Lung infections. 1. Role of bacteria in the pathogenesis and progression of acute and chronic lung infection.
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Acute Disease↗
Biomedical subjects
R A Stockley
Publications and source records attributed to R A Stockley.
IgG subclasses in the serum and sputum from patients with bronchiectasis.
BACKGROUND: IgG subclass deficiency is often associated with recurrent pulmonary infections. The prevalence of deficiency in a large well characterised group of patients with bronchiectasis has not previously been established. METHODS: Serum IgG subclass concentrations in 89 patients with bronchiectasis were compared with those obtained from a group of 82 age and sex matched normal healthy controls. Sputum IgG subclass concentrations were also assessed in 44 of the patients. Albumin was measured as a marker of protein transudation from plasma to determine the degree of local IgG subclass production. RESULTS: The serum concentrations of IgG1, IgG2 and IgG3 were increased in the patients compared with the control group whereas IgG4 concentrations were not. There was an overall incidence of deficiency of 1% for subclasses 1-3 and 5% for subclass 4 in patients with bronchiectasis based on observed concentrations being below the lower limit of the control group range. The concentrations of IgG subclasses in sputum were partly dependent upon the degree of inflammation as assessed by the macroscopic appearance of purulence. A comparison of the ratio of sputum:serum subclass concentration and sputum:serum albumin, however, revealed that all of the subclasses were present at greater concentrations than could be accounted for by transudation alone. CONCLUSIONS: A new normal control range for serum IgG subclasses has been established and from this range it was found that IgG subclass deficiency in a group of unselected patients with bronchiectasis was comparatively rare. A significant degree of local IgG production was also suggested in the lungs of these patients.
Adult↗
Commentary: bronchiectasis and inflammatory bowel disease.
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Bronchiectasis↗
The chemotactic activity of sputum from patients with bronchiectasis.
Persistent polymorphonuclear neutrophil (PMN) recruitment to airway is thought to be an important component of continuing inflammation and progression of chronic destructive lung diseases. Although chemoattractants are required for the PMN to migrate, the nature of the chemoattractants in the airways has not yet been clarified. We therefore investigated the contribution of interleukin-8 (IL-8) and leukotriene-B4 (LTB4) to the chemotactic activity of lung secretions by inhibiting their activity using a monoclonal antibody to IL-8 and an LTB4 receptor antagonist (LY293111 sodium). Fifty-nine sputum samples obtained from 19 patients with bronchiectasis were studied. In preliminary studies the chemotactic responses to IL-8 and LTB4 were found to be additive, and we were able to remove their contribution independently with the appropriate antibody and antagonist. The chemotactic activity of the secretions was related to the macroscopic appearance (mucoid, mucopurulent, and purulent), and this appeared to be related to an increase in IL-8 contribution. Chemotactic activity was reduced by antibiotic therapy and again that seemed to relate to a reduction in the IL-8 contribution. The contributions of LTB4 were similar among the three types of sputum in varying clinical states. These data suggest that LTB4 and IL-8 are important chemotactic factors in lung secretions from such patients, although IL-8 appears to play a more important role during acute exacerbations. These results may be useful in determining therapeutic strategies for chronic destructive lung diseases in the future.
Acute Disease↗
Proteolytic enzymes and airway diseases.
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Asthma↗
The distribution of antibodies to streptokinase.
To determine the distribution of antibodies to streptokinase that might be anticipated in patients requiring treatment with streptokinase, specific anti-streptokinase antibody titres were determined in a group of subjects from the general population and in a group of patients presenting with acute myocardial infarction. Enzyme-linked immunosorbent assays were developed to measure specific anti-streptokinase IgG and subclass IgG1 in 95 subjects from the general population and in 160 patients presenting with acute myocardial infarction. Low titres of IgG1 were found in both the general population (median = 5; range: 0-490) and in the myocardial infarction group (median = 7; range: 0-2000). A minority of subjects in both groups had high titres. The findings suggest that low titres of antibody are widespread in the population. The minority of subjects in both groups who had high titres may explain the infrequent type III immune reactions encountered with streptokinase.
Adult↗
Effect of fluticasone propionate on sputum of patients with chronic bronchitis and emphysema.
The effects of fluticasone propionate (FP) on sputum chemotactic activity, elastase inhibitory potential, albumin concentrations, and peripheral neutrophil function were studied in a group of patients with clinically stable, smoking-related chronic bronchitis and emphysema. Seventeen patients (50 to 75 yr of age) were entered into a double-blind, placebo-controlled study of 1.5 mg inhaled FP/d for 8 wk. Following treatment with FP the chemotactic activity of the sputum sol phase was lower than the corresponding values for the placebo group (p < 0.01). Values fell from a mean of 21.75 (+/- 1.58) during the run-in period to 18.37 (+/- 1.46; p < 0.01) after 4 wk and 17.63 (+/- 1.86; p < 0.05) after 8 wk treatment returning to 22.08 (+/- 1.26) cell/field after the washout period. The neutrophil elastase inhibitory capacity of the sputum sol phase increased (p < 0.025) with treatment from a mean of 0.177 microM elastase inhibited/L (+/- 0.05) pretreatment to 0.413 microM (+/- 0.054) after 4 wk and 0.415 microM (+/- 0.054) after 8 wk returning to 0.270 microM (+/- 0.07) after the washout period. Treatment with FP did not result in a change in the peripheral neutrophil functions studied or sputum albumin and myeloperoxidase concentrations. The results suggest that FP may play a protective role in these patients through a reduction in the chemotactic activity of lung secretions and potentially a reduction in the recruitment of neutrophils to the lung, and also by directly affecting the proteinase/antiproteinase balance, in favor of antiproteinases, within lung secretions.
