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R A Stockley

Publications and source records attributed to R A Stockley.

At least 19 recordsLinked to original sources
PubMed1992

The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils.

Apoptosis of neutrophils at sites of inflammation in vivo is thought to lead to their recognition and safe elimination by macrophages. Little is known, however, about the regulation of apoptosis in myeloid cells. We report here that the human promonocytic leukemic cell line, U937, and mature human neutrophils can be induced to become apoptotic when cultured with interleukin-6. Apoptosis of U937 cells, assessed morphologically and by the presence of DNA fragmentation, was increased significantly in a dose-dependent fashion by concentrations of 0.5-100 ng/ml interleukin-6. Apoptosis of U937 cells was evident after 48 h of incubation with 20 ng/ml interleukin-6, and the effect was eliminated by adsorption of interleukin-6 with a specific monoclonal antibody. Apoptosis was not evident in the presence of the differentiating agent phorbol 12-myristate 13 acetate; the induction of apoptosis in U937 cells was not therefore a consequence of differentiation. Apoptosis of mature neutrophils was enhanced after 24 h in culture with interleukin-6. Interleukin-6 might be an important factor in the normal resolution of inflammation through the induction of apoptosis of neutrophils.

Antibodies, Monoclonal↗
PubMed1992

Monocyte adherence to fibronectin: role of CD11/CD18 integrins and relationship to other monocyte functions.

Adherence of monocytes to extracellular matrix components is critical for their accumulation at sites of infection. To gain insight into the factors that regulate monocyte recruitment, we have studied monocyte adherence with regard to the regulatory effects of bacterial lipopolysaccharide (LPS) and the mechanisms involved; moreover, we have contrasted the phenotypes of adherent and nonadherent cells. Our results show that only a minor subpopulation of monocytes (20-25%) adhere spontaneously to fibronectin and that LPS stimulated a threefold increase in the proportion of adherent cells. Basal adherence and LPS-stimulated adherence of monocytes to fibronectin were substantially mediated by CD11/CD18 integrins. Further studies revealed that spontaneously adherent monocytes were 14-fold more actively phagocytic, released 1.6-fold more superoxide anion, and contained 20-fold more peroxidase activity than nonadherent cells, whereas LPS-adherent cells had an intermediate phenotype. These results indicate that LPS may enhance the accumulation of monocytes with an antimicrobial phenotype and thereby promote resolution of tissue infection.

Antigens, CD↗
PubMed1992

Use of an enzyme-linked immunosorbent assay to assess penetration of amoxicillin into lung secretions.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure total amoxicillin concentrations penetrating lung secretions, which were compared with "active" concentrations measured by conventional bioassay. An antibody was raised in rabbits to amoxicillin conjugated to bovine serum albumin and used in a competitive binding ELISA (sensitivity, 10 ng/ml; precision [coefficient of variation], 9%). The measurement of amoxicillin in lung secretions by using the ELISA method was verified by high-performance liquid chromatography. Amoxicillin concentrations were found to be similar in both whole sonicated sputum and sol-phase sputum obtained by ultracentrifugation following single oral doses of 3 g (4.6 mg/liter for sonicated and 4.7 mg/liter for sol-phase preparations) and 250 mg (0.23 mg/liter for both preparations). Eight patients with bronchiectasis received 500 mg of amoxicillin three times daily. On the second day of therapy (4 h after the morning dose), the mean concentration of amoxicillin in sputum was 0.88 mg/liter (standard error of the mean [SEM], 0.11) by ELISA and 0.40 mg/liter (SEM, 0.05) by bioassay, suggesting a significant degree of local inactivation. This difference between total and active amoxicillin levels was found to correlate significantly (r = 0.693; P less than 0.05) with beta-lactamase levels (mean, 29.5 mU/ml; SEM, 9.4). A pharmacokinetic study on day 3 revealed maximum levels in secretions 2 to 4 h after dosing (mean, 1.36 mg/liter; SEM, 0.26). At the end of successful therapy (day 14), total and active levels were lower (mean, 0.48 mg/liter; SEM, 0.11 [total]; mean, 0.21 mg/liter; SEM, 0.06 [active]); this result was associated with a reduction in lung inflammation (decreased serum-derived albumin in the lung secretions). In conclusion, antibiotic penetration is partly dependent on the degree of lung inflammation. The differences observed in total and active levels of amoxicillin and the relationship to beta-lactamase activity in sputum suggest why higher doses of antibiotic may be required to produce a therapeutic response in some patients.

Administration, Oral↗
PubMed1992

Increased adherence of monocytes to fibronectin in bronchiectasis. Regulatory effects of bacterial lipopolysaccharide and role of CD11/CD18 integrins.

