Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

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

Riana Cockeran, Helen C. Steel, Timothy J. Mitchell, Charles Feldman, Ronald Anderson
Riana Cockeran
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria,and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Helen C. Steel
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria,and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Timothy J. Mitchell
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Charles Feldman
Division of Pulmonology, Johannesburg Hospital and University of the Witwatersrand, Johannesburg, South Africa, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ronald Anderson
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria,and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.69.5.3494-3496.2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

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.

Although pneumolysin has been reported to increase the activity of phospholipase A2 in both neutrophils (4) and pulmonary endothelial cells (9), the effects of the toxin on the production of arachidonic acid-derived mediators of inflammation have not been described. In the current study, we have investigated the effects of recombinant pneumolysin on the production of prostaglandin E2 (PGE2) and leukotriene B4(LTB4) by human neutrophils in vitro.

Recombinant pneumolysin was expressed in Escherichia coliand purified from cell extracts as previously described (10). Protein homogeneity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stock toxin protein concentration was 0.21 mg/ml, which corresponds to 1.3 × 106 hemolytic units/ml, and the stock was essentially free of contaminating bacterial endotoxin (<2 pg/ml). The toxin was diluted in endotoxin-free Hanks' balanced salt solution (HBSS [pH 7.4]; 1.25 mM CaCl2; Highveld Biological Pty. Ltd., Johannesburg, South Africa), which was also added to the pneumolysin-free control systems described below, and used at fixed final concentrations of 8.37 and 41.75 ng/ml. Under the experimental conditions used in the present study, pneumolysin at these concentrations possesses either no (8.37 ng/ml) or minimal (41.75 ng/ml) cytotoxicity (4).

Purified neutrophils were prepared from heparinized (5 U of preservative-free heparin/ml) venous blood of healthy adult volunteers as described previously (4) by centrifugation of blood on Histopaque-1077 (Sigma Diagnostics, St. Louis, Mo.) to separate neutrophils from mononuclear leukocytes, followed by removal of erythrocytes using gelatin (3%) sedimentation and selective lysis with 0.84% ammonium chloride. The neutrophils were routinely of high purity (>90%) and viability (>95%). These cells (2 × 106) in HBSS were subjected to preincubation at 37°C for 10 min, followed by addition of pneumolysin (8.37 and 41.75 ng/ml [final concentrations]) or an equal volume (100 μl) of HBSS (control systems). The final volume in each tube was 1 ml. For systems in which the cells were activated with the synthetic chemotactic tripeptideN-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP; 1 μM [final concentration]; Sigma Chemical Co.), a preincubation period of 5 min at 37°C was used, after which pneumolysin was added, followed 5 min later by FMLP. In a preliminary kinetic experiment using cells from a single donor and incubation times of 1, 3, 5, and 10 min after the addition of pneumolysin only or FMLP with or without the toxin, PGE2 and LTB4 were detectable in the supernatants of pneumolysin-treated neutrophils with or without FMLP at 1 min and reached a plateau at 3 to 5 min (data not shown). For this reason, a fixed incubation time of 5 min was used for a series of subsequent experiments designed to investigate the effects of pneumolysin on the production of PGE2 and LTB4 by FMLP-treated and untreated neutrophils from 10 different donors.

After incubation, the reactions were terminated by the addition of 1 ml of ice-cold HBSS to each tube and the tubes were transferred to an ice bath. The tubes were then centrifuged to pellet the cells, and PGE2 and LTB4 in the supernatants were assayed by competitive binding radioimmunoassay procedures (Du Pont NEN Research Products, Boston, Mass.). The results are expressed as nanograms per 107 cells.

In an additional series of experiments using cells from three different donors, the extracellular calcium-chelating agent EGTA (5 mM [final concentration]) was added to the cells 5 s prior to the pneumolysin.

The results of each series of experiments are expressed as the mean ± the standard error of the mean (SEM). Levels of statistical significance were calculated by the Mann-Whitney U test (two tailed), and statistical significance was assigned aP value of <0.05.

