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Biomedical subjects

R M Senior

Publications and source records attributed to R M Senior.

At least 37 records · Page 2Linked to original sources
PubMed1998

Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by deposition of autoantibodies at the basement membrane zone. In an experimental BP model in mice, the subepidermal blistering is mediated by antibodies directed against the hemidesmosomal protein BP180 (collagen XVII, BPAG2), and depends on complement activation and neutrophil infiltration. Gelatinase B is present in BP blister fluid and can cleave BP180. In this study we investigated the role of gelatinase B in the immunopathogenesis of experimental BP using mice containing targeted disruption of the gelatinase B (MMP-9, 92 kD gelatinase) gene. Gelatinase B-deficient mice were resistant to the blistering effect of intracutaneous anti-mBP180 antibodies, although these mice showed deposition of autoantibodies at the basement membrane zone and neutrophil recruitment to the skin comparable to that observed in the control mice. Interleukin 8 given intradermally concomitantly with pathogenic anti-mBP180 elicited a significant neutrophil recruitment into the skin in gelatinase B-deficient mice, but blistering did not occur. However, gelatinase B-deficient mice reconstituted with neutrophils from normal mice developed blistering in response to anti-mBP180 antibodies. These results implicate neutrophil-derived gelatinase B in the pathogenesis of experimental BP and might lead to novel therapeutic strategies for BP.

Animals↗
PubMed1998

MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.

Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.

Animals↗
PubMed1998

Expression of laminin alpha3, alpha4, and alpha5 chains by alveolar epithelial cells and fibroblasts.

Laminins are principal components of basement membranes. Eleven laminin isoforms are known, each a heterotrimer composed of polypeptide chains designated alpha, beta, and gamma. Five alpha chains have been identified to date: alpha1, alpha2, alpha3, alpha4, and alpha5. Recent studies of fetal and adult mouse lung show prominence of alpha3, alpha4, and alpha5 in alveolar tissue, and point to differences in the cellular expression of these alpha chains in the developing alveolus. We examined isolated rat alveolar type II cells and lung fibroblasts for expression of laminins alpha3, alpha4, and alpha5. We found that laminin alpha3 was expressed only by alveolar epithelial cells, that laminin alpha4 was expressed only by lung fibroblasts, and that laminin alpha5 was expressed primarily by alveolar epithelial cells. Metabolic labeling and immunoprecipitation confirmed the production of laminin alpha4 by fibroblasts and laminin alpha5 by alveolar epithelial cells in culture. These studies indicate that different alveolar cell types contribute different laminin alpha chains to the laminin isoforms in alveolar basement membranes. Immunohistochemistry showed colocalization of these laminin alpha chains with the laminin beta1, beta2, and gamma1 chains, indicating the likelihood that laminins 6 to 11 are present in alveolar basement membranes.

Animals↗
PubMed1998

Heart rate variability reflects severity of COPD in PiZ alpha1-antitrypsin deficiency.

BACKGROUND: Analysis of heart rate variability (HRV) is a powerful method of assessing severity of conditions affecting the autonomic nervous system. STUDY OBJECTIVE: To determine if HRV is decreased and if HRV reflects severity in COPD. DESIGN: Prospective determination of HRV from 24-h outpatient Holter recordings. PATIENTS: Eighteen individuals with PiZ alpha1-antitrypsin deficiency: 13 with COPD and 5 with normal FEV1. HRV was also determined in 18 matched normal control subjects. Approximately 3 years after the initial recording, all COPD subjects were contacted to determine current status. MEASUREMENTS: Indexes of heart rate (HR) and HRV were compared for groups of patients with and without COPD and their control subjects. RESULTS: Mean and minimum HRs were higher in COPD patients. Virtually all indexes of HRV were significantly decreased in COPD patients. No differences were found in HR or HRV between PiZ individuals with normal FEV1 and their age-and gender-matched control subjects. Patients who had a change in status (ie, death, lung transplant, listed for transplant) had significantly higher daytime HRs, lower values for HRV indexes reflecting mixed sympathetic and parasympathetic modulation of HR, and reduced daytime high-frequency spectral power, an index of cardiac vagal modulation. Significant correlations (r=0.48 to 0.88) were found between FEV1 and these and other indexes of HRV. Most other indexes of HRV also tended to be lower for the group whose status had changed. CONCLUSION: PiZ alpha1-antitrypsin deficiency COPD is associated with abnormal cardiac autonomic modulation. Indexes of HRV appear to reflect severity and may have prognostic value in COPD patients.

Adult↗
PubMed1997

Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.

