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1.
Arthritis Rheum ; 43(2): 250-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10693863

ABSTRACT

OBJECTIVE: Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA. METHODS: Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA. RESULTS: Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3. CONCLUSION: TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.


Subject(s)
Arthritis, Rheumatoid/metabolism , Carrier Proteins/biosynthesis , Membrane Glycoproteins/biosynthesis , Synovial Membrane/chemistry , Bone Marrow Cells/metabolism , Bone Neoplasms/chemistry , Bone Neoplasms/metabolism , Bone Resorption/metabolism , Cathepsin K , Cathepsins/genetics , Gene Expression , Giant Cell Tumor of Bone/chemistry , Giant Cell Tumor of Bone/metabolism , Humans , Osteoclasts/physiology , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Reverse Transcriptase Polymerase Chain Reaction
2.
J Cell Biochem ; 72(3): 373-86, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10022519

ABSTRACT

The expression of the major matrix-degrading metalloproteinase, stromelysin (SL), is modulated by a variety of cytokines and growth factors. Interferon-gamma (IFN-gamma) is a potent modulator of SL expression, either inhibiting or activating expression in a cell-specific manner. We have investigated the mechanisms involved in the regulation of SL gene expression in cultured human fibroblasts by IFN-gamma. Reverse transcription-polymerase chain reaction (RT-PCR) assays confirmed the previously reported profound inhibitory response of SL mRNA expression to IFN-gamma [Amaldi et al., 1989]. For evaluation in transient gene expression assays, 1.2-kilobase (kb) pairs (-1214 to +14 relative to the transcription start site), and shorter, deletion mutant fragments of the SL promoter were cloned into appropriate chloramphenicol acetyltransferase transferase (CAT) expression vectors. The SL promoter along this region contains an active polyomavirus enhancer A-binding protein-3 (PEA-3) site at -216 and an activator protein-1 (AP-1) site at -70. Treatment of transfected neonatal foreskin fibroblasts with 300-500 U/ml IFN-gamma resulted in down-regulation of both basal and IL-1beta-induced CAT gene expression. IFN-gamma also decreased CAT expression when placed under the control of a synthetic multimeric AP-1 site construct. Gel-shift assay data indicate a decrease in specific binding to AP-1 oligonucleotide of nuclear extract from IFN-gamma and PMA/IFN-gamma-treated cells. The suppression of SL expression by IFN-gamma, in human fibroblasts therefore is mediated through the AP-1 element.


Subject(s)
Interferon-gamma/physiology , Matrix Metalloproteinase 3/physiology , Transcription Factor AP-1/physiology , Chloramphenicol O-Acetyltransferase/chemistry , Fibroblasts/physiology , Flow Cytometry , Gene Expression Regulation , Humans , Intercellular Adhesion Molecule-1/physiology , Interleukin-1/physiology , Models, Genetic , Mutagenesis , Polymerase Chain Reaction , Promoter Regions, Genetic , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/physiology
3.
J Rheumatol ; 25(2): 302-7, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9489823

ABSTRACT

OBJECTIVE: To investigate the safety and pharmacokinetics of a 28 day continuous subcutaneous infusion of recombinant human relaxin in patients with systemic sclerosis with diffuse scleroderma. METHODS: Thirty patients with stable diffuse scleroderma of moderate severity received recombinant human relaxin at 6, 12, 50, 100, and 200 microg/kg/day or placebo in a double blind, sequential panel, dose escalation study. RESULTS: All patients completed 28 days of study treatment. Steady state concentrations of serum relaxin were achieved by the 3rd day of infusion and were dose proportionate. Patients receiving 200 microg/kg/day achieved levels about 50-fold those of normal pregnancy. Pharmacokinetics of relaxin were nonlinear with hyperbolic increases of both t1/2 and volume of distribution and parallel decrease of elimination rate coefficient. An elimination transport system was suggested with saturation at serum relaxin concentration of 45 ng/ml. Adverse events included local infusion site rash and pain, minor bleeding episodes, and decreased hemoglobin concentration (mean reduction 1.1 g/dl). Standard measures of scleroderma were unchanged, although global assessment favored relaxin over placebo. CONCLUSION: Recombinant human relaxin in the doses used was safe and well tolerated. Longer term controlled trials are warranted to define the potential efficacy of relaxin in patients with diffuse scleroderma.