Regulated adherence of monocytes to extracellular matrix is a prerequisite for accumulation of mononuclear phagocytes during pulmonary infection and inflammation. We have obtained monocytes from patients with an inflammatory lung disease (bronchiectasis) and from control subjects and have compared their adherence to fibronectin. Spontaneous adherence of monocytes from the control subjects was 20 +/- 2%, whereas that of patients' cells was markedly higher and correlated with the severity of airway inflammation: 65 +/- 5% and 40 +/- 8% in patients with purulent and mucoid sputum, respectively. Endotoxin and cytokines from areas of airway disease are likely to be responsible for the observed monocyte activation, since: (1) endotoxin was detectable in all of the patients but in none of the control subjects; (2) LPS produced a dose-related increase in adherence of normal monocytes in vitro (maximal 65 +/- 2% adherence at 1 microgram/ml of LPS); (3) recombinant cytokines and LPS produced additive effects on monocyte adherence in vitro. The adherence of the patients' monocytes to fibronectin was substantially mediated by CD11/CD18 integrins, via both RGD-dependent and RGD-independent mechanisms. These data indicate that signals arising from foci of infection and inflammation can influence the adherence of monocytes, and they are likely to be determinants of the accumulation of mononuclear phagocytes in the lungs of patients with bronchiectasis.

Adult↗
PubMed1992

Regulation of alpha 1-antitrypsin synthesis by granulocyte macrophage colony-stimulating factor in the U937 promonocytic cell line.

Undifferentiated U937 cells possess low numbers (approximately 1,100 per cell) of receptors for granulocyte macrophage colony-stimulating factor (GMCSF). The receptors present are of at least two types with different affinities. A small number (less than 200 per cell) are of higher affinity (approximate Kd 97pM), and a larger number (approximately 900 per cell) are of lower affinity (approximate Kd 680pM). Following differentiation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA), the differentiated cells express a reduced number of receptors (approximately 680 per cell). These receptors are present as a single class with an approximate affinity of 230pM (Kd). The undifferentiated cells were responsive to GMCSF and this cytokine caused a seven-fold increase in accumulation of alpha 1-antitrypsin (alpha 1AT) compared to the control cells. This was accompanied by a similar increase in alpha 1ATmRNA, which suggests that the rate of alpha 1AT accumulation was regulated at the transcriptional level. After differentiation with PMA, alpha 1AT accumulation was not influenced by GMCSF, although the cells continued to bind the cytokine. These results imply that U937 cells do respond to GMCSF and this event involves binding of the ligand to a small number of specific cell surface receptors only present in the undifferentiated cells. Furthermore the response of these cells (in terms of alpha 1AT production) is associated with the type of GMCSF receptors expressed.

Cell Differentiation↗
PubMed1991

Effects of neutrophil adherence on the characteristics of receptors for tumor necrosis factor-alpha.

Human recombinant [125I]TNF-alpha was incubated with non-adherent human neutrophils, cells adherent to fibronectin-coated plastic, or adherent cells scraped into suspension (post-adherent). Binding of TNF to all cells increased with doses of added TNF but adherent cells bound little TNF. Binding of TNF by post-adherent cells was greater than when adherent, but still significantly less than that of non-adhered neutrophils, suggesting that TNF receptors were relocated on the adherent surface of neutrophils. Scatchard analysis showed that adherent cells expressed significantly fewer TNF receptors, but of higher affinity, than non-adherent cells. The results suggest that altered expression of TNF receptors might contribute to the differential effects of TNF on adherent and non-adherent neutrophils.

Cell Adhesion↗
PubMed1991

The effect of in vitro and in vivo dexamethasone on human neutrophil function.

There is a significant fall in PMN chemotaxis to the peptide FMLP in response to increasing concentrations of dexamethasone in vitro. The response fell in a dose related manner from a control value of 53.7 SE +/- 9.6 cells per high power field (cpf) to 47.3 SE +/- 8.1 at 10(-6) M (p less than 0.05) and 24.7 +/- 8.9 at 10(-3) M (p less than 0.025). A similar response was observed for the chemoattractants zymosan activated serum and the sol phase of purulent sputum. The effect was independent of protein synthesis or the period of incubation. Twelve milligrams of dexamethasone taken daily by 6 healthy volunteers resulted in a significant (p less than 0.025) reduction in the chemotactic response of PMN to 10(-8) M FMLP (from 29.5 +/- 1.55 to 13.7 +/- 1.8 cpf) which was apparent within 2 hours of taking the first dose. This effect was sustained for the three days on which dexamethasone was taken but returned to normal 7 days after the last dose had been administered. Dexamethasone therapy had no effect on unstimulated PMN superoxide anion production either in vitro or in vivo. The in vivo effect on neutrophil function occurred at mean serum dexamethasone concentrations of 1.26 (+/- 0.28) X 10(-7) M on day 1, 1.44 (+/- 0.15) X 10(-7) M on day 2 and 1.31 (+/- 0.13) X 10(-7) M on day 3. Thus we conclude that dexamethasone concentration which inhibit PMN chemotaxis in vivo are much lower than those required to exert the same effect in vitro.

