Abstract
The research question addressed in the current study was: does the pneumococcal pore-forming toxin, pneumolysin, mobilise matrix metalloproteinase (MMP) -8 and -9 from isolated human blood neutrophils at sublytic concentrations of 5, 10 and 20 ng·mL−1?
MMPs were measured in the supernatants of unstimulated neutrophils and of cells exposed to pneumolysin and the chemoattractant N-formyl-l-methionyl-l-leucyl-l-phenylalanine (f-MLP; 0.1 μM), individually and in combination, using ELISA procedures, and alterations in cytosolic Ca2+ concentrations were monitored using a fura-2 acetoxymethyl ester (fura-2/AM)-based spectrofluorimetric method.
Treatment of neutrophils with pneumolysin alone caused dose-related release of both MMPs, whereas f-MLP caused modest increases; the combination of both activators was, however, most effective. Pneumolysin/f-MLP-activated release of the MMPs from the cells was paralleled by increases in cytosolic Ca2+.
Exposure of human neutrophils to pneumolysin is accompanied by mobilisation of MMPs, which is potentiated by f-MLP. If operative in vivo, pneumolysin-mediated release of MMPs from neutrophils and other cell types may contribute to the pathogenesis of severe pneumococcal disease.
Pneumolysin, a cholesterol-binding pore-forming protein toxin, is a key virulence factor of the pneumococcus, which promotes dissemination of this microbial pathogen from the lungs to the vascular space via its pro-inflammatory and cytotoxic activities 1, 2. The pro-inflammatory activity of pneumolysin involves several distinct mechanisms. These include activation of immune and inflammatory cells via interaction of pneumolysin with Toll-like receptor 4 3, as well as by induction of Ca2+ influx, particularly into phagocytes and epithelial cells, as a consequence of sublytic action of the toxin 4, 5. In both cases, this results in activation of intracellular signalling cascades, which trigger the synthesis of a range of pro-inflammatory neutrophil- and monocyte/macrophage-activating chemokines/cytokines 6–8.
Although matrix metalloproteinases (MMPs), especially MMP-8 and -9, which are stored in neutrophil granules, have been implicated in lung and brain injury in bacterial pneumonia and meningitis, including pneumococcal meningitis 9–15, the potential of pneumolysin to activate the release of MMPs from these cells has not been investigated. In the present study, the ability of pneumolysin, alone and in combination with the chemoattractant N-formyl-l-methionyl-l-leucyl-l-phenylalanine (f-MLP), to activate the release of MMP-8 and MMP-9 from isolated human neutrophils was investigated.
MATERIALS AND METHODS
Unless otherwise indicated, all chemicals and reagents were obtained from the Sigma Chemical Co. (St Louis, MO, USA).
Pneumolysin
Recombinant pneumolysin was expressed in Escherichia coli and purified from cell extracts as described elsewhere 16. Protein homogeneity was confirmed by sodium dodecylsulphate-polyacrylamide gel electophoresis. The stock toxin protein concentration was 0.52 mg·mL−1, which corresponds to 6.1×105 haemolytic units·mL−1, as determined by one of us (T.J. Mitchell) using sheep erythrocytes, and the stock was essentially free (<2 pg·mL−1) of contaminating bacterial endotoxin. The toxin was diluted in Hank’s balanced salt solution (HBSS; Highveld Biological (Pty), Johannesburg, South Africa; pH 7.4; indicator-free; 1.25 mM CaCl2) and was used at fixed final concentrations of 5, 10 and 20 ng·mL−1, which have previously been found to sensitise or activate the pro-inflammatory activities of neutrophils in vivo 4.
Neutrophils
Permission to draw blood from healthy adult volunteers was granted by the Faculty of Health Sciences Research Ethics Committee of the University of Pretoria (Pretoria, South Africa), and prior informed consent was obtained from all participants.
Purified neutrophils were prepared from heparinised (5 units preservative-free heparil·mL−1) venous blood as described previously 4. The neutrophils, which were routinely of high purity (>90%) and viability (>95%) using a dye (0.1% trypan blue) exclusion procedure, were resuspended at 1×107 cells·mL−1 in PBS (0.15 M; pH 7.0) and held on ice until used.
