Patients with SSc who had elevated TWEAK levels less often had pulmonary fibrosis and decreased vital capacity than those with normal TWEAK levels. In the longitudinal study, SSc patients with inactive pulmonary fibrosis or without pulmonary fibrosis consistently exhibited increased TWEAK levels, while those with active pulmonary fibrosis showed decreased TWEAK levels during the followup period. CONCLUSION: TWEAK levels were increased in patients with SSc, and associated with a lower frequency of pulmonary fibrosis in patients with SSc. TWEAK could be a protective factor against the development of pulmonary fibrosis in this disease and as such would be a possible therapeutic target.

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.

OBJECTIVE: To determine serum concentrations of pentraxin 3 (PTX3) and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum PTX3 levels from 45 patients with diffuse cutaneous SSc (dSSc), 46 with limited cutaneous SSc (lSSc), and 20 healthy controls were examined by ELISA. PTX3 expression in the sclerotic skin from SSc patients was evaluated immunohistochemically. Normal and SSc fibroblasts were cultured and PTX3 levels in the culture medium were also examined by ELISA. RESULTS: Serum PTX3 levels were elevated in patients with SSc relative to controls. PTX3 levels in dSSc patients were significantly higher than in controls and lSSc patients. PTX3 expression in the sclerotic skin from SSc patients was more intense relative to normal skin. Elevation of serum PTX3 levels was associated with more frequent presence of pulmonary fibrosis, cardiac disease, and pitting scar/ulcer and increased serum immunoglobulin levels and erythrocyte sedimentation rates. PTX3 levels correlated positively with modified Rodnan total skin thickness score, and negatively with percentage vital capacity and percentage DLCO in patients with SSc. PTX3 levels also correlated positively with serum levels of 8-isoprostane, a marker of oxidative stress, and hyaluronan, recently identified as an endogenous ligand for Toll-like receptors. PTX3 production from cultured SSc fibroblasts was increased by stimulation with hyaluronan. CONCLUSION: These results suggest that elevated serum PTX3 levels are associated with the disease severity of SSc.

OBJECTIVE: To examine serum concentrations of insulin-like growth factor (IGF-1) and IGF binding protein (IGFBP-3), a major carrier protein for IGF-1, in patients with systemic sclerosis (SSc); and to relate the results to clinical features in SSc. METHODS: Serum IGF-1 and IGFBP-3 levels in 92 Japanese patients with SSc were measured by ELISA. Expression of IGF-1 and IGFBP-3 messenger RNA (mRNA) in the skin was quantified by real-time reverse transcription-polymerase chain reaction. RESULTS: Serum IGF-1 and IGFBP-3 levels were significantly elevated in patients with SSc compared with patients with systemic lupus erythematosus or healthy controls. IGF-1 levels were higher in patients with diffuse cutaneous SSc (dcSSc) than in patients with limited cutaneous SSc (lcSSc). Patients with increased IGF-1 levels had more severe skin involvement and pulmonary fibrosis. IGF-1 mRNA was upregulated in the affected skin of patients with SSc. There were no significant differences in serum IGFBP-3 levels between dcSSc and lcSSc. IGFBP-3 levels were not associated with skin thickness and pulmonary fibrosis. Patients with increased IGF-1 or IGFBP-3 had lower frequency of telangiectasia than patients with normal levels. CONCLUSION: These results suggest that both IGF-1 and IGFBP-3 are involved in the development of SSc. The role of IGF-1 appears to be different from that of IGFBP-3.

OBJECTIVE: .To determine levels of serum soluble OX40 (also termed CD134, a member of the tumor necrosis factor receptor superfamily) and their clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum soluble OX40 levels were examined by ELISA in 53 patients with SSc, 15 patients with systemic lupus erythematosus (SLE), and 32 healthy individuals. RESULTS: OX40 levels were significantly elevated in SSc patients (125.7 ± 5.7 pg/ml) compared to patients with SLE (80.7 ± 1.7 pg/ml; p < 0.005) and controls (88.2 ± 3.0 pg/ml; p < 0.0001). Elevated OX40 levels were found to be associated with disease duration of less than 2 years (p < 0.05). CONCLUSION: Our results suggest that serum soluble OX40 levels correlate with the early-onset of SSc disease.