Chemotaxis, Leukocyte↗
PubMed1991

Immunoglobulin response to intravenous streptokinase in acute myocardial infarction.

OBJECTIVE: To devise assays to assess and follow the specific antibody response in patients treated with streptokinase for acute myocardial infarction. DESIGN: Venous blood samples were collected before treatment with streptokinase started and subsequently at regular intervals over one year. Specific IgG and subclass IgG1 were assessed by an enzyme linked immunosorbent assay. SETTING: Coronary care unit in a general hospital. PATIENTS: 48 patients with acute myocardial infarction: 22 patients had venous blood samples taken at presentation only; serial blood samples were taken from 20 patients who then received thrombolytic therapy with streptokinase and six patients who were unsuitable for thrombolytic therapy. RESULTS: Titres of antibodies to streptokinase were low at presentation in 36 (75%) of the 48 patients. Serial measurements made in 20 patients showed the virtual disappearance of antibody within the first 24 hours. This was followed by a steady increase in the specific IgG1 titre, which peaked at day 14 before gradually declining. Values at one year remained significantly higher than baseline values. There was no evidence of an IgM response in the patients studied. CONCLUSION: Low titres of antibodies to streptokinase were widespread in the population. Antibody was consumed after treatment and the subsequent immunoglobulin rise suggested a secondary immune responses; the recently described neutralising capacity to streptokinase is probably related to this antibody.

Acute Disease↗
PubMed1991

Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium.

It has been suggested that proteinase enzymes could play an important role in the pathogenesis of chronic bronchial infections including bronchiectasis and cystic fibrosis (CF). Because Pseudomonas aeruginosa frequently colonizes the respiratory tract in bronchiectasis and CF, we examined the in vitro effects of human neutrophil elastase (HNE) and proteinase enzymes produced by P. aeruginosa (elastase: PE; alkaline proteinase: PAP) on the ciliary beat frequency (CBF) and ultrastructure of human nasal ciliated respiratory epithelium. HNE (500 micrograms/ml) progressively reduced CBF and caused marked epithelial disruption; lower concentrations (100 and 20 micrograms/ml) also caused epithelial disruption but without slowing CBF. The effects of HNE (500 micrograms/ml) were completely abolished by adding alpha 1-antitrypsin (5 mg/ml). There was no synergy between HNE and pyocyanin, a product of P. aeruginosa which slows CBF. PE in phosphate-buffered saline also caused epithelial disruption without slowing CBF; however, PE in medium containing divalent metal ions caused CBF slowing as well as epithelial disruption at 100 micrograms/ml. PAP (500 micrograms/ml) had almost no effect on ciliated epithelium. The effects of HNE and PE on nasal and bronchial epithelium obtained from the same patient were similar. Light and transmission electron microscopy revealed that HNE and PE were cytotoxic and caused detachment of epithelial cells from neighboring cells and the basement membrane. There was cytoplasmic blebbing of the cell surface and mitochondrial damage; however, no increase of abnormalities in the ultrastructure of cilia on living cells was seen. These results support the hypothesis that HNE and PE contribute to the delayed mucociliary clearance and epithelial damage that is observed in patients with chronic bronchial infection.

Bronchi↗
PubMed1991

Extracellular proteolysis of fibronectin by neutrophils: characterization and the effects of recombinant cytokines.

We have used 125I-labeled fibronectin (FN) as an extracellular substrate for neutrophils (PMN) in order to investigate the mechanism responsible for FN solubilization by PMN and the effects of recombinant cytokines on this process. Pure active alpha 1-antitrypsin (alpha 1AT), when added to PMN before or during, but not after, adherence to FN, inhibited solubilization of the substrate in a dose-dependent manner, but alpha 1AT that had been inactivated by proteolysis or oxidation and alpha 1AT Pittsburgh (alpha 1AT 358Met-Arg) had no significant effect. The solubilization of FN was also inhibited by the PMN elastase inhibitor N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone but not by the chymotrypsin and cathepsin G inhibitor N-Cbz-glycyl-glycyl-phenylalanine-chloromethylketone, nor by catalase or superoxide dismutase. The products of solubilization of FN by PMN, analyzed by sodium dodecyl sulphate polyacrylamide electrophoresis, were similar to those produced by pure PMN elastase but not cathepsin G. These results suggest that FN solubilization by PMN is caused largely by the pericellular activity of PMN elastase. The solubilization of FN by PMN was increased significantly by adding tumor necrosis factor-alpha, interleukin-1 alpha, or interferon-gamma to the adherent cells but without a significant general release of elastase into the culture supernatants. Granulocyte/macrophage colony-stimulating factor (GM-CSF) had no significant effect. None of the cytokines had any effect when preincubated with the cells in suspension, and non increased FN solubilization by PMN incubated with the optimal (10(-6) mol/liter) or suboptimal dose (10(-8) mol/liter) of the peptide formylmethionylleucylphenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1990

Changes in the expression of elastase and cathepsin B with differentiation of U937 promonocytes by GMCSF.