To determine which proteinases are responsible for the lung destruction characteristic of pulmonary emphysema, macrophage elastase-deficient (MME-/-) mice were subjected to cigarette smoke. In contrast to wild-type mice, MME-/- mice did not have increased numbers of macrophages in their lungs and did not develop emphysema in response to long-term exposure to cigarette smoke. Smoke-exposed MME-/- mice that received monthly intratracheal instillations of monocyte chemoattractant protein-1 showed accumulation of alveolar macrophages but did not develop air space enlargement. Thus, macrophage elastase is probably sufficient for the development of emphysema that results from chronic inhalation of cigarette smoke.

Animals↗
PubMed1997

Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis.

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P'1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.

Amino Acid Sequence↗
PubMed1996

Domain-specific interactions between entactin and neutrophil integrins. G2 domain ligation of integrin alpha3beta1 and E domain ligation of the leukocyte response integrin signal for different responses.

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the beta3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of alpha3beta1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

Antigen-Antibody Complex↗
PubMed1996

The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats.

Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity.

Amino Acid Sequence↗
PubMed1996

Entactin expression by rat lung and rat alveolar epithelial cells.

Although the composition of the subepithelial basement membrane of the alveolar septum has been studied in detail, there is relatively little information about which cells produce it. We examined intact rat lung and isolated rat alveolar type II cells for the expression of entactin, an integral basement membrane component that binds laminin and type IV collagen. By Northern analysis, late gestation and early neonatal rat lungs expressed high levels of entactin mRNA whereas lungs from adult animals had only minimal levels of entactin mRNA. These latter findings were confirmed by in situ hybridization, which showed prominent signal for entactin mRNA in cells in the alveolar walls of neonatal animals and no signal for entactin mRNA in the alveolar walls of lungs from adult animals. The entactin mRNA throughout the alveolar walls of neonatal animals was not limited to cells that expressed surfactant-associated protein C mRNA, a marker of alveolar type II cells. Freshly harvested adult alveolar type II cells and alveolar type II cells in culture for < 6 days expressed none to minimal entactin mRNA or protein. However, with longer periods in culture, both entactin mRNA and entactin protein synthesis were evident and progressively increased. In situ hybridization indicated that >60% of the alveolar epithelial cells expressed entactin mRNA with increasing time in culture. When cultured on Engelbreth-Holm-Swarm matrix, alveolar type II cells showed the same time course of entactin mRNA expression as cells cultured on plastic. Neonatal lung mesenchymal cells produced abundant entactin in culture, consistent with the likelihood that these cells are the principal source of entactin in alveolar walls in the developing lung. These results indicate that entactin production in the normal alveolar wall occurs primarily during lung development and that mesenchymal cells are probably the principal source of production. However, because adult alveolar epithelial cells synthesize entactin in culture, it is possible that alveolar epithelium contributes to the entactin in the alveolar subepithelial basement membrane.

Animals↗
PubMed1996

Postpneumonectomy lung growth: a model of reinitiation of tropoelastin and type I collagen production in a normal pattern in adult rat lung.

Elastic and collagen fibers confer recoil and tensile strength on the pulmonary vasculature, airways, alveolar walls, and pleura. These durable extracellular matrix components are primarily synthesized during lung development and growth, and are expressed at very low levels in healthy adult lung. However, reinitiation of elastin and collagen synthesis in diseases of adult lung, such as idiopathic pulmonary fibrosis, often leads to excessive or aberrant deposition of elastin and collagen which contribute to the pathophysiology of these diseases. We used an experimental model of postpneumonectomy lung growth to determine whether normal patterns of synthesis and deposition of these critical structural components can occur in the adult lung. Male Sprague-Dawley rats (250-300 grams) were subjected to left pneumonectomy and right lobectomy. The remaining lung tissue was harvested for analysis after 3, 7, or 14 days. Compensatory growth of the remaining right lung progressed throughout the time course. Total desmosine and hydroxyproline content increased in the postpneumonectomy lung, reflecting increased elastin and collagen accumulation, but both were normal in content per weight of lung tissue. Northern analysis demonstrated induction of tropoelastin and type I procollagen mRNA expression in lungs of pneumonectomy rats. In situ hybridization localized tropoelastin and type I procollagen mRNA expression to anatomical sites similar to those seen during lung development. These data indicate that the adult lung can reinitiate elastin and collagen production and deposit these extracellular matrix components in a normal pattern.

Animals↗
PubMed1995

Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate. Differential processing and stabilization of the carboxyl-terminal domain by tissue inhibitor of metalloproteinases (TIMP).