Administration, Inhalation↗
Role of inflammation in respiratory tract infections.
In respiratory tract infections, leukocytes are recruited to the lungs, where they help remove invading organisms through phagocytic clearance. In some instances, however, the normal inflammatory response may be as destructive as it is defensive. The neutrophils secrete a proteolytic enzyme, neutrophil elastase, which has emerged as a major culprit in the pathogenesis of inflammatory airway disease. This enzyme has been found to strip the bronchial epithelium, reduce ciliary beating, and stimulate excess mucus secretion, leading to mucus retention, bacterial proliferation, and recurrent infections. Neutrophil elastase also stimulates epithelial cell interleukin-8 secretion and produces other chemoattractant cleavage products that lead to further neutrophil recruitment. It also impairs host defenses by damaging the major opsonophagocytic receptor on the neutrophil and by weakening the efficacy of immunoglobulins. For this reason, an important goal in the treatment of respiratory tract infections should be to prevent or shorten the duration of neutrophil elastase release. A number of different approaches have been proposed or attempted in an effort to modulate the proteinase burden. These include direct inhibition of elastase and interference with the recruitment, adherence, and degranulation of neutrophils. An even more fundamental approach would start in the bone marrow, where attempts might be made to modulate or modify neutrophil precursors so as to limit the destructive potential of the mature neutrophils.
Bone Marrow Cells↗
The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.
Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.
Amino Acid Chloromethyl Ketones↗
Changes in protein kinase C isoenzyme expression associated with apoptosis in U937 myelomonocytic cells.
Spontaneously apoptotic U937 cells from exponentially growing cell cultures were enriched on discontinuous Percoll density gradients. Increased PKC-beta and reduced PKC-zeta expression were detected in apoptotic cells by Western blotting. Using confocal microscopy, changes in the level of PKC isoenzymes were confirmed and in addition alterations in the subcellular location of PKC isoenzymes were detected in apoptotic cells compared with nonapoptotic cells. The data indicate that the expression of specific PKC isoenzymes is modulated during apoptosis and that PKC-beta and PKC-zeta may play specific roles in the regulation of the apoptotic program.
Apoptosis↗
Simple method for quantifying viable bacterial numbers in sputum.
AIMS: To establish a simple method of quantitative culture for determining the viable bacterial numbers present in expectorated sputum samples. METHODS: Sputum samples were homogenised with dithiothreitol, sterile saline or glass beads to determine which method recovered the greatest number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacterial viability. Sputum samples homogenised with dithiothreitol were diluted in sterile saline and sampled using either standard bacteriological loops or a precision pipette to determine which method resulted in the least variation. RESULTS: Homogenisation of sputum using dithiothreitol increased the recovery of viable bacteria compared with sterile glass beads and/or saline, with no apparent effect on bacterial viability when incubated with culture broths. By inoculating agar plates with 10(-3), 10(-4) and 10(-5) dilutions of the homogenised sputum sample, all potential pathogens could easily be identified. A 10 microliter sample volume dispensed by precision pipette and spread with a "hockey stick" resulted in the least variation between plates (less than 16%) and an even distribution of bacterial colonies. Numbers of viable bacteria recovered from different aliquots of individual sputum samples were generally of the same order of magnitude. CONCLUSIONS: This method represents a relatively quick and simple technique for accurately quantifying viable bacteria present in sputum samples. The use of a small portion appears to be representative of the sample as a whole.
Colony Count, Microbial↗
Neutrophil elastase and cathepsin G protein and messenger RNA expression in bone marrow from a patient with Chediak-Higashi syndrome.
Aims-To determine whether neutrophil elastase and cathepsin G are expressed, at transcriptional or translational levels, in the bone marrow from a patient with Chediak-Higashi syndrome.Methods-Blood neutrophils were isolated from three patients with Chediak-Higashi disease and bone marrow was collected from one. Cell lysates were analysed for neutrophil elastase and cathepsin G activity by enzyme linked immunosorbent assay and western immunoblotting. Northern blotting was used to detect messenger RNA (mRNA) for cathepsin G, elastase and beta-actin in bone marrow extracts, and immunohistochemistry was used to localise the enzymes in marrow myeloid cells.Results-Elastase and cathepsin G were not detected in blood neutrophils from the patients with Chediak-Higashi disease, but were present in bone marrow cells, although immunohistochemistry showed they were not within cytoplasmic granules. The concentrations of elastase and cathepsin G in Chediak-Higashi bone marrow were about 25 and 15%, respectively, of those in normal marrow. Quantitative scanning of northern blots showed that elastase and cathepsin G mRNA, corrected for beta-actin mRNA, were expressed equally in normal marrow.Conclusions-Transcription of elastase and cathepsin G mRNA in promyelocytes of patients with Chediak-Higashi disease is normal, but the protein products are deficient in these cells and absent in mature neutrophils. This suggests that the translated proteins are not packaged into azurophil granules but are degaded or secreted from the cells.