OBJECTIVE: The relationship between systemic sclerosis (SSc) and interleukin 23 (IL-23), a cytokine associated with the differentiation of T lymphocytes, is unknown. We investigated serum IL-23 levels and their clinical association in patients with SSc. METHODS: Serum IL-23 levels were examined by ELISA in 63 patients with SSc, 15 patients with systemic lupus erythematosus (SLE), and 31 healthy individuals. SSc patients comprised 25 with limited cutaneous SSc and 38 with diffuse cutaneous SSc. RESULTS: Serum IL-23 levels were significantly elevated in SSc patients compared to patients with SLE (p < 0.05) and controls (p < 0.005). Elevated serum IL-23 levels were associated with the disease duration (p < 0.05) and the prevalence of pulmonary fibrosis (p < 0.05), although they were not associated with other clinical features, including the extent of skin sclerosis or the severity of pulmonary fibrosis. CONCLUSION: The results suggest that IL-23 is associated with induction of SSc and that blockade of IL-23 can be a potential therapeutic strategy in early SSc.

OBJECTIVE: Elevated serum concentrations of B cell-activating factor belonging to the tumor necrosis factor family (BAFF) are found in systemic sclerosis (SSc) and are associated with the severity of skin sclerosis. We investigated serum levels of a proliferation-inducing ligand (APRIL), a close homolog to BAFF, and its clinical association in patients with SSc as well as its correlation with BAFF. METHODS: Serum APRIL levels from 74 patients with SSc, 25 patients with systemic lupus erythematosus, and 25 healthy subjects were examined by ELISA. Clinical and laboratory measures were compared between SSc patients with elevated serum APRIL levels and those with normal levels. We assessed correlation of serum APRIL and BAFF levels in patients with SSc. RESULTS: Serum APRIL levels were elevated in SSc patients compared to controls. SSc patients with elevated serum APRIL levels had significantly higher incidences of pulmonary fibrosis than those with normal levels. Serum APRIL levels did not correlate with serum BAFF levels in SSc patients, and there were distinct profiles of SSc categorized by serum APRIL and BAFF levels. High APRIL levels served as a marker for involvement of pulmonary fibrosis and high BAFF levels served as a marker for severe skin sclerosis. CONCLUSION: The results suggest that elevated serum APRIL and BAFF levels were differentially associated with disease severity in SSc.

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule‐1 (ICAM‐1) delayed skin wound healing and mice lacking both L‐selectin and ICAM‐1 (L‐selectin/ICAM‐1−/−) show more delayed wound healing. Deficiency of both endothelial selectins (E‐selectin or P‐selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM‐1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L‐selectin/ICAM‐1−/−mice, wild‐type mice with both E‐ and P‐selectin blockade, and L‐selectin/ICAM‐1−/−mice with both E‐ and P‐selectin blockade. Wild‐type mice with both E‐ and P‐selectin blockade showed delayed wound healing that was comparable with that in L‐selectin/ICAM‐1−/−mice. Combined E‐ and P‐selectin blockade in L‐selectin/ICAM‐1−/−mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor‐β and interleukin‐6. Application of basic fibroblast growth factor (bFGF) but not platelet‐derived growth factor to the wounds significantly improved wound healing in L‐selectin/ICAM‐1−/−mice with both E‐ and P‐selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM‐1 and may provide critical information for the therapy of skin wounds.

