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Biomedical subjects

R M Senior

Publications and source records attributed to R M Senior.

At least 91 records · Page 5Linked to original sources
PubMed1985

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response.

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.

Animals↗
PubMed1984

Nicotine is chemotactic for neutrophils and enhances neutrophil responsiveness to chemotactic peptides.

Neutrophils contribute to chronic bronchitis and pulmonary emphysema associated with cigarette smoking. Nicotine was found to be chemotactic for human neutrophils but not monocytes, with a peak activity at approximately 31 micromolar. In lower concentrations (comparable to those in smokers' plasma), nicotine enhanced the response of neutrophils to two chemotactic peptides. In contrast to most other chemoattractants for neutrophils, however, nicotine did not affect degranulation or superoxide production. Nicotine thus may promote inflammation and consequent lung injury in smokers.

Chemotaxis, Leukocyte↗
PubMed1984

Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts.

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin -producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.

Age Factors↗
PubMed1984

Appearance of chemotactic responsiveness to elastin peptides by developing fetal bovine ligament fibroblasts parallels the onset of elastin production.

We studied chemotaxis to elastin peptides by bovine ligamentum nuchae fibroblasts to determine whether there is a developmental association between chemotactic responsiveness to elastin and expression of the elastin phenotype. Undifferentiated ligament cells demonstrate chemotactic responsiveness to platelet-derived growth factor and fibronectin, known chemoattractants for fibroblasts, but do not show chemotaxis to elastin peptides. After matrix-induced differentiation, however, young cells display a positive chemotactic response to elastin that persists even after the cells are removed from the matrix substratum. Matrix-induced chemotaxis to elastin could be inhibited selectively by incorporation of bromodeoxyuridine into DNA of undifferentiated cells before (but not after) contact with inducing matrix. These results show that the appearance of chemotaxis to elastin peptides parallels the onset of elastin synthesis and suggests that the acquisition of chemotactic responsiveness to elastin and expression of the elastin phenotype are affected by the same inducing elements or processes and may be closely coupled in development.

Animals↗
PubMed1984

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes.

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.

Animals↗
PubMed1984

Platelet-derived growth factor promotes polymorphonuclear leukocyte activation.

The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.

Calcium↗
PubMed1984

Cathepsin G in human mononuclear phagocytes: comparisons between monocytes and U937 monocyte-like cells.

Previous studies demonstrating that a continuous line of human monocyte-like cells (U937) and human monocytes contain elastase probably identical to human neutrophil elastase suggested the possibility that mononuclear phagocytes share other proteinases with neutrophils. The present work establishes that U937 cells contain cathepsin G, an enzyme heretofore found only in neutrophils. U937 cells contain approximately 10 micrograms of cathepsin G-like activity per 10(7) cells, about 25% of the cathepsin G activity in human neutrophils. Normal monocytes have minimal cathepsin G-like activity (approximately 0.1 microgram per 10(7) cells). The cathepsin G-like activity of U937 cells appears to be due to an enzyme that is the same as cathepsin G by several criteria: 1) it is a serine proteinase with activity like cathepsin G against a synthetic chymotrypsin substrate, succinyl-ala-ala-pro-phe-p-nitroanilide; 2) the proteolytic fragments it releases from fibronectin match those released by cathepsin G; 3) like cathepsin G, it can be purified by sequential Trasylol-Sepharose affinity chromatography and carboxymethyl-Sephadex ion exchange chromatography; 4) its amino acid composition and migration on SDS-polyacrylamide gel electrophoresis are indistinguishable from cathepsin G; and 5) it binds with antiserum raised to cathepsin G. The presence of cathepsin G in U937 cells, in much higher concentration than in normal monocytes, indicates either that the content of cathepsin G in monocytes decreases markedly during monocyte differentiation or that U937 cells differ from normal immature monocytes with respect to synthesis of this neutral proteinase.

Amino Acids↗
PubMed1983

Chemotactic activity of platelet alpha granule proteins for fibroblasts.

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.

Animals↗
PubMed1983

Neutral proteinases from human inflammatory cells. A critical review of their role in extracellular matrix degradation.

The cell biology and in vitro activity of neutral proteinases derived from inflammatory cells and the mechanisms by which these enzymes might injure connective tissue are outlined. Current work directed toward understanding local control of tissue degradation may lead to effective strategies for assessing and reducing connective tissue injury in human disease.

Animals↗
PubMed1982

Recruitment of osteoclast precursors by purified bone matrix constituents.

The osteoclast, the multinucleated giant cell of bone, is derived from circulating blood cells, most likely monocytes. Evidence has accrued that is consistent with the hypothesis that the recruitment of monocytes for osteoclast development occurs by chemotaxis. In the present study, we have examined the chemotactic response of human peripheral blood monocytes and related polymorphonuclear leucocytes to three constituents of bone matrix: peptides from Type I collagen, alpha 2-HS glycoprotein, and osteocalcin (bone gla protein). The latter two substances are among the major noncollagenous proteins of bone and are uniquely associated with calcified connective tissue. In chemotaxis assays using modified Boyden chambers, Type I collagen peptides, alpha 2HS glycoprotein, and osteocalcin evoke a dose-dependent chemotactic response in human monocytes. No chemotaxis is observed on PMNs despite their ontogenetic relationship to monocytes and their documented sensitivity to a broad range of other chemical substances. Our observations are consistent with the view that osteoclast precursors (monocytes) are mobilized by chemotaxis, and suggest that the chemoattractants responsible for this activity are derived from the bone matrix or, in the case of collagen and osteocalcin; directly from the osteoblasts which produce them.