Subject(s)
Relaxin/adverse effects , Relaxin/genetics , Relaxin/pharmacokinetics , Scleroderma, Systemic/drug therapy , Adult , Amino Acid Sequence , Double-Blind Method , Female , Hemoglobinometry , Humans , Male , Middle Aged , Molecular Sequence Data , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacokinetics , Treatment Outcome
4.
Semin Cutan Med Surg ; 17(1): 18-21, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9512102

ABSTRACT

The immune system abnormalities of scleroderma are diverse and in association with vascular and mesenchymal cell abnormalities, contribute to the organ system dysfunction and clinical expression of the disease. Recent insights into novel immune regulatory pathways, immune reactivity to self-antigens, and the potential association of persistent fetal cells and disease expression may serve to increase our understanding of this enigmatic disease.


Subject(s)
Autoimmune Diseases/immunology , Graft vs Host Disease/immunology , Scleroderma, Localized/immunology , Scleroderma, Systemic/immunology , Animals , Autoantigens/biosynthesis , Humans , Immunity, Cellular/physiology , Mice
5.
J Clin Endocrinol Metab ; 82(3): 839-46, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9062493

ABSTRACT

Glycodelin is a glycoprotein named for its unique carbohydrate structure. Glycodelin is produced by the secretory endometrium during the late luteal phase and returns to baseline during menses of the ensuing cycle, whereas in conceptive cycles it rapidly increases. Although progesterone and possibly estradiol are required for glycodelin production, they are not directly involved in the synthesis and release of this protein. Their role may be development of the endometrial secretory glandular elements, whereas other factors are required to initiate and maintain glycodelin secretion. The pattern of relaxin secretion during the luteal phase and early pregnancy is similar to that of glycodelin, but their profiles have not been determined simultaneously. To investigate the relationship of relaxin and glycodelin, two studies were conducted. In the first study, relaxin, glycodelin, and ovarian steroids were measured in daily serum samples from nonconceptive and conceptive natural cycles. Profiles of relaxin and glycodelin were closely associated, with the onset of relaxin preceding glycodelin secretion by 1-2 days in nonconceptive cycles, and the pregnancy-associated increases in each hormone differing by about 2 days. The second study tested the hypothesis that relaxin stimulates glycodelin secretion. Samples were obtained from patients injected with human relaxin for 28 days. In subjects demonstrating ovarian cyclicity, glycodelin secretion was elevated, but it was not detected in subjects without ovarian cyclicity or in placebo-treated control subjects. This study reveals a close temporal and quantitative relationship between relaxin and glycodelin profiles in the late luteal phase and early pregnancy. It also demonstrates that relaxin administration can stimulate glycodelin production from a developed endometrium. This is the first report of a nonsteroidal ovarian factor that controls glycodelin secretion, and these results suggest a function for relaxin during early pregnancy. Glycodelin is a potent inhibitor of sperm zona pellucida binding by virtue of its extensive carbohydrate structure, but it is normally at a nadir in the periovulatory period. The data demonstrate that relaxin can stimulate glycodelin secretion throughout the menstrual cycle, including the periovulatory period, when relaxin-induced glycodelin secretion could have a contraceptive effect.


Subject(s)
Glycoproteins/metabolism , Pregnancy Proteins/metabolism , Relaxin/physiology , Adolescent , Adult , Contraceptive Agents/pharmacology , Double-Blind Method , Estradiol/blood , Female , Glycodelin , Humans , Luteal Phase , Luteinizing Hormone/blood , Male , Menstrual Cycle/metabolism , Middle Aged , Pregnancy , Pregnancy Trimester, First/metabolism , Progesterone/blood , Recombinant Proteins , Relaxin/adverse effects , Relaxin/pharmacology
6.
J Clin Invest ; 98(12): 2739-45, 1996 Dec 15.
Article in English | MEDLINE | ID: mdl-8981919

ABSTRACT

Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.