OBJECTIVE: To investigate the distribution of anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies among patients with autoimmune diseases, and to analyze the clinical features of patients with dermatomyositis (DM) with anti-ARS antibodies. METHODS: Serum samples from 315 patients with autoimmune diseases or related disorders who had visited Kanazawa University Hospital or affiliated facilities were assessed for anti-ARS antibodies by immunoprecipitation. In particular, the association between anti-ARS antibodies and clinical features was investigated in detail in patients with DM. RESULTS: Anti-ARS antibody was positive in 16 (29%) of 55 patients with DM, 2 (22%) of 9 patients with polymyositis, and 7 (25%) of 28 patients with idiopathic pulmonary fibrosis. Although anti-ARS antibody was detected with high frequency (63%, 15/24) in DM patients with interstitital lung disease (ILD), the incidence of anti-ARS antibody was very low (3%, 1/31) in DM patients without ILD. Anti-ARS antibody-positive patients with DM had significantly higher incidences of ILD (94% vs 23%) and fever (64% vs 10%) than the antibody-negative patients. Some immunosuppressive agents, in addition to oral corticosteroids, were required more frequently in the antibody-positive patients with DM than the antibody-negative patients (88% vs 26%). Although 60% of DM patients with ILD simultaneously developed ILD and myositis, ILD preceded myositis in 33% of patients. CONCLUSION: Among patients with DM, anti-ARS antibodies are found in a subset with ILD. DM patients with anti-ARS antibodies appear to have a more persistent disease course that requires additional therapy compared to those without anti-ARS antibodies.

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.

Immune complex (IC)‐induced inflammation is mediated by inflammatory cell infiltration, a process that is highly regulated by expression of multiple adhesion molecules. The roles and interactions of ICAM‐1 and VCAM‐1, the major regulators of leukocyte firm adhesion, were examined in the cutaneous reverse‐passive Arthus reaction using ICAM‐1‐deficient (ICAM‐1−/−) mice and blocking mAb against VCAM‐1. Within 8 h, IC challenge of wild‐type mice induced edema, hemorrhage, interstitial accumulation of neutrophils and mast cells, as well as production of TNF‐α and IL‐6. All of these inflammatory parameters were reduced significantly in ICAM‐1−/−mice. The blockade of VCAM‐1 in wild‐type mice did not affect any inflammatory parameters. In contrast, ICAM‐1−/−mice treated with anti‐VCAM‐1 mAb had significantly reduced edema, hemorrhage, and neutrophil infiltration. Furthermore, VCAM‐1 blockade in ICAM‐1−/−mice suppressed cutaneous TNF‐α and IL‐6 production. Thus, VCAM‐1 plays a complementary role to ICAM‐1 in the cutaneous Arthus reaction by regulating leukocyte accumulation and proinflammatory cytokine production.

OBJECTIVE: To determine serum levels of soluble CD40 (sCD40) and clinical association in patients with systemic sclerosis (SSc). METHODS: Serum sCD40 levels were examined by ELISA in 49 patients with SSc, 15 patients with systemic lupus erythematosus, and 26 healthy individuals. sCD40 levels in plasma samples, which were obtained at the same time, were also determined. SSc patients were grouped into 22 patients with limited cutaneous SSc (lcSSc) and 27 patients with diffuse cutaneous SSc (dcSSc). RESULTS: There was no significant difference between sCD40 levels of sera and those of plasma. Serum sCD40 levels were significantly elevated in patients with SSc compared to patients with systemic lupus erythematosus and controls (p < 0.001). Serum sCD40 levels were higher in patients with lcSSc than in those with dcSSc (p <0.001). There was no correlation between sCD40 and sCD40 ligand levels in patients with SSc. CONCLUSION: Elevated serum sCD40 levels were associated with lcSSc. These results suggest that the blockade of CD40/CD40 ligand interaction could be a potential therapeutic strategy in SSc.

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 μg) 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 (∼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-γ mRNA expression decreased in E-selectin−/−mice treated with anti-P-selectin mAb relative to wild-type mice, while tumor necrosis factor-α 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-γ-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.