Matrix metalloproteinases
Neutrophils were resuspended in HBSS at a density of 2×106 cells·mL−1 and pre-incubated for 10 min at 37°C. This was followed by addition of pneumolysin at final concentrations of 5, 10 and 20 ng·mL−1, the chemoattractant f-MLP (0.1 μM final concentration) or pneumolysin in combination with f-MLP (the toxin was added to the cells followed 1 min later by f-MLP). The tubes were then incubated for 5 min at 37°C, after which an equal volume of ice-cold HBSS was added to each tube and the tubes placed in an ice bath in order to stop the reactions. The incubation time of 5 min was based on previous experience with f-MLP and pneumolysin, which demonstrated that the neutrophil responses to these agents were complete within this period 4, 17. The cells were then pelleted by centrifugation and the supernatants decanted and assayed for MMP-8 and -9 using ELISA procedures (Quantikine®; R&D Systems, Minneapolis, MN, USA), and the results expressed as nanograms per millilitre of supernatant.
Spectrofluorimetric measurement of Ca2+ fluxes
Fura-2 acetoxymethyl ester (fura-2/AM) was used as the fluorescent Ca2+-sensitive indicator in these experiments 4, 18. Following loading with fura-2/AM (2 μM; 1×107 cells·mL−1 for 25 min at 37°C), the cells were pelleted by centrifugation and resuspended in HBSS. The neutrophils (2×106 cells·mL−1) were then pre-incubated for 5 min at 37°C, after which the cells were transferred to disposable reaction cuvettes that were maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer (Hitachi, Tokyo, Japan), with the excitation and emission wavelengths set at 340 and 500 nm, respectively. After a stable baseline was obtained (1 min), the neutrophils were activated by addition of pneumolysin (5, 10 and 20 ng·mL−1 final concentration) and f-MLP (0.1 μM final concentration) individually and in combination (f-MLP added 1 min after pneumolysin). Alterations in fluorescence intensity were then measured over a 5–10-min period. The final volume in each cuvette was 3 mL, containing a total of 6×106 neutrophils.
Expression and statistical analysis of results
The results of the MMP experiments are expressed as mean±sem, and traces are shown for the fura-2/AM experiments. In both cases, n represents the number of different donors used in each series of experiments. Statistical analysis was performed by ANOVA, with subsequent Bonferroni multiple comparisons test.
RESULTS
Matrix metalloproteinases
The effects of exposure of neutrophils to pneumolysin and f-MLP individually and in combination are shown in figure 1⇓. Addition of pneumolysin to the cells resulted in release of MMP-8 (fig. 1a⇓) and -9 (fig. 1b⇓), which was dose-related and achieved significance at 20 ng·mL−1 toxin. Exposure of the cells to f-MLP resulted in modest increases in MMP release, whereas the combination of pneumolysin and chemoattractant was more effective than the individual agents alone.
Cytosolic Ca2+ concentrations
Cytosolic Ca2+ concentration results are shown in figure 2⇓. Addition of pneumolysin to neutrophils was followed by a lag phase, the duration of which was inversely related to the concentration of the toxin, followed by a dose-related increase in fura-2 fluorescence intensity, due to influx of Ca2+ 4, reaching sustained peak plateau levels, the magnitudes of which were related to the pneumolysin concentration (fig. 2a⇓). Addition of f-MLP to the cells was accompanied by the typical immediate increase in fura-2 fluorescence intensity compatible with mobilisation of Ca2+ from intracellular stores (fig. 2b⇓). This was followed by two successive phases, first a rapid decline in fluorescence intensity due to Ca2+ efflux, as well as resequestration of Ca2+ into stores, and, secondly, a more gradual decline due to store-operated influx of the cation 17. When the cells were exposed to the combination of pneumolysin and f-MLP, the magnitude of the abrupt f-MLP-mediated increase in fluorescence intensity remained unchanged. However, the decline in fluorescence intensity was slower and of lesser magnitude than that observed with f-MLP alone, being inversely related to the pneumolysin concentration and compatible with Ca2+ flooding of the cytosol (fig. 2b⇓).