Subject(s)
Pulmonary Fibrosis/metabolism , Relaxin/pharmacology , Animals , Bleomycin/pharmacology , Blotting, Western , Collagen/metabolism , Collagenases/metabolism , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/metabolism , Fibronectins/metabolism , Gene Expression Regulation/genetics , Histocytochemistry , Humans , Lung/cytology , Lung/drug effects , Lung Injury , Mice , Procollagen/metabolism , Pulmonary Fibrosis/therapy , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology
7.
J Biol Chem ; 271(44): 27936-41, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8910395

ABSTRACT

Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (Kd = 102 pM) to a specific receptor on THP-1 cells. Receptor density was low ( approximately 275 receptors/cell), but binding of relaxin triggered intracellular signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.


Subject(s)
Receptors, Peptide/metabolism , Relaxin/metabolism , Relaxin/pharmacology , Cell Line , Enzyme-Linked Immunosorbent Assay , Estrogens/pharmacology , Female , Hormones/pharmacology , Humans , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor II/pharmacology , Kinetics , Monocytes , Pregnancy , Progesterone/pharmacology , Proteins/pharmacology , Receptors, G-Protein-Coupled , Receptors, Peptide/drug effects , Receptors, Peptide/physiology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effects
8.
Curr Opin Rheumatol ; 8(6): 580-4, 1996 Nov.
Article in English | MEDLINE | ID: mdl-9018463

ABSTRACT

The recent literature on scleroderma emphasizes the importance of cytokine dysregulation, cell-cell interactions, and extracellular matrix overproduction in the pathogenesis of this disease. Advances in our understanding of these areas are discussed here.


Subject(s)
Cell Communication/physiology , Cytokines/physiology , Extracellular Matrix/physiology , Scleroderma, Systemic/etiology , Scleroderma, Systemic/physiopathology , Animals , Cell Division , Connective Tissue/physiology , Endothelium/physiology , Fibroblasts/physiology , Humans
9.
J Clin Invest ; 97(12): 2866-71, 1996 Jun 15.
Article in English | MEDLINE | ID: mdl-8675699

ABSTRACT

Current theories propose that systemic lupus erythematosus develops when genetically predisposed individuals are exposed to certain environmental agents, although how these agents trigger lupus is uncertain. Some of these agents, such as procainamide, hydralazine, and UV-light inhibit T cell DNA methylation, increase lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) expression, and induce autoreactivity in vitro, and adoptive transfer of T cells that are made autoreactive by this mechanism causes a lupuslike disease. The mechanism by which these cells cause autoimmunity is unknown. In this report, we present evidence that LFA-1 overexpression is sufficient to induce autoimmunity. LFA-1 overexpression was induced on cloned murine Th2 cells by transfection, resulting in autoreactivity. Adoptive transfer of the transfected, autoreactive cells into syngeneic recipients caused a lupuslike disease with anti-DNA antibodies, an immune complex glomerulonephritis and pulmonary alveolitis, similar to that caused by cells treated with procainamide. These results indicate that agents or events which modify T cell DNA methylation may induce autoimmunity by causing T cell LFA-1 overexpression. Since T cells from patients with active lupus have hypomethylated DNA and overexpressed LFA-1, this mechanism could be important in the development of human autoimmunity.