OBJECTIVE: To determine the efficacy of self-administered stretching of each finger in Japanese patients with systemic sclerosis (SSc). METHODS: Forty-five patients with SSc (32 with diffuse cutaneous SSc and 13 with limited cutaneous SSc) were given instructions on self-administered stretching and were directed to perform it every day. Individual fingers were maintained in a stretched position using the opposite hand for 10 seconds and this was repeated 3–10 times. To evaluate the effect of the stretching program, finger passive range of motion (ROM) was assessed using a goniometer on the first visit and after 1 month and 1 year of the stretching program. The Health Assessment Questionnaire (HAQ) was also assessed on the first visit and 1 year afterward. RESULTS: The total passive ROM was significantly improved in each finger after 1 month of finger stretching. The total passive ROM was further improved or maintained within 1 year after the first visit. Although ROM was less in patients with diffuse cutaneous SSc than in those with limited cutaneous SSc at the first visit, ROM increased significantly irrespective of disease duration or severity of skin sclerosis. Finger stretching may improve the finger function, since the HAQ score for hand functions such as eating and gripping was significantly decreased. CONCLUSION: Our original self-administered stretching program may be useful for improving finger joint motion in patients with SSc; future studies in various ethnic populations will be needed to determine the universal efficacy of this method.

Concanavalin A (Con A)‐induced hepatitis is a model for human T cell‐mediated hepatitis. We evaluated the role of L‐selectin and intercellular adhesion molecule‐1 (ICAM‐1) in this model by injecting Con A intravenously in mice lacking L‐selectin (L‐selectin−/−), ICAM‐1 (ICAM‐1−/−), or both (L‐selectin/ICAM‐1−/−). Blood and liver samples were collected 0, 8, 24, and 48 h after Con A treatment. Increases in plasma transaminase levels, which peaked 8 h after injection, were reduced significantly in L‐selectin−/−, ICAM‐1−/−, and L‐selectin/ICAM‐1−/−mice compared with wild‐type mice. Liver necrosis was more strongly inhibited in ICAM‐1−/−mice than in L‐selectin−/−mice but was most prominently reduced in L‐selectin/ICAM‐1−/−mice, in parallel with decreased plasma transaminase levels. The reduced severity of hepatitis in the mutant mice correlated with decreases in numbers of liver CD4+T cells but not numbers of CD8+T cells or neutrophils. Following Con A treatment, L‐selectin deficiency reduced liver mRNA expression of tumor necrosis factor‐α, and ICAM‐1 deficiency reduced expression of interleukin‐4. By contrast, reductions in liver macrophage inhibitor protein‐1α mRNA occurred in all mutant mice. These results indicate that L‐selectin and ICAM‐1 contribute cooperatively to the development of Con A‐induced hepatitis by regulating leukocyte infiltration and subsequent cytokine production.

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-γ 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-γ, 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.

OBJECTIVE: Skin sclerosis that progresses in the earlier disease phase in systemic sclerosis (SSc) spontaneously regresses thereafter. We investigated the relationship between changes of the serum cytokine profile and changes in skin fibrosis in patients with SSc. METHODS: Serum cytokine levels were examined by ELISA using 180 sera samples from 26 patients with early diffuse cutaneous SSc (dcSSc) with mean disease duration of 2.1 years. The mean followup period was 4.9 years (range 2–8). Cytokine mRNA expression in the affected skin was quantified by real-time reverse transcription-polymerase chain reaction. RESULTS: Modified Rodnan total skin thickness score decreased after 2, 4, and 6 years compared to that at first visit. Serum levels of the Th2 cytokines interleukin 6 (IL-6) and IL-10 and monocyte chemotactic protein-1 (MCP-1) were higher at first evaluation compared to healthy controls, while IL-4 levels were normal. Levels of all Th2 cytokines generally decreased as skin sclerosis regressed. Conversely, levels of serum IL-12, a Th1-inducing cytokine, were lower at first visit relative to controls, but increased by roughly 15-fold after 6 years to significantly higher levels than controls. Surviving dcSSc patients exhibited elevated IL-12 levels compared to deceased patients. Serum levels of transforming growth factor-ß1 (TGF-ß1), a fibrogenic cytokine, increased throughout followup, with slightly decreased levels at later timepoints. IL-12 mRNA expression was upregulated in affected skin from patients with late-stage dcSSc, while TGF-ß1 and MCP-1 expression was downregulated. CONCLUSION: These results suggest that a shift from Th2 to Th1 response correlates with improvement in skin fibrosis in SSc, and that IL-12 level is a serologically useful marker for disease activity and prognosis.