Bone Matrix↗
PubMed1982

Elastase of U-937 monocytelike cells. Comparisons with elastases derived from human monocytes and neutrophils and murine macrophagelike cells.

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized (14)C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [(3)H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at approximately 30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.

Amino Acid Chloromethyl Ketones↗
PubMed1982

Chemotaxis of monocytes and neutrophils to platelet-derived growth factor.

The platelet-derived growth factor (PDGF) is shown to be chemotactic for monocytes and neutrophils. Maximum monocyte chemotaxis to PDGF is fully equal to that achieved with C5a and occurs at congruent to 20 ng/ml (congruent to 0.7 nM). Maximum neutrophil chemotaxis is congruent to 60% that achieved with C5A but occurs at congruent to 1 ng/ml (congruent to 32 pM). The chemotactic activity of PDGF is blocked by specific antisera to PDGF and by protamine sulfate, a competitive inhibitor of PDGF binding to cell surfaces. In contrast to PDGF, epidermal growth factor shows no chemotactic activity for inflammatory cells at 0.5-100 ng/ml. The high level of chemotactic activity of PDGF suggests that in addition to its role as a mitogen for smooth muscle cells and fibroblasts, PDGF may be involved in attracting inflammatory cells to sites of platelet release.

Blood Platelets↗
PubMed1982

Chemotactic responses of fibroblasts to tropoelastin and elastin-derived peptides.

Fibroblasts are known to have chemotactic responses to two components of the extracellular matrix, collagen and fibronectin. To extend these observations to other extracellular connective tissue macromolecules and their proteolytic fragments, fibroblasts from adult human skin and from late-gestation (270 d), fetal bovine ligaments were studied for chemotactic responsiveness to tropoelastin and elastin-derived peptides. Bovine ligament tropoelastin and elastin-derived peptides, generated from either human aortic elastin with human neutrophil elastase or from bovine ligament elastin with pancreatic elastase, elicited chemotactic responses that were maximal at 0.2 micrograms/ml (3 X 10(-9) M) and 0.5-2.0 micrograms protein/ml, respectively. Fractionation of the elastin-derived peptides by gel filtration (Bio-Gel P-10) indicated that comparable levels of chemotactic activity were present in all fractions, and amino acid analysis of the fractions showed no relationship between chemotactic activity and desmosine concentration. Taken in conjunction with the observations on tropoelastin, it appears that fibroblast chemotaxis to elastin components does not involve the cross-links of elastin. These results demonstrate that the influences of the connective tissue matrix upon fibroblast migration might include elastin precursors and fragments of elastin.

Chemotaxis↗
PubMed1982

Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro.

Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, (125)I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by alpha(1)-proteinase inhibitor or alpha(2)-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render alpha(1)-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8+/-6.3 and 73.5+/-12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that alpha(1)-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of alpha(1)-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.

Anions↗
PubMed1982

Determination of oxidized alpha-1-proteinase inhibitor in serum.

When alpha-1-PI is oxidized, it loses its ability to inhibit porcine elastase, although it retains its TIC. Therefore the ratio of TIC to EIC increases in proportion to the degree of oxidation of the inhibitor. We have developed a rapid procedure for determining the percentage of oxidized alpha-1-PI in plasma or serum based on the ratio of TIC to EIC of alpha-1-PI in these tissues. The TIC/EIC ratio is not influenced by the other proteins in serum nor by the concentration of alpha-1-PI. When this technique was adapted to measure to proportion of oxidized alpha-1-PI in the serum of young adult, healthy smokers and nonsmokers, 23% oxidized inhibitor was found in the smokers' sera, whereas no oxidized alpha-1-PI was detectable in sera of nonsmokers. Thus smoking leads to oxidation damage to circulating alpha-1-PI. Reduction of the EIC of the alpha-1-PI in the serum of smokers was compensated by a 1.43-fold increase in their serum alpha-1-PI titers; however, these data do not exclude the possibility of local depletion of the absolute EIC in the extravascular space of the lungs. This assay for oxidized alpha-1-PI may be useful in studies of the relationship between oxidation of alpha-1-PI and the development of pulmonary emphysema.

Animals↗
PubMed1982

Characterization of the mannose/fucose receptor on human mononuclear phagocytes.

Cells of the mononuclear phagocyte system contain a cell surface receptor which mediates the uptake of mannose- and fucose-terminated glycoproteins. We have extended the initial studies to human alveolar and monocyte-derived macrophages in culture using two radiolabelled ligands, the synthetic glycoconjugate mannose-bovine serum albumin and the lysosomal glycosidase, beta-glucuronidase. Uptake (37 degrees C) of 125I-mannose-BSA by freshly isolated alveolar macrophages is saturable with increasing concentrations of ligand. Kuptake values in macrophages of smokers and nonsmokers are similar, and resemble earlier reported values using rabbit alveolar macrophages (Kuptake = 40 nM). Uptake of 125I-mannose-BSA in cultured smoker macrophages is identical to that found in fresh cells, and uptake is stable for 5-10 days in culture. Fucose- and mannose-BSA are the most effective inhibitors of uptake, while N-acetylglucosamine-BSA is inhibitory at slightly higher concentrations. Binding (4 degrees C) of 125I-mannose-BSA is likewise ligand concentration dependent (KD = 30 nM). Freshly isolated human monocytes from healthy subjects and patients with cystic fibrosis do not have mannose-specific uptake. However, after monocytes are in culture for 3 days, mannose-specific uptake appears and Kuptake values and specificity of uptake are identical with the results from the alveolar macrophages. No uptake of mannose-BSA could be found in the human monocyte-like cell line, U937.

Animals↗