Subject(s)
Lupus Vulgaris/chemically induced , Lymphocyte Function-Associated Antigen-1/physiology , T-Lymphocytes/physiology , Animals , Autoimmunity , Cells, Cultured , DNA/metabolism , Female , Lupus Vulgaris/immunology , Lymphocyte Function-Associated Antigen-1/genetics , Methylation , Mice , Mice, Inbred AKR , Mice, Inbred DBA , T-Lymphocytes/drug effects , Transfection
10.
Growth Factors ; 12(2): 151-7, 1995.
Article in English | MEDLINE | ID: mdl-8679249

ABSTRACT

Macrophages are a source of cytokines driving repair. Wound macrophages are derived from circulating monocytes. Monocyte chemotactic protein-1 (MCP-1) is a potent specific monocyte chemoattractant. Treatment of serum stimulated dermal fibroblasts with dexamethasone led to a dose dependent down-regulation of MCP-1 mRNA levels. Such an anti-inflammatory effect may partially explain the negative influence of glucocorticoid treatment on wound repair. Topical or parenteral of fibroblasts cultured in serum free media with TGF-beta increased MCP-1 mRNA levels. TGF-beta treatment of fibroblasts cultured in serum also partially overcame the dexamethasone mediated decrease in MCP-1 mRNA levels. In glucocorticoid treated animals TGF-beta may stimulate repair by an indirect pro-inflammatory action following transcriptional up-regulation of MCP-1.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Chemokine CCL2/genetics , Dexamethasone/pharmacology , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , RNA, Messenger/metabolism , Transforming Growth Factor beta/pharmacology , Autoradiography , Blood , Blotting, Northern , Cell Division , Cells, Cultured , Fibroblasts/drug effects , Humans , Platelet-Derived Growth Factor/pharmacology , Skin
11.
Circ Res ; 75(1): 181-9, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8013077

ABSTRACT

Vascular matrix remodeling occurs during development, growth, and several pathological conditions that affect blood vessels. We investigated the capacity of human smooth muscle cells (SMCs) to express matrix metalloproteinases (MMPs), enzymes that selectively digest components of the extracellular matrix (ECM), in the basal state or after stimulation with certain cytokines implicated in vascular homeostasis and pathology. Enzymatic activity associated with various proteins secreted in the culture media was detected by gelatin or casein sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Proteins were identified by immunoprecipitation and mRNA by Northern blotting. SMCs constitutively secreted a 72-kD gelatinase and the tissue inhibitors of MMPs (TIMPs) types 1 and 2. SMCs stimulated with interleukin-1 or tumor necrosis factor-alpha synthesized de novo 92-kD gelatinase, interstitial collagenase, and stromelysin. Several lines of evidence suggest that when stimulated by cytokines, SMCs produce activated forms of MMPs. Together, the constitutive and the cytokine-induced enzymes can digest all the major components of the vascular ECM. Moreover, since these mediators augment the production of MMPs without appreciably affecting the synthesis of TIMPs, locally secreted cytokines may tip the regional balance of MMP activity in favor of vascular matrix degradation.


Subject(s)
Cytokines/pharmacology , Extracellular Matrix/metabolism , Muscle, Smooth, Vascular/metabolism , Cells, Cultured , Collagenases/biosynthesis , Enzyme Activation , Extracellular Space/metabolism , Gelatinases/metabolism , Glycoproteins/biosynthesis , Humans , Interleukin-1/pharmacology , Intracellular Membranes/metabolism , Matrix Metalloproteinase 3 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/metabolism , Muscle, Smooth, Vascular/cytology , Tissue Inhibitor of Metalloproteinases
12.
Cell Immunol ; 156(1): 102-12, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7515330

ABSTRACT

Experimental autoimmune neuritis (EAN) is a T-cell-mediated autoimmune disease characterized by demyelination and mononuclear cell infiltration of the peripheral nervous system. It is induced in Lewis rats by administration of myelin P2 protein or a synthetic peptide (SP-26) corresponding to amino acid residues 53-78 of bovine P2 protein. The effects of transforming growth factor-beta 1 (TGF-beta 1) on the clinical signs, histological changes, cell-mediated immune responses, and secretion of interferon-gamma (IFN-gamma) by lymphoid cells of rats with EAN were examined. Systemic administration of TGF-beta 1 markedly inhibited the clinical signs and histological changes of EAN when given intraperitoneally every other day for Days 0 through 18. In addition, it decreased proliferative responses and reduced the delayed-type hypersensitivity (DTH) response to SP-26 compared to control rats. The reduction in clinical severity correlated with skin test unresponsiveness (DTH) to the disease-inducing agent (SP-26) as well to decreased cellular responsiveness to the antigen in vitro. The decrease in cellular responsiveness extended to a decrease in at least one T cell lymphokine, IFN-gamma. The profound effect of TGF-beta on disease progression in EAN, a T-cell-mediated process, is consistent with a direct effect of this growth factor on T lymphocytes.