DISCUSSION
As is the case with the primary granule protease, elastase, the secondary and tertiary granule MMPs, MMP-8 and -9, are essential for the protective functions of neutrophils. Elastase not only facilitates transendothelial migration of neutrophils 19 but also protects against Gram-negative bacterial pathogens by degrading major outer membrane proteins 20, and the MMPs are necessary for extracellular matrix degradation and neutrophil migration 21, 22. However, if the release of neutrophil-derived proteolytic enzymes, particularly MMPs, is poorly regulated and excessive, as may occur during hyperacute inflammatory responses, the risk of injury to bystander cells and tissues is likely to be considerable 9–15.
Although pneumolysin is considered to be a key player in promoting extrapulmonary dissemination of the pneumococcus and mediating neurological damage in severe pneumococcal infection, via both cytotoxic and pro-inflammatory mechanisms 1, 2, the potential of the toxin to induce the release of MMPs from human neutrophils has not, to our knowledge, been addressed. In the present study, we investigated the effects of exposure of isolated human neutrophils to pneumolysin, at concentrations of 5–20 ng·mL−1, in the presence and absence of the chemoattractant f-MLP on the release of MMP-8 and -9. Pneumolysin concentrations of up to 9 μg·mL−1 and 180 ng·mL−1 have been detected in pneumococcal culture fluids in vitro and in the cerebrospinal fluid of patients with pneumococcal meningitis, respectively 23, 24. N-formylated polypeptide chemoattractants are produced by the pneumococcus 25, 26, and may act in concert with pneumolysin to augment the pro-inflammatory activities of neutrophils, as well as those of monocytes/macrophages 4.
Although exposure of neutrophils to either f-MLP or pneumolysin, in particular, resulted in release of MMP-8 and -9 from the cells, prior treatment with pneumolysin followed by addition of f-MLP resulted in augmentation of MMP release, reaching levels which were significantly higher than those attained with the toxin or chemoattractant individually. Although proportionately similar, the absolute concentrations of MMP-8 released from neutrophils activated with pneumolysin and f-MLP, individually and in combination, were less than those of MMP-9, which appeared to reflect the total intracellular concentrations of these enzymes. The higher intracellular level of MMP-9 observed in the present study may reflect the relative intracellular distribution of MMP-8 and -9, with the former being located exclusively in secondary granules, and the latter in both secondary and tertiary granules 27.
Exocytosis of primary, secondary and tertiary neutrophil granules are Ca2+-dependent events, with the latter two granule types being more readily mobilised than the primary granules, the threshold value for cytosolic Ca2+ required for significant release being 200–300 nM 28, 29. In the present study, this threshold value was attained and exceeded, with the release of both MMPs being closely correlated with pneumolysin/f-MLP-mediated increases in neutrophil cytosolic Ca2+, with the combination of f-MLP and pneumolysin resulting in sustained increases in cytosolic Ca2+ levels.
Depending on local concentrations, pneumolysin appears to play a dual role in pneumococcal host defences 2. At low concentrations, the toxin, possibly acting in concert with bacterially derived chemoattractants, may promote anti-pneumococcal host defences, preventing colonisation of the airways 5. In severe pneumococcal infection, conversely, excessive production of the toxin may promote hyperacute, damaging inflammatory responses 1.
Notwithstanding the relatively small sample size used in the present study and the requirement for confirmation in animal models of experimental infection, the current findings demonstrate that exposure of human neutrophils to extremely low concentrations of pneumolysin results in the release of MMP-8 and -9, which is potentiated by f-MLP, a mimic of pneumococcal chemoattractants. If operative during severe pneumococcal infection, this mechanism may contribute to the pathology of lung and brain injury, underscoring the potential value of antimicrobial agents, especially macrolides, which target the synthesis of pneumolysin 30, 31.
Statement of interest
A statement of interest for T.J. Mitchell can be found at www.erj.ersjournals.com/misc/statements.dtl
- Received January 15, 2009.
- Accepted March 18, 2009.
- © ERS Journals Ltd