The human promonocytic cell line, U937, when treated for up to 72h with 12,O,tetradecanoyl-phorbol-13-acetate or granulocyte-macrophage colony-stimulating factor, exhibited increased phagocytic activity and expression of the marker p150/95. There was an associated increase in the monocyte proteinase cathepsin B and its mRNA but decreased cellular levels of neutrophil elastase and elastase mRNA. Granulocyte-macrophage colony-stimulating factor therefore causes differentiation of U937 cells, with appropriate effects on the synthesis of leukocyte proteinases.

Cathepsin B↗
PubMed1990

Assessment of soluble parameters in sputum.

The concentrations of soluble protein in lung secretions are dependent upon the degree of inflammation, local production and cell activation. Infections and corticosteroid therapy will alter the protein content of lung secretions as a result of their effect on lung inflammation.

Humans↗
PubMed1990

Inflammatory indices for chronic bronchitis and COAD. Proteases and antiproteases.

The study of lung secretion proteinases and their inhibitors, both functionally and quantitatively, provide information concerning the degree of inflammation in the lung. Interpretation of individual parameters may be difficult in isolation. However, a general pattern of response provides supportive evidence of a reduction in lung inflammation, including 1) reduction in plasma inhibitor concentrations, 2) increase in local inhibitor concentrations, 3) increase in enzyme inhibition, and 4) reduction in enzyme activity. Thus such changes provide an objective basis for the assessment of drug therapy.

Bronchitis↗
PubMed1990

IgA subclasses in sputum from patients with bronchiectasis.

The concentrations of IgA1 and IgA2 were measured in the serum and sputum from 27 clinically stable patients with bronchiectasis. Of the 27 patients, nine were regularly producing mucoid secretions, nine mucopurulent and nine purulent secretions. No significant differences were observed in serum IgA2 concentrations between the patient groups, although IgA1 concentrations were higher in the serum from the patients producing mucopurulent sputum than those producing mucoid or purulent sputum. The sputum concentrations of both IgA1 and IgA2 increased significantly with purulence. Furthermore, there was a significant increase in the proportion of IgA2 as the secretions became more purulent. The sputum concentrations of both IgA subclasses indicated significant local synthesis, with increased production (especially of IgA2) in the presence of inflammation due to infection.

Bronchiectasis↗
PubMed1990

Effect of non-steroidal anti-inflammatory drugs on neutrophil chemotaxis--an in vitro and in vivo study.

We have studied the effects of the two non-steroidal anti-inflammatory drugs (NSAIDs), nabumetone and indomethacin, on neutrophil chemotaxis in vitro and in vivo. When used in therapeutic concentrations in vitro, neither agent had any effect on the chemotactic response of neutrophils isolated from healthy volunteers. This was true for the three chemotactic agents studied: FMLP, zymosan activated serum and purulent sputum. Nabumetone and indomethacin decreased neutrophil chemotaxis over a period of 2 weeks in 12 normal subjects in vivo. The average chemotactic response to 10(-8) mol/l FMLP for all 12 during the control period was 42.1 +/- 6.1 cells per high power field and this fell to 26.1 +/- 4.9 (P less than 0.025) after 7 days and to 15.6 +/- 2.5 (P less than 0.005) after 14 days. The results were similar for both drugs analysed independently. The results suggest that NSAIDs have no effect on the chemotactic response of mature cells in vitro, but suppress chemotaxis progressively when given in vivo. This may be explained by an effect of NSAIDs on maturing cells prior to release into the circulation.

Adult↗
PubMed1990

Neutrophil activation--an important cause of tissue damage during liver allograft rejection?

Although neutrophils have been implicated as mediators of tissue damage in a variety of conditions, their role in graft rejection has not previously been studied. Peripheral neutrophil activation was assessed sequentially in 16 patients following liver transplantation by measuring the ability of neutrophils to respond to the chemotactic peptide FMLP, to release superoxide radicals, and to cause extracellular proteolysis. All three functions increased 1-2 days before episodes of acute rejection and only returned to normal after treatment with high-dose corticosteroids. Three patients who did not develop rejection showed no increase in neutrophil function. Lymphocytes isolated from patients with acute rejection produced factors that were both chemotactic for and capable of activating normal neutrophils. This study shows that neutrophil activation occurs during acute liver allograft rejection, possibly in response to lymphokine secretion, and suggests an important, and hitherto unrecognized, mechanism of graft damage during rejection.

Adult↗