Subject(s)
Myelin P2 Protein , Neuritis, Autoimmune, Experimental/immunology , Transforming Growth Factor beta/pharmacology , Amino Acid Sequence , Animals , Female , Hypersensitivity, Delayed/immunology , Interferon-gamma/biosynthesis , Interleukin-2/pharmacology , Lymphocyte Activation/drug effects , Molecular Sequence Data , Myelin Basic Protein/chemistry , Myelin Basic Protein/immunology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Rats , Rats, Inbred Lew , Spleen
13.
J Immunol ; 152(8): 4149-56, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-7511670

ABSTRACT

Angiogenesis is important in the proliferation of inflammatory synovial tissue. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that is also angiogenic in vivo. We examined the potential role of VEGF in mediating chemotaxis and proliferation of endothelial cells in rheumatoid arthritis (RA) using samples of synovial tissue and synovial fluid from 55 arthritic patients. Synovial fluid VEGF by ELISA was higher in RA synovial fluids (386 +/- 122 ng/ml) (SE) compared with osteoarthritis (OA) synovial fluids (< 0.8 ng/ml) (p < 0.05) or synovial fluids from patients with other arthritides (6.6 +/- 2 ng/ml). In addition to its known mitogenic properties, we found that human rVEGF was chemotactic for HUVECs at concentrations above 0.02 nM. Incubation of RA synovial fluids with neutralizing anti-VEGF resulted in 23 to 66% (mean 53 +/- 4%) inhibition of HUVEC chemotaxis. Conditioned medium from four of five RA synovial tissue explants was mitogenic for bovine adrenal capillary endothelial cells. Anti-VEGF neutralized from 19 to 42% (mean 28 +/- 4%) of this mitogenic activity. To determine the cellular source of VEGF in synovial tissue, we employed immunohistochemistry. VEGF+ cells were rarely (< 1%+) found in normal synovial tissues. In contrast, RA and OA synovial tissues exhibited VEGF+ lining cells (8% and 13%, respectively). A few synovial tissue macrophages were VEGF+ in both RA and OA (5% and 2%, respectively). These results elucidate a newly described function for VEGF as a potent chemotaxin for endothelial cells as well as a role for VEGF in RA-associated endothelial migration and proliferation.


Subject(s)
Arthritis, Rheumatoid/physiopathology , Endothelial Growth Factors/physiology , Lymphokines/physiology , Neovascularization, Pathologic , Cell Division , Chemotaxis , Endothelium, Vascular/cytology , Humans , Osteoarthritis/physiopathology , Synovial Fluid/physiology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
14.
Exp Cell Res ; 211(2): 212-8, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8143767