The effects of pneumolysin on the production of PGE2 and LTB4 are shown in Fig. 1. Exposure of neutrophils to pneumolysin in the absence of FMLP increased the production of both PGE2 and LTB4, which, in the case of PGE2, was statistically significant at both concentrations of the toxin, but only at the highest concentration for LTB4. FMLP per se significantly (P < 0.05) increased the production of both PGE2 and LTB4by neutrophils, an effect which was strikingly and significantly (P < 0.05) potentiated by pretreatment of the cells with pneumolysin at both 8.37 and 41.75 ng/ml.

Fig. 1.
  • Open in new tab
  • Download powerpoint
Fig. 1.

Effects of pneumolysin (Pln; 8.37 and 41.75 ng/ml) on the production of LTB4 and PGE2 by unstimulated and FMLP (1 μM)-activated neutrophils. The results of 9 (PGE2) or 10 (LTB4) experiments using cells from different donors are expressed as the mean ± the SEM. Asterisks indicate P < 0.05 for comparison with the appropriate pneumolysin-free control systems.

The effects of removal of extracellular Ca2+ by treatment of the cell-suspending medium with EGTA (5 mM) on the production of PGE2 and LTB4 by pneumolysin-treated neutrophils with or without FMLP are shown in Fig.2. EGTA completely attenuated the pneumolysin-mediated enhancement of the production of PGE2and LTB4 by FMLP-treated and untreated neutrophils but did not affect the production of these bioactive lipids in control systems with or without FMLP in the absence of pneumolysin. In the presence of EGTA, the pneumolysin-treated systems both with and without FMLP did not differ significantly from the corresponding pneumolysin-free control systems in PGE2 and LTB4 production.

Fig. 2.
  • Open in new tab
  • Download powerpoint
Fig. 2.

Effects of EGTA (5 mM) on pneumolysin (Pln; 8.37 and 41.75 ng/ml)-mediated enhancement of production of LTB4 and PGE2 by unstimulated and FMLP (1 μM)-activated neutrophils. The results of two (PGE2) or three (LTB4) experiments using cells from different donors are expressed as the mean ± the SEM.

We have previously reported that pneumolysin at concentrations similar to those used in the present study, which had either no or minimal effects on cellular energy metabolism and viability, augmented the activity of phospholipase A2 in neutrophils (4). In the current study, most likely as a consequence of its enhancing effects on phospholipase A2, pneumolysin caused a dose-related increase in the production of both PGE2 and LTB4 by human neutrophils. Although this was evident with the toxin per se, it was most striking with the combination of pneumolysin and FMLP, which is most likely to mimic the situation at sites of inflammation.

Pneumolysin-mediated augmentation of the proinflammatory activities of human neutrophils is achieved by a pore-forming mechanism resulting in influx of extracellular Ca2+ (4). In the current study, inclusion of the Ca2+-chelating agent EGTA in the cell-suspending medium completely attenuated the pneumolysin-mediated enhancement of PGE2 and LTB4 production by neutrophils, probably by preventing Ca2+-dependent activation of phospholipase A2(4, 9).

Receptor-mediated proadhesive, chemotactic, secretory, and pro-oxidative interactions of LTB4 with phagocytes, particularly neutrophils, have been well documented (3), and potentiation of these proinflammatory activities by pneumolysin may contribute to hyperacute inflammation during pneumococcal infection (7). Pneumolysin-mediated augmentation of the production of PGE2 by neutrophils may also contribute to dysregulated inflammatory responses by increasing vascular permeability, thereby facilitating influx of proinflammatory polypeptides, as well as by delaying neutrophil apoptosis (8).

In addition to augmentation of the production of proinflammatory cytokines, reactive oxidants, including nitric oxide, proteolytic enzymes, and adhesion molecules (2, 4, 6), data from the current study demonstrate that pneumolysin also increases the production of the proinflammatory lipids PGE2 and LTB4 by human neutrophils. Acting in concert these various proinflammatory agents may promote hyperacute inflammation during infection with Streptococcus pneumoniae. The relationship between the proinflammatory activities of pneumolysin and microbial persistence is more complex, with several possible scenarios. Exaggerated inflammatory responses may favor accelerated eradication of the microbial pathogen. Alternatively, hyperactivation of phagocytes may result in premature auto-oxidative inactivation of their protective functions (1), as well as inhibition of the proliferative responses of neighboring lymphocytes (5), favoring microbial persistence.

