Helen C. Steel

Proinflammatory interactions of pneumolysin with human neutrophils.

The effects of pneumolysin on the proinflammatory activity of human neutrophils, as well as on cation fluxes in these cells, have been investigated. Superoxide production, release of elastase, CR3 expression, phospholipase A2 activity, and alterations in membrane potential were measured by use of lucigenin-enhanced chemiluminescence and colorimetric, flow cytometric, radiometric, and spectrofluorimetric procedures, respectively; and cation fluxes were measured by use of 45Ca2+ and 86Rb+ and by fura-2 spectrofluorometry. Pneumolysin at concentrations >1.67 ng/mL caused influx of Ca2+ and increased phospholipase A2 activity and CR3 expression, which were associated with enhanced superoxide production and release of elastase after activation of the cells with the chemotactic tripeptide FMLP. At the same concentrations, pneumolysin caused efflux of K+ and membrane depolarization. The effects of pneumolysin on cation fluxes were not attributable to inhibition of Ca2+-adenosine triphosphatase (ATPase) or Na+, K+-ATPase. Pneumolysin potentiates the proinflammatory activities of neutrophils by a pore-forming mechanism resulting in Ca2+ influx.

Pathogen- and host-directed anti-inflammatory activities of macrolide antibiotics.

Macrolide antibiotics possess several, beneficial, secondary properties which complement their primary antimicrobial activity. In addition to high levels of tissue penetration, which may counteract seemingly macrolide-resistant bacterial pathogens, these agents also possess anti-inflammatory properties, unrelated to their primary antimicrobial activity. Macrolides target cells of both the innate and adaptive immune systems, as well as structural cells, and are beneficial in controlling harmful inflammatory responses during acute and chronic bacterial infection. These secondary anti-inflammatory activities of macrolides appear to be particularly effective in attenuating neutrophil-mediated inflammation. This, in turn, may contribute to the usefulness of these agents in the treatment of acute and chronic inflammatory disorders of both microbial and nonmicrobial origin, predominantly of the airways. This paper is focused on the various mechanisms of macrolide-mediated anti-inflammatory activity which target both microbial pathogens and the cells of the innate and adaptive immune systems, with emphasis on their clinical relevance.

Overview of Community-Acquired Pneumonia and the Role of Inflammatory Mechanisms in the Immunopathogenesis of Severe Pneumococcal Disease

Community-acquired pneumonia (CAP) remains a leading cause of morbidity and mortality among the infectious diseases. Despite the implementation of national pneumococcal polyvalent vaccine-based immunisation strategies targeted at high-risk groups, Streptococcus pneumoniae (the pneumococcus) remains the most common cause of CAP. Notwithstanding the HIV pandemic, major challenges confronting the control of CAP include the range of bacterial and viral pathogens causing this condition, the ever-increasing problem of antibiotic resistance worldwide, and increased vulnerability associated with steadily aging populations in developed countries. These and other risk factors, as well as diagnostic strategies, are covered in the first section of this review. Thereafter, the review is focused on the pneumococcus, specifically the major virulence factors of this microbial pathogen and their role in triggering overexuberant inflammatory responses which contribute to the immunopathogenesis of invasive disease. The final section of the review is devoted to a consideration of pharmacological, anti-inflammatory strategies with adjunctive potential in the antimicrobial chemotherapy of CAP. This is focused on macrolides, corticosteroids, and statins with respect to their modes of anti-inflammatory action, current status, and limitations.

Pneumolysin Potentiates Production of Prostaglandin E2 and Leukotriene B4 by Human Neutrophils

ABSTRACT Exposure to pneumolysin (8.37 and 41.75 ng/ml) caused a calcium-dependent increase in the generation of prostaglandin E2 and leukotriene B4 by both resting and chemoattractant-activated human neutrophils in vitro. These interactions of pneumolysin with neutrophils may result in dysregulation of inflammatory responses during pneumococcal infection.

Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease.

The relationship between activation of NADPH‐oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N‐formyl‐ l‐methionyl‐ l‐leucyl‐ l‐phenylalanine (FMLP; 1 μm) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store‐operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 μm) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH‐oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.

Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Background and purpose:  The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1–2 µmol·L−1), on Ca2+‐dependent pro‐inflammatory activities, cytosolic Ca2+ fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (1 µmol·L−1) and platelet‐activating factor (200 nmol·L−1).