ABSTRACT

Recessive dystrophic epidermolysis bullosa (RDEB) is a mutilating disease of the skin characterized by recurrent blistering and erosions that result from compromised integrity of the basement membrane zone. In this study, fibroblasts derived from the skin of RDEB patients were characterized for expression of the major metalloproteinases, particularly interstitial collagenase. Consistent with previous reports on increased collagenase protein levels in fibroblasts from some RDEB patients, we found that steady-state levels of collagenase mRNA were significantly increased in fibroblast strains derived from three of five RDEB patients compared to fibroblasts obtained from normal donors. Stromelysin mRNA was elevated in the same three fibroblast strains, whereas expression of neither the 72- nor the 92-kDa type IV collagenases was different from that of controls. Tissue inhibitor of metalloproteinases was expressed in RDEB fibroblasts at levels similar to those observed in normal fibroblasts. To investigate the mechanism behind the steady-state elevation in collagenase and stromelysin expression, AP-1 expression and activation were studied. Although levels of Jun expression were not different from those seen in normal fibroblasts, AP-1 activity, as assessed by ability to bind to a TPA response element-containing oligonucleotide, was endogenously elevated in RDEB fibroblasts compared to normal fibroblasts. Transfection studies using a plasmid construct containing the collagenase promoter linked to a CAT reporter gene demonstrated that RDEB fibroblasts were able to support active transcription of the promoter compared to normal fibroblasts. These studies support the hypothesis that RDEB fibroblasts contain chronically activated AP-1, and perhaps other transactivating factors, that contribute to the cellular phenotype of collagenase and stromelysin overexpression.


Subject(s)
Collagenases/genetics , Epidermolysis Bullosa Dystrophica/genetics , Epidermolysis Bullosa Dystrophica/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-jun/metabolism , Cells, Cultured , Collagenases/metabolism , Epidermolysis Bullosa Dystrophica/etiology , Gene Expression Regulation , Genes, Recessive , Humans , Matrix Metalloproteinase 3 , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Up-Regulation
15.
Exp Cell Res ; 210(2): 166-71, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8299714

ABSTRACT

Wound healing and other inflammatory processes are driven by a complex series of interactions among cells, the extracellular matrix, and secreted products of various cell types. Cytokines, such as interleukin-1 and transforming growth factor-alpha, are present at wound sites and contribute to the proinflammatory milieu of these sites. In the present study, we have investigated the effect of these cytokines, individually and in concert, on fibroblast expression of matrix metalloproteinases, which contribute to extracellular matrix remodeling, and of prostaglandin E2, which alters vascular tone and permeability. The metalloproteinases, procollagenase (matrix metalloproteinase-1) and prostromelysin (matrix metalloproteinase-3), are induced by exposure of dermal fibroblasts to interleukin-1, not stimulated by transforming growth factor-alpha, but are synergistically induced by the combination of cytokines. The 92-kDa type IV procollagenase (matrix metalloproteinase-9, progelatinase B), is also stimulated in synergistic fashion. Prostaglandin E2 is induced in rheumatoid synovial fibroblasts by interleukin-1 beta, not altered by transforming growth factor-alpha, and is synergistically released by the combination of the two cytokines. Fibroblast proliferation, which is also a component of normal wound healing, is also synergistically stimulated by the action of the two cytokines in concert. These results indicate that interleukin-1 beta and transforming growth factor-alpha synergize to elicit a number of phenotypic responses in fibroblasts which are relevant to normal wound healing and chronic inflammation.


Subject(s)
Dinoprostone/metabolism , Interleukin-1/pharmacology , Metalloendopeptidases/biosynthesis , Synovial Membrane/metabolism , Synovial Membrane/pathology , Transforming Growth Factor alpha/pharmacology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cell Division/drug effects , Cells, Cultured , Collagenases/analysis , Collagenases/biosynthesis , DNA/biosynthesis , Drug Synergism , Enzyme Induction , Enzyme Precursors/analysis , Enzyme Precursors/biosynthesis , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression/drug effects , Humans , Metalloendopeptidases/analysis , Molecular Weight , RNA, Messenger/biosynthesis , Reference Values , Skin/cytology , Skin/drug effects , Skin/metabolism , Synovial Membrane/drug effects , Thymidine/metabolism , Transcription, Genetic/drug effects
16.
Growth Factors ; 11(3): 205-13, 1994.
Article in English | MEDLINE | ID: mdl-7734146