We describe a patient with drug‐induced hypersensitivity syndrome (DIHS) associated with human herpesvirus 6 (HHV‐6) and cytomegalovirus (CMV) infection induced by sulfasalazine. Two weeks after starting sulfasalazine to treat a rectal ulcer, the patient developed disseminated macular erythema accompanied by fever, liver injury, and lymphadenopathy. Seroconversion of antibodies to HHV‐6 was observed. Systemic steroid treatment was not effective against the eruptions. Five months after the onset, he presented with an acute febrile disease. The detection of CMV antigen on peripheral blood leukocytes and positive staining for CMV on cutaneous endothelium indicated active CMV infection. Furthermore, he developed a bacteremia of methicillin resistant Staphylococcus aureus.An associatiation the CMV reactivation with DIHS was suggested, although there remains the possibility that the systemic steroid treatment precipitated CMV reactivation. Recently, HHV‐6 has been documented to have immunomodulating effects and to be associated with CMV reactivation. Therefore, we should pay attention to the possibility of CMV reactivation in patients with DIHS in whom the immunomodulating virus of HHV‐6 has been reactivated.

OBJECTIVE: The vascular damage systemic sclerosis (SSc) consists mainly of microvascular changes, but recently macrovascular changes with dyslipidemia were recognized. In systemic lupus erythematosus (SLE), autoantibody to lipoprotein lipase (LPL), a key enzyme that hydrolyzes triglycerides, suggested a role of autoimmunity for elevated serum triglyceride levels and atherosclerosis. We investigated the prevalence and levels of anti-LPL antibodies, their clinical correlation, and their functional significance in patients with SSc. METHODS: Serum samples from patients with diffuse cutaneous SSc (dSSc; n = 55), limited cutaneous SSc (lSSc; n = 75), SLE (n = 21), and dermatomyositis (DM; n = 21) and healthy controls (n = 41) were examined by ELISA. The presence of anti-LPL antibody was evaluated by immunoblotting analysis using purified LPL. To determine the functional relevance of anti-LPL antibody in vivo, we assessed whether anti-LPL autoantibody was able to inhibit LPL activity using the LPL activity kit. RESULTS: ELISA revealed that IgG or IgM anti-LPL antibodies were detected in 35% of SSc patients, while they were also positive in 67% of SLE patients and 43% of DM patients. The presence of IgG anti-LPL antibody was associated with elevated serum triglyceride levels, greater extent of skin fibrosis, and more frequent presence of lung fibrosis, heart involvement, and anti-topoisomerase I antibodies. The presence of anti-LPL autoantibody was confirmed by immunoblotting analysis. LPL activity was inhibited by IgG anti-LPL antibodies in sera from SSc patients with elevated serum triglyceride levels. CONCLUSION: Our results suggest that anti-LPL autoantibody contributes to elevated serum triglyceride levels by inhibiting LPL enzyme activity in patients with SSc.

