Hiroaki Ueki

Effects of Histamine on Collagen Synthesis by Cultured Fibroblasts Derived from Guinea Pig Skin

SummaryFibroblast-like cells derived from guinea-pig skin were cultured for 3 h in the presence of various concentrations of histamine. The total protein synthesized was determined by the incorporation of radioactive proline, and the collagenous protein synthesized was measured by the incorporation of labeled hydroxyproline in the cell layer and medium. Synthesis of both total and collagenous protein increased in the presence of histamine in the concentration range of 101–103 μg/ml. The ratio of collagen to total protein synthesized also increased at these concentrations. However, in no case was an increase found when H1 antagonist (chlorpheniramine) and H2 antagonist (cimetidine) were added with the histamine. DNA synthesis was not affected by histamine at the concentrations used. These results suggest that histamine increases the synthesis of collagen by fibroblast-like cells through H1 and H2-receptors.

A novel autoantibody reactive with carbonic anhydrase in sera from patients with systemic lupus erythematosus and Sjögren's syndrome.

Carbonic anhydrase (CA) is an extremely basic zinc metalloenzyme with a wide phyletic distribution, and the enzyme is important for the regulation of acid-base status. A novel autoantibody reactive with carbonic anhydrase was demonstrated. Several different classes of CA are known in mammals. Using the immuno blotting method and and immun-dot analysis, we found this autoantibody to be reactive with CA in the sera from patients with Sjögrens syndrome (20.8%), including a patient with Sjögrens syndrome and renal tubular acidosis, and in patients with systemic lupus erythematosus (31.6%). The autoantibody varied in the extent of its cross-reactivity among human CA I (or B), human CA II (or C), bovine CA I, bovine CA II, rabbit CA, and dog CA. The titers continued to float and tended to parallel disease activity. Positive reactivity of autoantibody was observed on eccrine sweat glands and the distal tubules of the kidney by the indirect immunofluorescent method.

Over‐expression of the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) derived from silicosis patients

Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, is considered to be involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Recently, a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds FasL and inhibits FasL‐induced apoptosis, has been identified. Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities. We have found that serum soluble Fas (sFas) levels are elevated in silicosis patients and that sFas message is dominantly expressed in PBMC derived from these patients. This study examined DcR3 gene expression in PBMC derived from patients with silicosis, SLE, or progressive systemic sclerosis (PSS), and compared it with that in healthy volunteers (HV). The relative expression level of the DcR3 gene was examined in PBMC derived from 37 patients with silicosis without clinical symptoms of autoimmune disease, nine patients with SLE, 12 patients with PSS, and 28 HV using the semiquantitative multiplex‐reverse transcriptase‐polymerase chain reaction (MP‐RT‐PCR). The correlation between the relative expression level of the DcR3 gene and multiple clinical parameters for respiratory disorders and immunological abnormalities in individuals with silicosis was analysed. The DcR3 gene was significantly over‐expressed in cases of silicosis or SLE when compared with HV. In addition, the DcR3 relative expression level was positively correlated with the serum sFas level in silicosis patients. It is unclear, however, whether over‐expression of the DcR3 gene in silicosis is caused by chronic silica exposure, merely accompanies the alteration in Fas‐related molecules, or precedes the clinical onset of autoimmune abnormalities. It will be necessary to study these patients further, establish an in vitro model of human T cells exposed recurrently to silica compounds, and resolve whether the increase in DcR3 mRNA expression is a cause or consequence of disease.

Epidemiological Analysis of Prognosis of 496 Japanese Patients with Progressive Systemic Sclerosis (SSc)

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.

SOLUBLE FAS MRNA IS DOMINANTLY EXPRESSED IN CASES WITH SILICOSIS

Although it is well known that cases with silicosis exhibit various immunological abnormalities, the mechanisms involved in the occurrence of immuno‐dysfunction or dysregulation induced by silica compounds have not yet been determined. Fas is a well‐known cell surface molecule that is involved in the apoptosis pathway that belongs to the tumour necrosis factor‐receptor family. Soluble Fas (sFas) is produced as an alternatively spliced product of the Fas gene and protects cells from apoptosis due to antagonization of the binding between membrane form of the Fas gene (mFas) and the Fas ligand. To determine the role of the Fas/Fas ligand system in silica‐induced immunological abnormalities, we investigated Fas and Fas‐ligand message expression levels using the multiplex reverse transcription–polymerase chain reaction (RT–PCR) method with peripheral blood mononuclear cells from silicosis cases with no clinical symptoms of autoimmune diseases. Although the relative expression levels of the Fas or Fas‐ligand genes were not remarkably altered in these cases, we observed the sFas message was dominantly expressed compared with mFas expression. These results suggest that self‐recognizing clones in cases with silicosis survive for decades, escaping the exclusion mechanisms induced by apoptosis. Then they cause the appearance of autoantibodies and the acquisition of autoimmune diseases sequentially.