ABSTRACT

Collagen metabolism is a balance between synthesis and lysis. Both are under tight regulatory control. TGF-beta reverse the impairment of healing seen after glucocorticoid treatment in vivo. Both TGF-beta and glucocorticoids are known to regulate collagen metabolism directly. We have examined the effect of dexamethasone and of TGF-beta individually and in combination on the regulation of procollagen type 1, interstitial collagenase and tissue inhibitor of metallo-proteinase-1 (TIMP-1) synthesis at both the protein and mRNA levels in low passage human dermal fibroblasts. Dexamethasone treatment decrease synthesis of procollagen and caused a dose dependent down-regulation of TIMP-1 synthesis. Interstitial collagenase synthesis by fibroblasts was detectable but low. Thus, glucocorticoid treatment of fibroblasts tilts the balance of collagen metabolism away from accumulation. TGF-beta had opposing effects, stimulating both procollagen and TIMP-1 synthesis at the protein and mRNA levels. TGF-beta was able to cause a dose-dependent reversal of the glucocorticoid induced decrease in procollagen and TIMP-1 synthesis. Stimulation of healing in glucocorticoid treated animals by TGF-beta may be by the direct action of this agent upon fibroblast collagen metabolism.


Subject(s)
Collagen/metabolism , Dexamethasone/pharmacology , Fibroblasts/drug effects , Transforming Growth Factor beta/pharmacology , Blotting, Northern , Cell Count/drug effects , Cell Division/drug effects , Cells, Cultured , Collagen/biosynthesis , Collagen/drug effects , Collagenases/biosynthesis , Collagenases/drug effects , Dexamethasone/antagonists & inhibitors , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/biosynthesis , Enzyme Precursors/drug effects , Fibroblasts/metabolism , Glycoproteins/biosynthesis , Glycoproteins/drug effects , Humans , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/drug effects , Procollagen/biosynthesis , Procollagen/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Skin/drug effects , Skin/metabolism , Tissue Inhibitor of Metalloproteinases , Wound Healing/physiology
17.
Am Rev Respir Dis ; 148(6 Pt 1): 1671-6, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8256918

ABSTRACT

A common side effect of radiotherapy is the development of fibrosis in the irradiated tissue. To study the mechanisms of this fibrogenic response, we developed a model system of whole-lung radiation in the rat and studied the evolution of injury by assessment of the cells and protein recovered by lavage. Once the pattern of injury was known, we attempted to modulate this reaction by administering the cytokine interferon-gamma (IFN-gamma). Rats received 15 Gy radiation to the whole thorax and were studied by lung lavage at intervals of 1 to 35 days after radiation. The effect of radiation was an initial (24 h) leak of protein, unaccompanied by cellular alterations, that resolved by 48 h. This was followed 2 wk later by a phase of inflammatory cell recruitment and more significant protein leak. A third phase of increase in inflammatory cells and further increase in protein flux was noted at Day 35. A significant cellular infiltrate was seen in lung sections obtained from animals treated in parallel experiments. IFN-gamma was given by osmotic pump from Day 0 to Day 35. This treatment significantly attenuated the PMN recruitment and protein leak (p < 0.002 and 0.01, respectively) at Days 25 and 35. Histologic sections demonstrated reduced alveolar cellularity and exudate at Day 25 (p < 0.05); however, significant numbers of inflammatory cells and exudate were present in irradiated and IFN-gamma-treated animals at Day 35. These data indicate that inflammatory cell recruitment may play a role in the lung injury following radiation. Furthermore, these preliminary data indicate that a cytokine blocks this reaction.


Subject(s)
Bronchoalveolar Lavage Fluid , Interferon-gamma/therapeutic use , Radiation Pneumonitis/diagnosis , Radiation Pneumonitis/therapy , Animals , Bronchoalveolar Lavage Fluid/cytology , Chemotaxis, Leukocyte , Lung/pathology , Male , Neutrophils/physiology , Radiation Pneumonitis/pathology , Radiation Pneumonitis/physiopathology , Rats , Recombinant Proteins , Specific Pathogen-Free Organisms
18.
J Clin Invest ; 92(6): 2841-9, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8254038