Immune complex (IC)‐induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. To assess the contribution of P‐selectin glycoprotein ligand‐1 (PSGL‐1) and selectins in the pathogenetic process, the cutaneous reverse‐passive Arthus reaction was examined in mice treated with monoclonal antibodies (mAb) to PSGL‐1 or P‐ and/or E‐selectin. Edema and hemorrhage were significantly reduced in mice treated with anti‐P‐selectin mAb compared with control mice while they were not inhibited in mice treated with anti‐E‐selectin mAb. It is remarkable that blocking PSGL‐1 by mAb resulted in significant, further reduction in edema and hemorrhage compared with blocking anti‐P‐ or anti‐E‐selectin. However, blockade of E‐ and P‐selectins exhibited more significant reduction relative to PSGL‐1 blockade. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells. Reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated with the decreased production of tumor necrosis factor α and interleukin‐6. The results of this study indicate that PSGL‐1 contributes to the Arthus reaction mainly as a ligand of P‐selectin and partly as a ligand of E‐ and/or L‐selectin by regulating neutrophil and mast‐cell recruitment and that PSGL‐1 would be a therapeutic target for human IC‐mediated diseases.

OBJECTIVE: To clarify the clinical significance of surfactant protein-D (SP-D) and KL-6 in the diagnosis and monitoring of pulmonary fibrosis (PF) in patients with systemic sclerosis (SSc), and to evaluate the differences between SP-D and KL-6. METHODS: Serum SP-D and KL-6 concentrations were determined by ELISA in 42 SSc patients. In a retrospective longitudinal study, 83 serum samples from 6 SSc patients were analyzed during a followup period of 0.6–6.3 years. RESULTS: SP-D and KL-6 concentrations at the first visit were higher in patients with SSc, especially those with PF, compared with healthy controls. Increased concentrations of SP-D were associated with decreased DLCO and decreased vital capacity in SSc patients more strongly than those of KL-6. The sensitivity and specificity for PF were 91% and 88% for SP-D and 39% and 100% for KL-6, respectively. In the longitudinal study, both SP-D and KL-6 concentrations were associated with activity of PF in patients with SSc. SP-D concentrations changed more rapidly than KL-6 concentrations, in parallel with the PF activity. CONCLUSION: SP-D was a more sensitive marker for PF than KL-6. By contrast, KL-6 showed higher specificity than SP-D. Combined use of these 2 serum markers would be more helpful to diagnose and monitor the PF activity in patients with SSc than single use of each marker.

Oesophageal complications are common in systemic sclerosis (SSc). However, the ability to determine the severity of oesophageal complications according to SSc type and skin lesion has not been evaluated.

OBJECTIVE: B cell activation, fibrosis, and expression of adhesion molecules on endothelial cells are regulated by soluble CD40L (sCD40L)/CD40 interactions. Since these effects are characteristic in patients with systemic sclerosis (SSc), serum concentrations of sCD40L were determined in patients with SSc. METHODS: Fifty-two Japanese patients with SSc were examined. They were grouped into 24 patients with limited cutaneous SSc (lSSc) and 28 with diffuse cutaneous SSc (dSSc). Serum sCD40L levels were examined by ELISA. As a disease control, serum samples from 20 patients with systemic lupus erythematosus (SLE) were also examined. In addition, a retrospective longitudinal study was performed in 71 serum samples from 18 patients with SSc. RESULTS: Serum sCD40L levels were elevated in SSc patients compared with healthy controls (p < 0.001). Levels of sCD40L in patients with SSc were higher than in patients with SLE (p < 0.001) that had elevated sCD40L levels compared with healthy controls. Among SSc subsets, there were no differences in sCD40L levels between lSSc and dSSc. sCD40L levels correlated positively with C-reactive protein levels in SSc patients (p < 0.0001, r = 0.449). In a cross-sectional study and a longitudinal study, serum sCD40L levels in dSSc patients were persistently elevated, although those in lSSc patients were temporarily elevated at the early phase of the disease process. CONCLUSION: Patients with SSc exhibited elevated sCD40L levels that may correlate with disease activity. These results suggest that CD40/CD40L interactions may be potential therapeutic targets in SSc.

