Search

Hepatic stellate cells (HSC) are the main producers of type I collagen in fibrotic liver, and transforming growth factor-β (TGF-β) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of α2(I) collagen gene (COL1A2) that is essential for its basal and TGF-β-stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF-β-responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF-β signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor-1 (PAI-1) genes and low response to TGF-β in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated from the cytoplasm to the nucleus upon TGF-β treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation. Increased COL1A2 and PAI-1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether, the results correlate abnormality in TGF-β/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC. © 2001 Wiley-Liss, Inc.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily, which regulate the differentiation of osteoprogenitor cells. Here we show that among members of the BMP family, BMP-4 and growth/differentiation factor 5 (GDF-5) induce osteoblast differentiation through the activation of three receptor- regulated Smads (i.e. Smad1, Smad5 and Smad8). By contrast, BMP-6 and BMP-7 induce alkaline phosphatase activity through Smad1 and Smad5, but not through Smad8. Consistent with these findings, BMP-4 induced phosphorylation and nuclear translocation of Smad1, Smad5 and Smad8, but BMP-6 activated only Smad1 and Smad5. BMP-4 and GDF-5 are known to bind to activin receptor-like kinase 3 (ALK-3) and/or ALK-6 (also termed BMP type IA and type IB receptors, respectively), whereas BMP-6 and BMP-7 preferentially bind to ALK-2. Compared with the effects induced by only one of the type I receptors, the combination of constitutively active forms of ALK-2 and ALK-3 (or ALK-6) more strongly induced alkaline phosphatase activity in C2C12 cells. Moreover, addition of BMP-4 and BMP-6 to C2C12 cells resulted in higher alkaline phosphatase activity than that of only one of these BMPs. The combination of ALK-2 and ALK-3 also induced higher transcriptional activity than either receptor alone. Thus, ALK-2 and ALK-3 (or ALK-6) might synergistically induce osteoblast differentiation of C2C12 cells, possibly through efficient activation of downstream signaling pathways.

Background:Recent studies have reported that TH1 and TH2 cells express CXCR3 and CCR4, respectively. Objective:Our goal was to assess the association of CCR4 and CXCR3 expression with TH2 and TH1 cells and association of CCR4 and CXCR3 expression with inflammation in patients with atopic dermatitis (AD). Methods:Intracellular cytokine production and chemokine receptor expression in blood T cells were examined by flow cytometry. Immunohistochemical expression of chemokine receptors was also investigated in chronically lesional skin. Results:CCR4+and CXCR3+CD4+T cells predominantly produced IL-4 and IFN-γ, respectively. Although the frequency of CXCR3+cells among CD4+CD45RO+T cells was similar for patients with AD (n = 29) and healthy control subjects (n = 19), patients with severe AD (n = 14) had a reduced frequency of CXCR3+cells. In contrast, the frequency of CCR4+cells and the CCR4/CXCR3 ratio were higher in patients with AD (n = 22) than healthy control subjects (n = 16) and correlated with disease severity of AD. The frequency of CCR4+cells correlated positively with eosinophil numbers and serum IgE levels, whereas the frequency of CXCR3+cells correlated inversely with eosinophil numbers. The frequency of CCR4+or CXCR3+cells was similar in patients with psoriasis (n = 6) and healthy control subjects. Immunohistochemical analysis showed that the frequency of CCR4+cells among CD4+T cells in chronically lesional skin of patients with AD (n = 9) was higher than that of patients with psoriasis (n = 4). Conclusion:Our data suggest the association of CCR4 expression with TH2 cells, the predominance of CCR4+cells in blood from patients with AD, and an important role of CCR4 in the migration of TH2 cells from blood into AD lesional skin. (J Allergy Clin Immunol 2001;107:353-8.)

Inflammatory cells play a crucial role in wound healing, but the role of adhesion molecules including L-selectin and intercellular adhesion molecule-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisional wounds in mice lacking L-selectin, ICAM-1, or both. The loss of ICAM-1 inhibited wound healing, keratinocyte migration from the edges of the wound toward the center, and granulation tissue formation. By contrast, L-selectin deficiency alone did not affect any of these parameters. However, the loss of both L-selectin and ICAM-1 resulted in inhibition of keratinocyte migration and granulation tissue formation beyond those caused by loss of ICAM-1 alone. Treatment of platelet-derived growth factor to the wounds normalized delayed wound healing in ICAM-1−/−mice, but not in L-selectin/ICAM-1−/−mice. Therefore, although ICAM-1 contributes to wound repair to a greater extent than L-selectin, a role for L-selectin was revealed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiltration of neutrophils and macrophages in ICAM-1−/−and L-selectin/ICAM-1−/−mice. These results demonstrate a distinct role of ICAM-1 and L-selectin in wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site.

Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. We have isolated the mouse Smad6promoter and identified the regions responsible for transcriptional activation by BMPs. The proximal BMP-responsive element (PBE) in the Smad6promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for DrosophilaMad and Medea. We generated a luciferase reporter construct (3GC2-Lux) containing three repeats of the GC-rich sequence derived from the PBE. BMPs and BMP receptors induced transcriptional activation of 3GC2-Lux in various cell types, and this activation was enhanced by cotransfection of BMP-responsive Smads, i.e.Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE was observed. These results indicate that the expression of Smad6is regulated by the effects of BMP-activated Smad1/5 on the Smad6promoter.

The major obstacle preventing effective treatment of melanoma is the biological heterogeneity of tumor cells. This study was performed to determine clonal genetic heterogeneity within primary melanoma and the evolution of these heterogeneous sub-clones during disease progression. DNA samples were obtained from 44 morphologically distinct areas identified within 10 primary tumors and from 15 metastases in the same patients. Loss of heterozygosity (LOH) analyses were performed using 17 microsatellite markers that mapped to chromosomes 6q, 9p, 10q and 18q, the most frequently deleted in melanoma. Of 10 primary tumors, 8 were revealed to have intratumoral genetic heterogeneity in terms of LOH of the 4 chromosome arms examined, 7 containing at least 2 different sub-clones harboring LOH of different chromosome areas, while the remaining one tumor showed prominent intratumoral genetic heterogeneity consisting of at least 6 genetically distinct sub-clones. LOH of 6q was detected only in a sub-set of multiple microdissected samples in most of the primary tumors, but was most frequently detected in metastases, suggesting that loss of this chromosome arm occurred late and played an important part in metastatic progression. Comparison of LOH between sub-clones within primary tumors and within metastases showed the divergence of metastatic clones from dominant populations within the primary tumor in 5 patients, whereas in the remaining three patients parent sub-clones were not identified, or constituted only a minor sub-population within the primary tumors. These results, showing considerable genetic heterogeneity in sporadic melanoma, have profound implications for the choice of future therapeutic strategies. Int. J. Cancer 85:492–497, 2000. © 2000 Wiley-Liss, Inc.

For the first time, we performed an epidemiological study of SSc in Japan to study the factors influencing prognosis, survival rate and cause of death of Japanese SSc patients and to compare our data with those from foreign countries.

BACKGROUND AND DESIGN: Recently, we detected antihistone antibodies (AHAs) in patients with localized scleroderma. However, the exact antigen specificity of AHAs in this disease is still unknown. Therefore, we determined the reactivity of AHAs with five individual histones and the correlation of AHAs with rheumatoid factor in localized scleroderma by means of enzyme-linked immunosorbent assay. Twenty patients with localized scleroderma who had IgG and/or IgM AHAs, as determined by enzyme-linked immunosorbent assay, were examined. These patients were classified into the following three subgroups: patients with generalized morphea (n=11), patients with linear scleroderma (n=6), and patients with morphea (n=3). RESULTS: In generalized morphea, IgG AHAs strongly reacted with histones H1, H2A, and H2B; and IgM AHAs strongly reacted with HI and H2B, as determined by means of enzyme-linked immunosorbent assay. The pattern of reactivity in linear scleroderma and morphea was similar to that in generalized morphea. A homogeneous immunofluorescent pattern on HEp-2 cells, which was produced by localized scleroderma sera, was completely abolished by absorption with total histones. By employing a latex agglutination test, IgM rheumatoid factor was detected in 60% of the 20 patients with localized scleroderma and at a frequency of 82% in those with generalized morphea. However, an absorption test of rheumatoid factor activity with human IgG revealed no cross-reactivity of AHAs with rheumatoid factor. CONCLUSIONS: Our data suggest that AHAs in localized scleroderma are directed against native chromatin, since H1, H2A, and H2B occupy a relatively exposed portion of chromatin.(Arch Dermatol. 1994;130:1273-1277)

BACKGROUND AND DESIGN: Recently, we reported that the serum procollagen type I carboxyterminal propeptide (P1CP) level of patients with systemic sclerosis was elevated and correlated with disease severity. In this study, the serum level of P1CP was measured using the enzymelinked immunosorbent assay in 39 patients with localized scleroderma and in 30 control subjects. RESULTS: The mean P1CP level in the patients was significantly higher than that in the normal control subjects. In 30% of the patients with localized scleroderma, the serum P1CP level was considered to be elevated (>305 ng/mL; ie, 2 SDs above the mean control value). The mean serum P1CP level in the patients with generalized morphea was significantly higher than in the pa- tients with morphea or linear scleroderma. In addition, the serum P1CP level in the patients with localized scleroderma was correlated with the number of sclerotic lesions, and it was negatively correlated with the duration of the disease. Anti—single-stranded DNA antibody and antihistone antibody were detected significantly more frequently in the patients with elevated P1CP than in the patients with normal P1CP. CONCLUSION: These findings suggest that the serum P1CP level is a useful indicator of disease severity in patients with localized scleroderma, as it has been found to be in those with systemic sclerosis.(Arch Dermatol. 1994;130:1269-1272)

Skin fragments of a giant molluscum pendulum and so‐called adenomata sebacea (AS) in the face of a 21‐year‐old female with Pringle's disease were explanted and the cell divisions of outgrown and subcultured non‐epithelial cells (NECs) were observed at suitable intervals with a phase contrast microscope. In both tissues, a number of NECs, especially large and medium sized ones, showed distinct chromosome dysarrangements, including segmental arrangement, eccentric arrangement, and arrangement failure, when chromosomes were arranged in the central area of individual cells in metaphase, causing extreme lagging in the subsequent cell division.