Cysteinyl leukotriene receptor-1 antagonists as modulators of innate immune cell function

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

Pneumolysin‐mediated activation of NFκB in human neutrophils is antagonized by docosahexaenoic acid

This study was designed to investigate the relationship between influx of extracellular Ca2+, activation of NFκB and synthesis of interleukin‐8 (IL‐8) following exposure of human neutrophils to subcytolytic concentrations (8·37 and 41·75 ng/ml) of the pneumococcal toxin, pneumolysin, as well as the potential of the omega‐3 polyunsaturated fatty acid, docosahexaenoic acid, to antagonize these events. Activation and translocation of NFκB were measured using a radiometric electrophoretic mobility shift assay, while influx of extracellular Ca2+ and synthesis of IL‐8 were determined using a radioassay and an ELISA procedure, respectively. Exposure of neutrophils to pneumolysin was accompanied by influx of Ca2+, activation of NFκB, and synthesis of IL‐8, all of which were eliminated by inclusion of the Ca2+‐chelating agent, EGTA (10 m m), in the cell‐suspending medium, as well as by pretreatment of the cells with docosahexaenoic acid (5 and 10 µg/ml). The antagonistic effects of docosahexaenoic acid on these pro‐inflammatory interactions of pneumolysin with neutrophils were not attributable to inactivation of the toxin, and required the continuous presence of the fatty acid. These observations demonstrate that activation of NFκB and synthesis of IL‐8, following exposure of neutrophils to pneumolysin are dependent on toxin‐mediated influx of Ca2+ and that these potentially harmful activities of the toxin are antagonized by docosahexaenoic acid.

Dissociation of the PAF-receptor from NADPH oxidase and adenylate cyclase in human neutrophils results in accelerated influx and delayed clearance of cytosolic calcium

The magnitude and duration of the abruptly occurring increases in cytosolic Ca2+ in human neutrophils following activation with PAF (20 and 200 nM) and FMLP (1 μM), have been compared and related to alterations in NADPH oxidase activity, membrane potential and intracellular cyclic AMP. Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, transmembrane fluxes of Ca2+ by radiometric procedures, and NADPH oxidase activity and cyclic AMP by chemiluminescence and radioimmunoassay respectively. Activation of neutrophils with both PAF (200 nM) and FMLP (1 μM) was accompanied by an abrupt increase in cytosolic Ca2+, which was of similar magnitude for each activator (393±9 and 378±17 nM respectively). Unlike FMLP‐activated cells in which Ca2+ was rapidly removed from the cytosol, peak levels of cytosolic Ca2+ were sustained for longer (0.14±0.02 vs 1.16±0.04 min, P0.0001) and declined at a slower rate in PAF‐treated neutrophils. The prolonged elevation of cytosolic Ca2+ in PAF‐treated cells was due to accelerated store‐operated influx of extracellular cation and was attenuated by dibutyryl cyclic AMP (4 mM), the Ca2+‐chelator, EGTA (5 mM), and SKF 96365 (10 μM). In contrast to FMLP, basal levels of superoxide production and cyclic AMP were unaltered in PAF‐activated neutrophils, while only moderate membrane depolarization was detected. These observations demonstrate that mechanisms which restore Ca2+ homeostasis to FMLP‐activated neutrophils, viz. activation of NADPH oxidase and adenylate cyclase, are not operative in PAF‐treated cells, presenting the potential hazard of Ca2+ overload and hyperactivity.

Effects of cigarette smoke condensate on pneumococcal biofilm formation and pneumolysin

Although the well-recognised predisposition of cigarette smokers to the development of severe pneumococcal disease may be attributable to impairment of local host defences, less is known about the direct effects of smoke exposure on airway pathogens, or their virulence factors. In the current study, we have investigated the effects of cigarette smoke condensate (CSC) on biofilm formation by Streptococcus pneumoniae, and on the pore-forming activity of its major toxin, pneumolysin. Biofilm formation following exposure of the pneumococcus to CSC (20–160 &mgr;g·mL−1) was measured using a crystal violet-based spectrophotometric procedure, while the pore-forming activity of recombinant pneumolysin was determined by a fura-2/acetoxymethyl ester-based spectrofluorimetric procedure to monitor the uptake of extracellular Ca2+ by isolated human neutrophils. Exposure of the pneumococcus or pneumolysin to CSC resulted in significant dose-related augmentation of biofilm formation (p≤0.05 at 80 and 160 &mgr;g·mL−1) and substantial attenuation of the pore-forming interactions of pneumolysin, respectively. Augmentation of biofilm formation and inactivation of pneumolysin as a consequence of smoking are likely to favour microbial colonisation and persistence, both being essential precursors of pneumococcal disease.