Elevated soluble Fas/APO- 1 (CD95) levels in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours

Soluble Fas (sFas) is produced as translation products of alternative mRNA splicing, and antagonizes the membranous Fas molecule in Fas/Fas ligand interactions. We investigated the serum sFas levels in 64 Japanese silicosis patients with no clinical symptoms of autoimmune diseases or malignant tumours, using ELISA for sFas. The serum sFas levels in the silicosis patients were significantly higher than those in healthy volunteers. Elevated serum sFas levels were also detected in patients with systemic lupus erythematosus but, unexpectedly, no difference was observed in sFas levels between progressive systemic sclerosis patients and healthy volunteers. On the other hand, there was no significant difference in the expression of Fas on peripheral blood lymphocytes between the patients with silicosis and age‐matched healthy volunteers. These observations provided the first evidence that serum sFas levels are elevated in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours. It remains to be clarified whether patients with elevated sFas levels have a tendency to develop autoimmune diseases later, or whether some other distinct factor(s) is necessary to initiate the progression of autoimmune diseases.

Intramolecular epitope spreading among anti-caspase-8 autoantibodies in patients with silicosis, systemic sclerosis and systemic lupus erythematosus, as well as in healthy individuals

Dysregulation of apoptosis through the Fas‐Fas ligand pathway is relevant in autoimmune disease onset. We recently reported elevated serum levels of sFas in patients with silicosis, systemic sclerosis (SSC) and systemic lupus erythematosus (SLE), and proposed a block of apoptosis in the pathogenesis. The disturbance of apoptosis in lymphocytes including autoreactive clones could induce autoantibody production. Since autoantibodies directed against unknown antigens are present in the sera of these patients, the sera samples were examined for the presence of autoantibodies directed to caspase‐8.

Serum levels of soluble Fas ligand in patients with silicosis.

Certain patients with silicosis have been reported to exhibit immunological abnormalities such as the appearance of antinuclear antibodies and the occurrence of autoimmune diseases. Fas ligand (FasL) is a type II membrane protein which induces apoptosis by binding to its membrane receptor, Fas. FasL is converted to a soluble form by a metalloproteinase‐like enzyme. We have already found serum soluble Fas (sFas) levels in silicosis patients as well as in patients with systemic lupus erythematosus (SLE) to be significantly higher than those in healthy volunteers. To examine further the role of the Fas/FasL system in silica‐induced immunological abnormalities, we investigated serum soluble FasL (sFasL) levels in silicosis patients with no clinical symptoms of autoimmune diseases, using ELISA for sFasL. Although the serum sFasL levels in patients with SLE were significantly higher than those in healthy volunteers and showed a slight positive correlation with serum sFas levels, those in silicosis patients exhibited no significant difference from those in healthy volunteers, and there was no correlation with serum sFas levels. However, sFasL levels were elevated in silicosis patients with slight dyspnoea or normal PCO2 among various clinical parameters of silicosis. It may be speculated that the immunological disturbances presented by the abnormalities of apoptosis‐related molecules in silicosis patients do not occur with a similar degree of respiratory involvement. Further studies are required to clarify which kinds of factors are involved in silicosis patients who exhibit immunological abnormalities.

Reduced collagenase gene expression in fibroblasts from hypertrophic scar tissue.

Summary The major histopathological feature of hypertrophic scar lesions is fibrosis. characterized by excessive accumulation of collagen. The purpose of this study was to determine if there is not only increased expression of collagen but also decreased expression of collagenase in hypertrophic scar fibroblasts. We compared the expression of mRNA for of α1 (I) and α1 (III) collagen, and collagenase in cultured fibroblasis from different portions of hypertrophic scars and normal dermis. the hypertrophic scar fibroblasts. increased levels of α1 (I) and α1 (III) collagen mRNAs were observed in fibroblasts from the edge and outside of scar tissue, while normal levels were noted in fibroblasts from the centre of this tissue. In contrast. decreased levels of collagenase mRNA were found in the hypertrophic scar fibroblasts. The reductions were centre (25% of the control) greater than the edge (43% of the control) greater than the outside (84% of the control). The changes in the collagenase mRNA levels of the hypertrophic scar fibroblasts correlated well with decreased collagenolytic activity as determined by the degradation rate of fluorescent isothiocyanate‐labelled type I collagen in fibroblast culture supernatant. These results suggest that decreased expression of collagenase in hypertrophic scar fibroblasts may be one possible cause for the excessive accumulation of collagen in the skin lesions of hypertrophic scars.

T‐cell subsets in lesions of systemic and discoid lupus erythematosus

In 6 patients with untreated systemic lupus erythematosus (SLE) in the progressive stage, and in 6 with discoid lupus erythematosus (OLE), an analysis of inflammatory infiltrates was performed in situ using the avidin‐biotin‐peroxidase complex (ABC) method with monoclonal antibodies. In all patients, over 75% of the infiltrates reacted with the pan T‐cell antibody OKT3, but only sporadically with that of B‐cell OKB7. In addition, a large number of the infiltrates were OKT8‐positive, indicating that they were in an activated state. Many OKT8‐positive cells were seen infiltrating the epidermis especially in the vicinity of basal keratinocytes. Staining for T‐cell subsets revealed that the proportion of OKT8‐positive cells (suppressor/ cytotoxic) was from 2 to 3 fold higher than that of OKT4‐positive cells (helper/inducer) in lesions of SLE. On the contrary, in OLE, a predominance of OKT4‐pos‐itive cells (the OKT4/OKT8 ratio was from 1:1 to 3:1) was observed. Thus, our results provide further evidence that these 2 main types of LE show quite contrary findings on immunohistochemical analysis of T‐cell subsets, and that besides the humoral immune mechanism, the cell‐mediated immune mechanism may be involved in the pathogenesis of these disorders.