ABSTRACT

The role of intravenously administered recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) on the healing of incisional wounds in rats with impaired healing due to age or glucocorticoid administration was investigated. The administration of methylprednisolone to young adult rats decreased wound breaking strength to 50% of normal control. Breaking strength of incisional wounds from 19-mo-old rats was decreased approximately 27% compared with wounds from normal healing young adult rats. A single intravenous administration of rhTGF-beta 1 (100 or 500 micrograms/kg) increased wound breaking strength from old rats or young adult rats with glucocorticoid-induced impaired healing to levels similar to normal healing control animals when determined 7 d after injury. Even though the circulating half-life of systemically administered rhTGF-beta 1 is < 5 min, a sustained stimulatory effect on extracellular matrix secretion was evident in glucocorticoid-impaired rats when rhTGF-beta 1 was administered at the time of wounding, 4 h after wounding, or even 24 h before wounding. These observations indicate a previously unrecognized potential for the active form of TGF-beta 1 to profoundly influence the wound healing cascade after brief systemic exposure.


Subject(s)
Aging/physiology , Methylprednisolone/pharmacology , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology , Wound Healing/physiology , Wounds and Injuries/physiopathology , Analysis of Variance , Animals , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/ultrastructure , Extracellular Matrix/drug effects , Extracellular Matrix/pathology , Extracellular Matrix/ultrastructure , Humans , Injections, Intravenous , Male , Microscopy, Electron , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/therapeutic use , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/therapeutic use , Wound Healing/drug effects , Wounds and Injuries/pathology
19.
J Bone Miner Res ; 8(11): 1407-14, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8266832

ABSTRACT

Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Fracture Healing/drug effects , Skull/metabolism , Transforming Growth Factor beta/metabolism , Animals , Iodine Radioisotopes/metabolism , Male , Rabbits , Skull/injuries , Transforming Growth Factor beta/pharmacology
20.
Cell Immunol ; 150(2): 298-310, 1993 Sep.
Article in English | MEDLINE | ID: mdl-7690307

ABSTRACT

Neuritogenic T cells specific for SP-26, a synthetic peptide (residue 53-78) of myelin P2 protein that causes experimental autoimmune neuritis (EAN), use the same T cell receptor (TCR) V gene family (V beta 8) that can induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Tolerance to autoregulatory T cells may be induced in rats by intravenous (iv) administration of antigen-coupled splenocytes; however, the mechanisms that lead to altered immune reactivity are not well understood. Here we demonstrate that SP-26, when coupled to syngeneic spleen cells and administered iv, either before or after disease induction, markedly inhibited development and expression of clinical signs and histological changes of EAN. The induction of tolerance by this method was peptide-specific and MHC-restricted. We showed previously that T cells involved in EAN utilize the T cell antigen receptor V beta 8, whereas less than 5% of normal rat peripheral T cells express V beta 8. We have examined T lymphocytes from tolerized rats to determine the presence or absence of V beta 8(+)-bearing cells in order to determine the mechanism of tolerance. V beta 8 cells were undetectable by Northern blot analysis in the lymph nodes of unimmunized animals but easily detected in SP-26-primed and tolerized rats. In addition, spleen cells isolated from tolerized animals were anergic and failed to proliferate in response to SP-26, but retained responsiveness to IL-2 and Con A stimulation. Thus, the peptide-specific unresponsiveness that can be induced in rats with EAN, a T-cell-mediated process that is MHC-restricted and utilizes the T cell receptor V beta 8, occurs while V beta 8 transcripts remain readily detectable in spleen and lymph node cells. The detection of V beta 8-bearing T cells requires the development of antibodies specific for this rat surface protein.


Subject(s)
Immune Tolerance , Myelin Basic Protein/immunology , Myelin P2 Protein , Neuritis, Autoimmune, Experimental/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Female , Immunization , Lymphocyte Activation , Molecular Sequence Data , Rats , Rats, Inbred Lew , Rats, Wistar , Receptors, Antigen, T-Cell, alpha-beta/genetics
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