Notes

Editor: E. I. Tuomanen

FOOTNOTES

    • Received 16 January 2001.
    • Returned for modification 5 February 2001.
    • Accepted 22 February 2001.
  • Copyright © 2001 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Baehner R. L.,
    2. Boxer L. A.,
    3. Allen J. M.,
    4. Davis J.
    Autooxidation as a basis for altered function by polymorphonuclear leukocytes.Blood501977327335
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Braun J. S.,
    2. Novak R.,
    3. Gao G.,
    4. Murray P. J.,
    5. Shenep J. L.
    Pneumolysin, a protein toxin of Streptococcus pneumoniae, induces nitric oxide production from macrophages.Infect. Immun.67199937503756
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Cleasson H. E.,
    2. Dahlén S.-E.
    Asthma and leukotrienes: antileukotrienes as novel antiasthmatic drugs.J. Int. Med.2451999205227
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Cockeran R.,
    2. Theron A. J.,
    3. Steel H. C.,
    4. et al.
    Proinflammatory interactions of pneumolysin with human neutrophils.J. Infect. Dis.1832001604611
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. El-Hag A.,
    2. Lipsky P. E.,
    3. Bennett M.,
    4. Clark R. A.
    Immunomodulation by neutrophil myeloperoxidase and hydrogen peroxide: differential susceptibility of human lymphocyte functions.J. Immunol.139198624062413
    OpenUrlAbstract
  6. 6.↵
    1. Houldsworth S.,
    2. Andrew P. W.,
    3. Mitchell T. J.
    Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin 1 β by human mononuclear phagocytes.Infect. Immun.62199415011503
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Kadioglu A.,
    2. Gingles N. A.,
    3. Grattan K.,
    4. Kerr A.,
    5. Mitchell T. J.,
    6. Andrew P. W.
    Host cellular immune response to pneumococcal lung infection in mice.Infect. Immun.682000492501
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Rossi A. G.,
    2. Cousin J. M.,
    3. Dransfield I.,
    4. Lason M. F.,
    5. Chilvers E. R.,
    6. Haslett C.
    Agents that elevate CAMP inhibit neutrophil apoptosis.Biochem. Biophys. Res. Commnn.2171995892899
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Rubins J. B.,
    2. Mitchell T. J.,
    3. Andrew P. W.,
    4. Niewoehner D. E.
    Pneumolysin activates phospholipase A in pulmonary artery endothelial cells.Infect. Immun.62199438293836
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Saunders F. K.,
    2. Mitchell T. W.,
    3. Walker J. A.,
    4. Andrew P. W.,
    5. Boulnois G. J.
    Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae, does not require a thiol group for in vitro activity.Infect. Immun.57198925472552
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
Pneumolysin Potentiates Production of Prostaglandin E2 and Leukotriene B4 by Human Neutrophils
Riana Cockeran, Helen C. Steel, Timothy J. Mitchell, Charles Feldman, Ronald Anderson
Infection and Immunity May 2001, 69 (5) 3494-3496; DOI: 10.1128/IAI.69.5.3494-3496.2001

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Pneumolysin Potentiates Production of Prostaglandin E2 and Leukotriene B4 by Human Neutrophils
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Pneumolysin Potentiates Production of Prostaglandin E2 and Leukotriene B4 by Human Neutrophils
Riana Cockeran, Helen C. Steel, Timothy J. Mitchell, Charles Feldman, Ronald Anderson
Infection and Immunity May 2001, 69 (5) 3494-3496; DOI: 10.1128/IAI.69.5.3494-3496.2001
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • Notes
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Dinoprostone
Leukotriene B4
neutrophils
Streptolysins

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522