Inhibition of type I collagen production by dermal fibroblasts upon contact with activated T cells: different sensitivity to inhibition between systemic sclerosis and control fibroblasts

OBJECTIVE: To assess the role of T lymphocyte-fibroblast contact in type I collagen production by cultured dermal fibroblasts from normal individuals and from patients with diffuse systemic sclerosis (SSc). METHODS: Cell membranes were prepared from activated CD4+ and CD8+ T cells, or type 1 T helper (Th1) clones, and added to confluent fibroblast monolayers. Type I collagen production was measured in culture supernatants, and messenger RNA (mRNA) levels of type I procollagen alpha1 (pro alpha1[I]) and matrix metalloproteinase 1 (MMP-1) were evaluated by Northern hybridization analysis. RESULTS: Dose-dependent inhibition of type I collagen production was observed with CD4+ and CD8+ T cells from both SSc patients and controls. Inhibition of type I collagen was significantly less pronounced in fibroblasts from SSc patients than in fibroblasts from controls (P < 0.02). Inhibition was not reversed by the addition of exogenous transforming growth factor beta, interleukin-4, interleukin-1 receptor antagonist, anti-tumor necrosis factor, anti-CD40, or indomethacin, whereas anti-interferon-gamma (IFNgamma) reversed Th1-mediated inhibition. This inhibitory activity was specific for type I collagen, since mRNA levels of pro alpha1(I) were decreased, whereas mRNA levels of MMP-1 were strongly increased. CONCLUSION: The production of type I collagen by skin fibroblasts is specifically down-regulated by membranes from activated T cells. The contact-dependent regulatory activity exerted by T cells on fibroblasts depends, at least in part, on the presence of membrane-associated IFNgamma. However, SSc fibroblasts are more resistant to inhibition than are fibroblasts from normal individuals.     

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.     

Immunolabelling of collagen types I, III and IV, laminin and fibronectin in the human lens capsule

PURPOSE: To localize collagen types I, III, and IV, laminin and fibronectin in the anterior human lens capsule. MATERIAL AND METHODS: Twenty-one anterior capsules were sampled by capsulorhexis during extracapsular cataract extraction (mean age 71.5). All capsules were labelled by an immunostaining specific for each antibodies. Immunostaining of four capsules was revealed with immunoperoxydase and seventeen using indirect immunofluorescence. RESULTS: Labelling of collagen types I and III was observed throughout the entire thickness of the capsule for each technique, the strongest labelling was found in the base of the epithelial cells with immunofluorescence. Collagen type IV was observed at the base of the epithelial cells whichever technique was used. Laminin could be detected in the inner layer of the capsule, using immunoperoxydase or immunofluorescence. No specific labelling was found for fibronectin using the two techniques. CONCLUSIONS: Different kinds of collagens have been found in capsules, more particularly the type III. The latter does not appear on other ocular basement membrane. Because of this uneven distribution in the capsule's thickness, each collagen might have a specific function.     

Evidence for a structural mutation of procollagen type I in a patient with the Ehlers-Danlos syndrome type VII

We have found that the collagen from a patient with the Ehlers-Danlos syndrome type VII contained a polypeptide chain, pN alpha 2, not present in collagen prepared from normal tissue. Fibroblasts cultured from the patient's skin produced type I procollagen in which the NH2-terminal propeptide of pro alpha 2 was cleaved to about half of normal values by chick procollagen neutral protease which removes the NH2-terminal propeptides from procollagen (N-protease). The NH2-terminal propeptide on the pro alpha 2 chain of the patient's procollagen was also more resistant than procollagen from control fibroblasts to digestion by pepsin or alpha-chymotrypsin. assays for procollagen N-protease indicated that the patient's fibroblsts contained about the same level of enzymic activity as normal fibroblasts. These results suggest that the patient's fibroblasts synthesize both an abnormal pro alpha 2 chain and a normal pro alpha 2 chain. The abnormality probably consists of a structural mutation in or near the site at which procollagen N-protease cleaves the pro alpha 2 chain. The results presented here appear to provide the first example of a mutation in a structural gene for collagen. Since equal amounts of pN alpha 2 and alpha 2 are found in the protein in neutral salt extracts of the patient's tissue, as well as in newly synthesized collagen produced by cultured skin fibroblasts, and since both parents are phenotypically normal and express exclusively normal collagen chains, the patient is likely to be a sporadic heterozygote, arisen by new mutation, with one normal and one abnormal gene coding for pro alpha 2.     

Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress

PURPOSE: Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions. METHODS: The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry. RESULTS: In both species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM. CONCLUSIONS: Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.     

Analysis of forensic psychiatric opinions issued in Warsaw in 1968 during legal proceedings for deprivation of civil rights

The isolation and characterization of type III collagen from adult human lung parenchyma are described. The identity of this molecule as type III collagen has been established on the basis of (a) demonstration of intramolecular disulfide cross-links in the helical portion of the molecule, (b) amino acid analysis characteristic for type III collagen, and (c) composition and size of isolated cyanogen bromide peptides alpha1(III)-CB3, alpha1(III)-CB5, and alpha1(III)-CB8. The molecular weight of lung alpha1(III) was determined as 93 000 by Agarose chromatography, but its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels was slower than that of type I alpha chains which also have a molecular weight of 93 000.     

Purification and characterization of an allergy-induced melanogenic stimulating factor in brownish guinea pig skin

We have demonstrated recently that phenylazonaphthol (PAN) allergy-induced hyperpigmentation in brownish guinea pig skin is associated with the concomitant appearance of a melanogenic soluble factor(s) that activates the intracellular signal transduction system, including phosphatidylinositol turnover subsequent to ligand-receptor binding in cultured guinea pig melanocytes. In this study we have purified and characterized the PAN-induced melanogenic stimulating factor (PIMSF) that occurs in allergy-associated hyperpigmented skin. By successive column chromatography on TSK 2000SW, Mono Q, and octadecyl-NPR, the PIMSF was purified to homogeneity with a single band of apparent molecular mass of 7.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific bioactivity of PIMSF increased by 5,195-fold over the original skin homogenate. In cultured guinea pig melanocytes, this purified PIMSF had the potential of activating an intracellular signal transduction system such as inositol 1,4,5-trisphosphate formation and intracellular calcium levels through a pertussis toxin-sensitive G protein-coupled receptor. PIMSF consistently caused a rapid translocation of cytosolic protein kinase C (PKC) to membrane-bound PKC within 5 min of treatment with a return to the basal level after 120 min. The stimulating effects of PIMSF on proliferation and melanization of cultured guinea pig melanocytes were abolished completely by a PKC down-regulating agent (phorbol 12,13-dibutyrate). PIMSF was similar in molecular mass to rat growth-related oncogene alpha (GRO-alpha; molecular mass of 7.9 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had immunocross-reactivity with GRO-alpha upon Western immune blotting analysis. Further, the stimulatory effect of purified PIMSF on DNA synthesis of cultured guinea pig melanocytes was suppressed markedly by the addition of anti-rat GRO-alpha antibody, implying that the PIMSF is apparently identical to GRO-alpha. These findings suggest that PAN allergy provides a new mechanism of hyperpigmentation in which biological factors such as the GRO-alpha superfamily generated within allergy-induced skin stimulate melanocytes through activation of the PKC-related signal transduction pathway.     