A missense mutant of Smad2, Smad2D450E, that was not phosphorylated by transforming growth factor β (TGF‐β) signaling, was identified in colorectal cancer. Previously, we constructed a mutant Smad3, Smad3D407E, which has an Asp to Glu mutation in the corresponding position of Smad2D450. Smad3D407E was not phosphorylated by the constitutively active form of the TGF‐β type I receptor, and inhibited the phosphorylation of co‐expressed wild‐type Smad2 and Smad3. In the present study, we examined the inhibitory effects of Smad2D450E on TGF‐β signaling and found that there are considerable differences between Smad2D450E and Smad3D407E. Smad2D450E suppressed the phosphorylation of Smad2, but did not affect the phosphorylation of Smad3, while Smad3D407E blocked the phosphorylation of both Smad2 and Smad3. Consistent with these results, Smad2D450E reduced hetero‐oligomer formation of Smad2 with Smad4, but not of Smad3 with Smad4, while Smad3D407E reduced hetero‐oligomer formation of both Smad2 and Smad3 with Smad4. However, Smad2D450E reduced the binding of Smad3 to a target DNA element as well as Smad2‐binding, and Smad2D450E had inhibitory effects on the transcriptional activity of several targets, as Smad3D407E did. These results suggested that Smad2D450E might block the Smad3‐mediated TGF‐β signaling in a hitherto unknown manner after the phosphorylation and hetero‐oligomer formation, such as in the process of nuclear translocation or transcriptional regulation, which we could not identify previously by using Smad3D407E. (Cancer Sci 2004; 95: 12–17)

We have recently reported that S1P (sphingosine-1-phosphate) differentially regulates cellular Rac activity and cell migration in either a positive or a negative direction via distinct G-protein-coupled receptor subtypes, i.e. S1P1/Edg1 (endothelial differentiation gene) and S1P2/Edg5 respectively, when each of the S1P receptor subtypes is expressed in CHO (Chinese-hamster ovary) cells. In B16F10 mouse melanoma cells, in which S1P2, but not the other S1P receptor subtypes, is endogenously expressed, S1P inhibited cell migration with concomitant inhibition of Rac and stimulation of RhoA in dose-dependent manners. Overexpression of S1P2 in the melanoma cells resulted in potentiation of S1P inhibition of both Rac and cell migration. In contrast, overexpression of S1P1 led to stimulation of cell migration, particularly at the lower S1P concentrations. Treatment of B16F10 cells with S1P inhibited lung metastasis 3 weeks after injection into mouse tail veins. Intriguingly, overexpression of S1P2 greatly potentiated the inhibition of metastasis by S1P, whereas that of S1P1 resulted in aggravation of metastasis. Suppression of cellular Rac activity by adenovirus-transduced expression of N17Rac, but not N19RhoA, strongly inhibited cell migration in vitro and lung metastasis in vivo. These results provide the first evidence that G-protein-coupled receptors could participate in the regulation of metastasis, in which ligand-dependent, subtype-specific regulation of the cellular Rac activity is probably critically involved as a mechanism.

Studies have suggested that psoriasis vulgaris is mediated by type 1 T cells. In this study, we examined both chemokine receptor expression and intracellular cytokine production by circulating T cells to check the type 1/type 2 balance in psoriasis. CCR4+and CXCR3+T cells predominantly produced interleukin-4 and interferon-γ, respectively. The frequency of interferon-γ-producing cells and that of CXCR3+cells in circulating CD4+T cells were similar for psoriatic patients and healthy control subjects. By contrast, the frequency of CCR4+CD8+T cells and CCR4/CXCR3 ratio in circulating CD8+T cells were significantly higher in psoriatic patients than in healthy control subjects. Analysis of intracellular cytokine production also indicated relative increase of type 2 CD8+T (Tc2) cells in peripheral blood from psoriatic patients. The frequency of circulating Tc2 cells directly correlated with Psoriasis Area and Severity Index. Immunohistochemical analysis showed that not only CXCR3+CD8+T cells but also a similar number of CCR4+CD8+T cells infiltrated the psoriatic epidermis and dermis. Our findings suggest an increase in Tc2 cell number in blood from psoriatic patients, and the association of Tc2 cells with inflammation in psoriasis.

