Riana Cockeran

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.

Pneumolysin Activates the Synthesis and Release of Interleukin-8 by Human Neutrophils In Vitro

The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-alpha by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-alpha were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-alpha, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 microg/mL) or depletion of Ca(2+) from the cell-suspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.

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.

The role of pneumolysin in the pathogenesis of Streptococcus pneumoniae infection.

In addition to being cytotoxic for eukaryotic cells, recent research has clearly indicated that pneumolysin at sub-cytolytic concentrations potentiates the proinflammatory activities of neutrophils and macrophages. Together these cytotoxic and proinflammatory activities of the toxin are likely to contribute to the virulence of the pneumococcus, particularly in facilitating adherence, invasion and dissemination of this important microbial pathogen. Pneumolysin-based vaccine strategies, although in the early stages of development and evaluation, show promise in reducing the severity of pneumococcal disease.

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.

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.

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.

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 &mgr;M) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca2+ fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) (1 &mgr;M) in combination with cytochalasin B (CB; 3 &mgr;M). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca2+ fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca2+, as well as release of elastase and production of superoxide and LTB4, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

Platelet-activating factor and lyso-PAF possess direct antimicrobial properties in vitro

The effects of platelet‐activating factor (PAF) and lyso‐platelet‐activating factor (L‐PAF) at concentrations of 0.25–20 μg/ml on potassium transport and growth of gram‐positive and gram‐negative bacteria have been investigated in vitro and compared with those of lysophosphatidylcholine (LPC). Potassium transport was determined using 86Rb+ as tracer, while growth was measured according to the extent of uptake of radiolabeled amino acids. All of the test phospholipids caused dose‐related inhibition of 86Rb+‐uptake and growth of gram‐positive bacteria, the order of potency being PAF>LPC>L‐PAF. Gram‐negative bacteria, on the other hand, were less sensitive to the inhibitory effects of the phospholipids on K+ transport and growth. Some, but not all, of the gram‐positive and gram‐negative bacteria were able to degrade LPC, but not PAF or L‐PAF, demonstrating that enzymatic degradation of phospholipids does not explain the differential sensitivity to these agents. The bioactive phospholipids LPC, PAF and L‐PAF may represent an oxygen‐independent antimicrobial host defense system operative primarily against gram‐positive bacteria.