The matrix metalloproteinase 92-kDa gelatinase is a major product of inflammatory cells. Macrophages synthesize and secrete this proteinase as a proenzyme in association with tissue inhibitor of metalloproteinases (TIMP) (92TIMP), whereas neutrophils store and release it from secondary granules as a TIMP-free proenzyme (92TIMP-free). Metalloproteinase proenzymes can be activated in vitro by a variety of agents, including organomercurials and proteinases, resulting in loss of an 8-10-kDa NH2-terminal domain which disrupts the interaction of a conserved cysteine residue with the catalytic zinc molecule. We report that the activation and processing of 92-kDa gelatinase differs depending on its association with TIMP and the nature of the activating agent. We observed that 92TIMP undergoes classic activation to 82 kDa by stromelysin, whereas exposure to 4-aminophenylmercuric acetate (APMA) results in a final product of 83 kDa that still contains the "prodomain" cysteine. Association with TIMP appears to stabilize the COOH-terminal domain, whereas 92TIMP-free is converted by APMA to a final product of 67 kDa lacking the COOH-terminal portion. In the continued presence of APMA, which maintains cysteine-zinc disruption, the 67-kDa species is at least as active as the classic 82 kDa. In contrast, activation of 92TIMP-free by stromelysin initially generates the 82-kDa form which is followed by final conversion to a 50-kDa species that lacks the catalytic domain of the parent molecule. Therefore, although stromelysin activation of 92TIMP-free is initially efficient, the active 82-kDa form is short-lived and is replaced by an inactive 50-kDa product. This complex pattern of activation of the 92-kDa gelatinase may serve to restrict its proteolytic capacity following exposure to stromelysin and may serve to regulate proteinase activity in vivo.

Amino Acid Sequence↗
PubMed1995

Elastin in lung development and disease.

Elastic fibres are present in lung structures including alveoli, alveolar ducts, airways, vasculature and pleura. The rate of lung elastin synthesis is greatest during fetal and neonatal development, and is minimal in the healthy adult. We have determined that glucocorticoids up-regulate fetal lung tropoelastin expression while concomitantly accelerating terminal airspace maturation. Because there is minimal turnover of elastin in healthy adult lung, the elastin incorporated into the lung early in development supports lung function for the normal lifespan. However, in the adult lung, in pathological circumstances such as emphysema or pulmonary fibrosis there may be reactivation of elastin expression. We have found in silica-induced pulmonary fibrosis that expression of tropoelastin is primarily increased in the walls and the septal tips of the alveolus, with modest increases in other compartments which normally express tropoelastin during development. This finding suggests that the mesenchymal cell of the alveolar wall increases tropoelastin expression during fibrotic disorders. In emphysema and fibrosis, elastin is present in abnormal-appearing, probably non-functional, elastic fibres, suggesting that the adult lung cannot recapitulate the elastic fibre assembly mechanisms operative during normal lung growth.

Adult↗
PubMed1995

Binding and degradation of elastin by the staphylolytic enzyme lysostaphin.

Lysostaphin is a bacterial zinc metalloproteinase that degrades staphylococcal cell wall peptidoglycans. We have shown that lysostaphin also binds tightly to elastin and contains elastolytic activity. The objective of this investigation was to further characterize the biochemical mechanism of elastolysis by lysostaphin. Binding of lysostaphin to elastin was demonstrated with elastin peptide affinity chromatography. Elastolysis by lysostaphin was studied using a tritium release assay with tritium borohydride-reduced elastin as substrate. Proteolysis of elastin by lysostaphin was maximal at neutral to slightly basic pH and proceeded linearly for 10 hr before reaching saturation. The elastolytic activity was not affected by inhibitors of cysteine or serine proteinases, but was inhibited by o-phenanthroline, EDTA, and by the addition of exogenous zinc. Inhibition by zinc was not due to an alteration in enzyme-ligand interaction since zinc-containing buffers did not impair the ability of lysostaphin to bind elastin. Lysostaphin elastolysis was not inhibited by trypsin treatment of the enzyme, which has been shown to inactivate the enzyme's staphylolytic properties. It is therefore concluded that: (1) lysostaphin binds and degrades elastin; (2) the elastolytic activity of lysostaphin is distinct from its staphylolytic activity; (3) sequence similarities with matrix metalloproteinases suggest the Ala-Ala-Thr-His-Glu sequence in the amino terminus of lysostaphin to be involved in elastin degradation; (4) our studies are important in establishing that metalloproteinases from staphylococci can participate in elastin degradation.

Amino Acid Sequence↗
PubMed1995

Interactions of pulmonary surfactant protein D (SP-D) with human blood leukocytes.