Survey of echinococcosis in Hokkaido and measure against it

Exposure to hyperoxia results in lung injury and a decrease in lung collagen. Retinol is known to influence collagen gene expression, and retinol deficiency has been shown to potentiate hyperoxic lung injury. To investigate the combined effects of retinol deficiency and hyperoxia on lung collagen expression, retinol-deficient rats were exposed to acute hyperoxia, and expression of the alpha-1 chains of type I procollagen [pro alpha 1 (I)] and type III procollagen [pro alpha 1 (III)] were determined using Northern hybridization analyses and immunohistochemical staining. Hyperoxia alone reduced pro alpha 1 (I) mRNA by 60 +/- 4% (p < .05) and pro alpha 1 (III) mRNA by 30 +/- 5% (p < .05), and retinol deficiency alone reduced pro alpha 1 (I) mRNA abundance by 49 +/- 8.8% (p < .05) and pro alpha 1 (III) mRNA abundance by 14 +/- 7.5% (p = not significant), respectively. Retinol deficiency plus hyperoxia did not cause any further reduction in procollagen mRNA than that seen with oxygen exposure alone. Immunohistochemical staining demonstrated decreased staining for type I collagen in retinol-deficient animals. Hyperoxic exposure resulted in decreased connective tissue staining and increased alveolar wall staining for type I collagen. Retinol deficiency and hyperoxia together resulted in a marked increase in alveolar exudates staining for type I collagen. No changes in type III collagen staining were seen. These findings demonstrate that while retinol deficiency does not potentiate hyperoxia-induced reductions in procollagen mRNA, it is associated with alterations in collagen staining in distal lung and immunohistologic evidence of collagen fragments in alveolar exudates.     

Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium

PURPOSE: We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK). METHODS: The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy. RESULTS: Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon. CONCLUSIONS: The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.     

Effect of contact lens wear on the conjunctival mucous system

Biopsy specimens from the upper tarsal conjunctiva of 20 contact lens wearers with a clinically evident increase in mucus and ten non-lens wearing subjects were examined by light microscopy and scanning and transmission electron microscopy to determine the effect of contact lens wear on the mucous cell system(s). Three types of crypts associated with mucous secretion were found in all specimens: those with intracellular openings (type I, 0.1 to 0.2 mum) associated with non-goblet mucous secretory cells; those with small intercellular openings (type II, 1 to 2 mum) usually associated with goblet cells, and those with intraepithelial and intrastromal crypts with large intercellular openings (type III, 10 to 60 mum) lined with goblet and non-goblet mucous secretory cells. Contact lens wearers had increased numbers of non-goblet cells with mucous secretory vesicles lining the surface of the conjunctiva and the epithelial infoldings of type III crypts than did the normal subjects. We conclude that increased mucous secretion in contact lens wearers is associated with an increased number of cells and number of secretory vesicles involved in the non-goblet cell mucous system.     

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase. Purification, characterization, and further evidence for identity

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities were co-purified from the rat kidney supernatant fraction. The resulting preparation was determined to be nearly homogenous by analytical disc gel electrophoresis at pH 8.9 and 7.5 isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of approximately 85,000 was determined on Sephadex G-200 chromatography and sucrose density gradient analysis. The enzyme was determined to be comprised of two subunits of approximately the same molecular weight (45,500 +/- 850) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An isoelectric pH of 6.56 +/- 0.06 was determined by focusing on polyacrylamide gels. Further evidence is provided to support the idea that the alpha-aminoadipate aminotransferase and kynureine aminotransferase activities are properties of a single protein: (a) co-purification of the two activities from the rat kidney supernatant fraction with the ratio of their specific activities remaining constant, (b) similar chromatographic behavior, (c) a similarity in their dependence on added pyridoxal-P for activity, and (d) a similar pattern of heat inactivation.     

The effect of a preparation from the horns of the saiga antelope on the goal-directed drinking behavior of rats

We analyzed 79 consecutive patients with aneurysms and found a patient who lacked type III collagen. Collagen was extracted from the skin, and the lack of type III collagen was determined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only 1 of the 79 patients was found to lack type III collagen. This patient was a 55-year-old man who had sustained an abdominal aortic aneurysm and aortic dissection. He did not show any of the typical clinical symptoms of Ehlers-Danlos syndrome type IV, such as hyperextensible skin and joints. He had none of the usual risk factors nor any clear family history of the syndrome. Furthermore, his collagen fibrils demonstrated a homogeneous appearance. This case may represent a new form of type III collagen deficiency.     