Explant cultures were prepared using normal‐looking skin and the so‐called adenoma sebaceum (AS) from the face of a 42‐year‐old male with tuberous sclerosis, Pringle's disease (PD), and the DNA content of the subcultured non‐epithelial cells (NECs), stroma cells, were investigated per nucleus and per cell by a flow cytofluorometer. The DNA histogram per nucleus of the NECs from the normal‐looking skin of the patient did not reveal any remarkable differences from that of the normal control group.In contrast, the DNA histograms per nucleus of cells from AS in the patient showed distinct and different patterns in comparison with those of the normal control group, including an increase in width and decrease in height of 2C peak, vanishing of 4C peak, and formation of a new small peak at the left end of the histogram.On the other hand, the DNA histogram per cell of the NECs from the normal‐looking skin of the patient displayed a slightly different pattern from that per nucleus, with a distinct increase in width and a faint indentation.Furthermore, the DNA histograms per cell of the NECs from AS exhibited drastic changes such as remarkable flattening and increase in the width of the 2C peak with a newly formed small peak at the left end of this histogram. However, these changes also varied according to the population density of large dendritic cells in the culture dish, even though these NECs were derived from the same AS tissue.The above‐mentioned findings suggest that the amount of DNA per nucleus and probably per cell might not be equal among atypical NECs from AS in the patient even in the same G0•G1phase.

Explant cultures were attempted from the so‐called adenoma sebaceum in the face and a plaque lesion on the neck observed in three genetically related cases (three males twelve, fifteen, and 42 years old) with Pringle's disease, tuberous sclerosis. Then the DNA content of outgrown non‐epithelial cells (NEC), or stroma cells, derived from the respective tissues (10,000 nuclei and 15,000 cells) were investigated by a flow cytofluorometer.

Cell adhesion to extracellular matrix proteins is a dynamic process leading to dramatic changes in the cell phenotype. Integrins are one of the major receptor families that mediate cell-matrix contact. Evidence that integrins can act as signal transducing molecules has accumulated over the past few years. We report here that p44erk-1and p42erk-2mitogen-activated protein (MAP) kinases are rapidly phosphorylated on tyrosine residues upon adhesion of human skin fibroblasts to fibronectin or upon cross-linking of β1 integrins with antibody. The tyrosine phosphorylation of both kinases is associated with increased enzymatic activity. Pretreatment of the cells with cytochalasin D, which selectively disrupts the network of the actin filaments, completely inhibits this adhesion-mediated MAP kinase activation. Thus, our findings indicate that ligation of β1 integrins induces an increase in both tyrosine phosphorylation and enzymatic activity of p44erk-1and p42erk-2MAP kinases, and that the integrity of the actin cytoskeleton is essential in this process. Since MAP kinase behaves as a convergence point for diverse receptor-initiated signaling events at the plasma membrane, this serine/threonine kinase plays a key role and helps to account for the diversity of integrin-dependent cell functions.

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.

Localized scleroderma is generally considered to be limited to the skin and to the subcutaneous tissue beneath the cutaneous lesions. Previous studies have revealed that this disease is accompanied by various immunologic abnormalities. Recently, we discovered that the major antigens of antinuclear antibodies (ANA) detected in localized scleroderma are nuclear histones.1 In addition, we found that the serum levels of interleukin (IL) 2, IL-4, IL-6, and soluble IL-2 receptor were significantly elevated in patients with localized scleroderma, which suggests that T-cell activation is also involved in its pathogenesis.2Interleukin 8 is known to be chemotactic for neutrophilic granulocytes and T lymphocytes and is produced by a variety of cell types, such as epithelial cells, fibroblasts (including dermal fibroblasts), and endothelial cells.3,4Recently Reitamo et al5 reported that IL-8 was detected in 21 (19%) of 108 serum samples of patients with systemic sclerosis (SSc) but not in

TO THE EDITOR.— Multiple sclerosis is a chronic demyelinating disease that affects the central nervous system. The characteristic pathologic feature is the breakdown of the myelin sheath, with relative sparing of axons. Recently, an immunologic disorder has been found, and it has been said that an autoimmune basis may be related to the pathogenesis of the disease. In this study, we report a case of systemic sclerosis associated with multiple sclerosis. REPORT OF A CASE.— A 42-year-old woman was healthy until 1974, when she developed dysesthesia in the left leg. Paraplegia in both legs and gait disturbance appeared in 1975, and paropsis and dysuria were noted in 1976. Results of a physical examination revealed reduced light reflex of the left eye, decreased sensation below the C8 level, and muscle weakness in her left leg. Hyperactivity of the deep tendon reflexes and Babinski's sign were detected. Multiple sclerosis was diagnosed and