Journal Article↗
The isolation and characterization of non-typeable Haemophilus influenzae from the sputum of adult cystic fibrosis patients.
The role of non-typeable Haemophilus influenzae in cystic fibrosis (CF) remains unclear. We wanted, therefore, to determine the presence and characteristics of non-typeable H. influenzae in sputum samples from patients with CF. In order to do this, we have assessed sputum samples from 55 consecutive clinically stable patients seen routinely at an adult CF out-patient clinic. Quantitative bacterial culture was performed using a selective media containing cefsoludin, and isolates were characterized by biotyping and outer membrane protein profile analysis. In 17 (30%) of these samples, non-typeable H. influenzae was isolated and was present in similar viable numbers (mean 7.7 x 10(8) colony-forming units (cfu).mL-1; SEM 3.1) to Pseudomonas aeruginosa (mean 8 x 10(8) cfu.mL-1: SEM 2.4). All non-typeable H. influenzae isolates recovered were beta-lactamase negative and sensitive to a range of antibiotics. Several biotypes and outer membrane protein profiles were observed, with no apparent association between these two phenotypic characteristics. The study showed that large numbers of non-typeable H. influenzae are often present in sputum from adult patients with CF. Further longitudinal studies of outer-membrane protein profile analysis are required to determine the dynamics of non-typeable H. influenzae colonization in individual patients and the clinical significance.
Adolescent↗
The pathogenesis of chronic obstructive lung diseases: implications for therapy.
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Humans↗
In vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion.
Bronchiectasis is associated with sputum containing high levels of the proteolytic enzyme elastase, which is thought to be involved in the pathogenesis of the disease. Agents which inhibit neutrophil function and interfere with neutrophil elastase release may have a beneficial effect on the development and progression of such diseases. We have studied the effects of the nonsteroidal anti-inflammatory agent indomethacin on neutrophil function in nine patients with clinically stable bronchiectasis. All patients remained clinically stable during the study. We observed a significant reduction in peripheral neutrophil chemotaxis to 10 nmol.L-1 N-formyl-methionyl-leucyl-phenylalanine (FMLP) from a mean of 19.86 (SEM 1.35) to 8.46 (0.68) cells.field-1 after 4 weeks of therapy. There was also a significant reduction in fibronectin degradation both by resting and FMLP-stimulated neutrophils, from a mean of 1.90 (0.19) micrograms x 3 x 10(5) cells at the start of therapy to 0.87 (0.08) micrograms after 4 weeks, and from 3.17 (0.35) micrograms to 1.48 (0.05) micrograms, respectively. There was no effect on spontaneous or stimulated superoxide anion generation by neutrophils. Despite the marked changes in peripheral neutrophil function, no adverse effect was observed on viable bacterial load in the bronchial secretions. In addition, there was no difference in sputum albumin, elastase or myeloperoxidase levels, and only minor changes in the chemotactic activity of the sputum. These results suggest that nonsteroidal anti-inflammatory agents have a major effect on peripheral neutrophil function but do not appear to have an adverse effect on bacterial colonization of the airways.
Adult↗
The effect of the Z mutation on the ability of alpha 1-antitrypsin to prevent neutrophil mediated tissue damage.
Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.
Connective Tissue↗
Overt and subclinical reactions to streptokinase in acute myocardial infarction.
The aims of this study were (1) to assess the possibility of predicting allergic reactions to streptokinase (SK) by measuring pretreatment antibody titers and by intradermal skin testing, and (2) to determine if SK is associated with subclinical changes in renal function. Specific anti-SK immunoglobulin G (IgG) and subclass IgG1 were assessed by enzyme-linked immunosorbent sorbent assays, and renal function was assessed by measurement of serum urea and creatinine in 204 patients with acute myocardial infarction. Twenty-six patients had 24-hour proteinuria loss and creatinine clearance assessed at presentation. Median IgG titer at presentation was 6 (range 0 to 10,000), and increased to 60 (range 0 to 18,000; p < 0.0001) on day 6. Fifteen of 180 patients (8.3%) had minor allergic reactions to SK; the median titer on admission for these patients was 5 (range 0 to 60), identical to those who tolerated SK uneventfully. No change was seen in serum urea or creatinine; for those treated with SK, the median value for proteinuria loss at day 0 was 0.45 g/liter (range 0.1 to 2), and decreased by day 5 to 0.1 g/liter (range 0.1 to 0.8; p = 0.0027). No significant proteinuria was seen in those who did not receive SK. The reactions to SK were minor, and could not be predicted on the basis of IgG titers at presentation. Significant proteinuria was found in the first 24 hours in SK-treated patients, but not in those who did not receive SK, and it resolved by day 5.
Drug Hypersensitivity↗
Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.
The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.
Calcium↗