Procollagen and collagen produced by normal bovine corneal stroma fibroblasts in cell culture

Procollagen and collagen were isolated from the culture medium of normal bovine corneal stromal fibroblasts. DEAE-cellulose chromatography was used to separate the collagen molecules from the different procollagens present. One collagen and four procollagen peaks were isolated and biochemically characterized. All the procollagen fractions and the collagen fraction yielded, after limited pepsin or chymotrypsin digestion followed by CNBr digestion, molecules that correspond to (alpha 1)2 alpha 2 exclusively. Thus only type I collagen is found in the growth medium of of bovine corneal stromal fibroblast cultures. Each of the individual procollagen peaks contained pro-alpha chains having molecular weights of 120,000, 150,000, 165,000, 180,000, and 190,000 daltons, according to their elution position on DEAE-cellulose. The presence of type I procollagen molecules having pro-alpha chains of 165,000, 180,000, and 190,000 daltons has not previously been reported and probably represents higher-molecular-weight precursor intermediates. The amino acid compositions of the different procollagen fractions are unique, and each contains relatively large amounts of cysteine and tryptophan. Carbohydrate analysis, cyanogen bromide peptide analysis, electron microscopy of SLS-crystallities, and SDS-polyacrylamide gel electrophoresis were used to further characterize the procollagen and collagen molecules.     

Line-shape analysis of speed-dependent collisional width inhomogeneities in CO broadened by Xe, N2, and He

The electrophoretic method of Sch?gger and von Jagow (Anal. Biochem. 199, 233-231 (1991) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10(6) fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.     

Ceramide, a mediator of interleukin 1, tumour necrosis factor alpha, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells

OBJECTIVES: To examine the effects of ceramide, which is a lipid second messenger of cell surface receptors, including tumour necrosis factor alpha (TNF alpha), interleukin 1 (IL1), and Fas receptors, on rheumatoid arthritis (RA) synovial cells. METHODS: Synovial cells from RA patients and normal skin fibroblasts were cultured with cell permeable ceramide (C2-ceramide). Apoptosis was assessed by microscopic observation of morphological changes, nuclear staining, and DNA electrophoresis. DNA synthesis was examined by thymidine incorporation. RESULTS: C2-ceramide induced reversible morphological changes of synovial cells such as cell rounding within four hours. Subsequently, irreversible nuclear changes characteristic to apoptosis were observed at 48 hours. DNA synthesis was not promoted. The addition of ceramide exerted similar effects on cultured dermal fibroblasts. CONCLUSION: Ceramide induced apoptosis in RA synovial cells. Ceramide could be a second messenger specific for apoptosis of RA synovial cells.     

The use of the radioimmunoassay in the characterization of antibodies to basement membrane collagen

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.     

Extracellular matrix of opacified anterior capsule after endocapsular cataract surgery

BACKGROUND: We determined the types and distribution of glycosaminoglycans (GAGs) and collagens, in anterior capsular opacification after endocapsular phacoemulsification and aspiration (ECPEA) and intraocular lens implantation. METHODS: Opacified anterior capsules were removed from human eyes after ECPEA. Immunohistochemical staining was performed to determine GAGs with monoclonal antibodies to chondroitin, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), and keratan sulfate (KS); collagens with monoclonal antibodies to types I, II, and III collagens; and cellular characteristics with monoclonal antibodies to vimentin, desmin, alpha-smooth muscle actin, and cytokeratin. Decorin mRNA and type I collagen mRNA were detected by in situ hybridization. RESULTS: In the capsules, the C6S, DS, KS, and types I and III collagens were similar to the chemical components found at the adhesion site of the anterior and posterior capsules after extracapsular cataract extraction, and cellular components contained vimentin, desmin, alpha-smooth muscle actin, cytokeratin, decorin mRNA, and type I collagen mRNA. CONCLUSIONS: The GAGs and collagens in opacified anterior capsule after ECPEA were similar to those found during wound healing, although KS is present in normal anterior segment tissue during development and only in the cornea postnatally. These chemical components may be produced by myofibroblast-like cells presumably transformed from lens epithelial cells.