Surfactant protein D (SP-D) is believed to contribute to nonimmune host defense within the alveoli and distal airways of the lung. SP-D molecules can bind to specific carbohydrates on the surface of bacterial, fungal, and viral organisms and can also interact with membrane glycoconjugates expressed by alveolar macrophages. Because neutrophils (PMN) and monocytes are recruited into the airspaces in association with many types of infection or lung injury, we examined the interactions of these cells with purified natural and recombinant SP-Ds, using a modified Boyden chamber assay and checkerboard analysis. Natural or recombinant rat SP-D (approximately 10(-9) to 10(-13) M) showed dose-dependent effects on human PMN and monocyte migration with a maximal response at a SP-D concentration of 5 ng/ml (approximately 10(-11) M). The migratory response was comparable to that obtained with the optimum concentration of FMLP (10(-8) M). HL-60 cells, after induction of differentiation with DMSO, responded to SP-D with the same dose-response as neutrophils. The effects of SP-D were abrogated by the simultaneous addition of SP-D to the upper chamber or by the addition of antibodies to the carboxy-terminal lectin domain. Migration toward SP-D was markedly inhibited (< 10% of controls) by 10 mM maltose but was not significantly inhibited by to 50 mM lactose. These studies establish that SP-D can bind to specific sites on neutrophils and monocytes and strongly suggest that these interactions involve the saccharide binding domains of SP-D.

Animals↗
PubMed1994

Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of alpha 1-antitrypsin.

alpha 1-antitrypsin, the primary physiologic inhibitor of human leukocyte elastase, is proteolytically inactivated by several matrix metalloproteinases including interstitial collagenase, stromelysin and 92 kDa gelatinase. In this report, we describe the catalytic effects of matrilysin, a recently identified metalloproteinase, upon alpha 1-antitrypsin. Matrilysin was found to be approximately 30-fold more effective than 92kDa gelatinase, 70-fold more effective than collagenase, and 180-fold more effective than stromelysin. Cleavage of alpha 1-antitrypsin by matrilysin produced two fragments of approximately 50 kDa and 4 kDa. The single cleavage occurred at the Phe352-Leu353 peptide bond, a locus within alpha 1-antitrypsin's active-site loop. These results suggest that apart from its activity against extracellular matrix, matrilysin provides a mechanism for the regulation of leukocyte elastase activity through its capacity to degrade alpha 1-AT.

Amino Acid Sequence↗
PubMed1993

Degradation of entactin by matrix metalloproteinases. Susceptibility to matrilysin and identification of cleavage sites.

Entactin is the basement membrane protein which bridges laminin and type IV collagen. Entactin is known to be degraded by serine proteinases, but its susceptibility to matrix metalloproteinases has not been determined. We have studied the capacity of three matrix metalloproteinases (interstitial collagenase, 92-kDa gelatinase, and matrilysin) to degrade entactin. While all three metalloenzymes cleaved entactin, matrilysin was approximately 100-fold as effective as collagenase and 600-fold as effective as 92-kDa gelatinase. The Km of matrilysin for entactin was 8.9 x 10(-7) M. A Vmax of 21 molecules of entactin degraded/molecule of matrilysin/min at 37 degrees C was observed. An Arrhenius plot relating matrilysin's catalytic activity to temperature was linear from 15 to 37 degrees C and indicated an activation energy of 10,060 calories/mol. Matrilysin produced multiple, but distinct, cleavages in entactin resulting in peptide fragments ranging from 115 to 29 kDa. The precise sites of cleavage of six fragments were determined by Edman degradation. Cleavage sites consistently occurred amino-terminal to leucine or isoleucine. These data indicate that entactin is a substrate for matrix metalloproteinases. The effectiveness of matrilysin is noteworthy, however, particularly in relation to the minimal ability of other much more well described matrix metalloproteinases to attack this substrate. Our results suggest a potentially important role for matrilysin in disruption of basement membranes by tumor or inflammatory cells.

Amino Acid Sequence↗
PubMed1992

PDGF AA homodimers are potent chemoattractants for fibroblasts and neutrophils, and for monocytes activated by lymphocytes or cytokines.

The A-chain homodimers of the platelet-derived growth factor (PDGF AA) are widely expressed in normal and transformed cells. The mitogenic properties of PDGF AA are well established; however, the chemotactic potential of PDGF AA remains controversial. We now show that PDGF AA is a strong chemoattractant for human monocytes, granulocytes, and fetal bovine ligament fibroblasts. However, highly purified (greater than 98%) monocytes require the addition of lymphocytes or IL-1 for chemotactic responsiveness to PDGF AA but not for full chemotactic activity with formyl-methionyl-leucyl-phenylalanine (fMLP) or C5a. These results indicate that PDGF AA is a potent chemoattractant. These results also indicate that monocytes require activation either by lymphocytes or exogenous cytokines in order to respond chemotactically to PDGF AA but not to fMLP or C5a and suggest roles of the lymphocyte and cytokine in the chemotactic response of the monocyte to PDGF AA in vivo.

Animals↗