OBJECTIVE: To determine whether serum concentrations of 2 CXC chemokines, interleukin 8 (IL-8) and growth-regulated oncogene-a (GRO-a), which are potent chemoattractants and activators for neutrophils, are elevated and whether they correlate with clinical features in patients with systemic sclerosis (SSc). METHODS: Serum samples from patients with diffuse cutaneous SSc (dSSc; n = 36), limited cutaneous SSc (lSSc; n = 42), systemic lupus erythematosus (SLE; n = 15), dermatomyositis (DM; n = 15), and healthy controls (n = 35) were examined by ELISA. RESULTS: Serum IL-8 was detected significantly more frequently in patients with dSSc (61%) and lSSc (55%) relative to healthy controls (6%), patients with SLE (7%), and those with DM (13%). Similarly, serum GRO-a concentrations in SSc patients were significantly increased compared with controls, patients with SLE, or those with DM. Elevated IL-8 concentrations significantly correlated with decreased % DLCO and rheumatoid factor positivity, while increased GRO-a levels were significantly associated with decreased % DLCO and % vital capacity, involvement of kidney and muscle, the presence of anti-topoisomerase I antibody, and increased serum IgG levels. CONCLUSION: Our results suggest that the elevation of serum levels of the CXC chemokines IL-8 and GRO-a is specific to SSc and that the elevation of CXC chemokines, particularly GRO-a, correlates with the involvement of internal organs, especially pulmonary damage.

OBJECTIVE: To assess whether the functional disability in Japanese patients with systemic sclerosis (SSc) can be adequately evaluated by the Health Assessment Questionnaire (HAQ) developed in the United States. METHODS: The HAQ was completed by 121 Japanese patients with SSc, in whom SSc-specific physical examinations and laboratory tests were performed at the same time. Clinical findings associated with the disability index (DI) and individual components of the HAQ were examined using Student's t tests and Pearson's correlation tests. Logistic regression analysis was used to identify clinical findings that independently contributed to the increase in the HAQ-DI score. RESULTS: Japanese patients with SSc had significant functional disability, especially in the categories of eating and gripping, but the degree of disability was much less than was reported in previous studies carried out in the US. The increase in the HAQ-DI score was strongly correlated with increased total skin score, reduced oral aperture, reduced hand extension, increased finger flexion, subcutaneous calcinosis, flexion contractures, increased erythrocyte sedimentation rates, decreased percent vital capacity, and vascular involvement (p < 0.001 for all correlations). Multivariate logistic regression analysis showed that hand extension was the most important and an independent correlate of the HAQ-DI. CONCLUSION: Our multicenter, cross-sectional study has demonstrated that the self-administered HAQ is a valuable assessment tool of functional disability in Japanese SSc patients, who have social customs different from Americans, but functional disability measured by the HAQ is potentially influenced by ethnic variability.

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-α 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.

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)-β1 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-β1. This suggests that these adhesion molecules are potential therapeutic targets for inhibiting human pulmonary fibrosis.

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor-βs (TGF-βs) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proα2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the −17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial β-galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF-β1, -β2, -β3, basic fibroblast growth factor, platelet-derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF-β exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF-β1 and -β2 significantly increased the number of fibroblasts which were positive for X-Gal staining, TGF-β3 treatment did not change the number of β-galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF-β1 and those with TGF-β3. These findings suggest that TGF-β1 and -β3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other. J. Cell. Physiol. 190: 375–381, 2002. © 2002 Wiley-Liss, Inc.