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Am J Physiol Lung Cell Mol Physiol 290: L833-L840, 2006. First published December 9, 2005; doi:10.1152/ajplung.00333.2005
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Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent

Jessica G. Moreland1,2 and Gail Bailey1,2

1Division of Critical Care, Department of Pediatrics, and 2The Inflammation Program, The University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa

Submitted 28 July 2005 ; accepted in final form 2 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The recruitment of polymorphonuclear leukocytes (PMN) from the vascular space to the alveolar air space is an early event in host defense against pneumococcal pneumonia. Pneumolysin is a virulence factor for Streptococcus pneumoniae, but a specific role for pneumolysin in neutrophil-endothelial cell interactions has not been investigated. Using a Transwell system, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to wild-type S. pneumoniae (D39) and a pneumolysin-deficient mutant (plnA–) incubated on the abluminal surface of the monolayer. S. pneumoniae induction of PMN migration was dose dependent and elicited by ≥105 D39. Mutants lacking pneumolysin had dramatically reduced potency for eliciting PMN migration compared with the parent strain (5 x 106 plnA– elicits 18.6% PMN migration vs. 55.5% for 5 x 106 D39). The disparity between D39 and plnA– persisted in ethanol-fixed bacteria, consistent with the properties of pneumolysin. Neither conditioned medium from D39 nor purified pneumolysin elicited PMN migration to the same extent as the intact D39, suggesting that the role of pneumolysin in eliciting PMN migration requires a more complex interaction between the organism, the endothelium, and the PMN. Both D39 and plnA– adhered to, and translocated across, the endothelium in the abluminal to luminal direction and elicited similar levels of IL-8 production. Neither strain elicited upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin, and they did not cause translocation of NF-{kappa}B to the nucleus. These findings demonstrate a novel role for pneumolysin in pneumococcus-induced PMN recruitment across the pulmonary endothelium.

endothelial cells; inflammation; pneumonia; virulence factors

AMONG BACTERIAL CAUSES OF community-acquired pneumonia, Streptococcus pneumoniae produces the most severe pneumonia and is frequently associated with bacteremia (12, 17, 31). Moreover, S. pneumoniae is the leading cause of bacterial pneumonia, otitis media, and bacteremia in children and the most common cause of pneumonia in adults (6, 29). In vivo, the initial host-pathogen interaction in the lower airway involves binding of S. pneumoniae to alveolar epithelial cells. Pneumococcal cell surface phosphorylcholine appears to direct this binding by acting as a direct ligand for the platelet-activating factor receptor (PAFR) (7, 9), which is upregulated on the epithelium in response to inflammatory stimuli. Bacterial translocation occurs after ligand-receptor pairing and endocytosis, and the bacteria are expelled on the abluminal surface (17). This is the presumed mechanism for progression from pneumonia to bacteremia and indicates that a direct interaction occurs between the intact organism and the microvascular endothelium.

S. pneumoniae has many virulence factors implicated in the pathogenesis of pneumococcal disease, including pneumolysin, an intracellular thiol-activated toxin with cytolytic and complement-activating properties (3, 19). Pneumolysin-negative mutants demonstrate reduced virulence in mice after intranasal challenge or intraperitoneal delivery. Effects of pneumolysin on host cells may be either direct (i.e., pore formation in host cell membranes) and/or indirect (i.e., activation of the complement system) (8, 14). In a murine model of bronchopneumonia, pneumolysin-negative mutants had reduced survival in the lungs during the onset of pneumonia and decreased invasion and survival in the bloodstream. The host response to these mutants was muted, with reduced neutrophil influx and diminished inflammatory response (11). However, whether this reflected simply a decrease in the severity of infection because of reduced bacterial growth or also a specific impairment in the induction of polymorphonuclear leukocytes (PMN) migration and accompanying inflammation could not be discerned. In addition, purified pneumolysin delivered into the lungs via intranasal inoculation in a murine model elicited a dose-dependent influx of PMNs with accompanying inflammatory response (24).

Neutrophil migration from the vascular space to the air space is an early and prominent component of the host innate response to pneumococcal invasion (17), and the pneumococcal-endothelial cell interactions that influence neutrophil transendothelial migration have not been well defined. However, recent data indicate that neutrophil recruitment to the lung and the early development of bacteremia were specifically influenced by the cytolytic activity of the toxin pneumolysin (10).

The present study has focused specifically on interactions between the intact pneumococcus and the pulmonary microvascular endothelium and the role of the virulence factor pneumolysin in eliciting neutrophil transendothelial migration. Using our in vitro transmigration model, we demonstrate that pneumolysin is required to induce the maximal neutrophil transendothelial migratory response. Effects of pneumolysin are most clearly manifest during direct exposure of the endothelium to intact pneumococci and not through secretion of bacterial products into the surrounding media. Both pneumolysin-deficient mutants and the wild-type D39 adhere to and translocate across the endothelium to a similar extent and induce similar secretion of the neutrophil chemokine IL-8 by the endothelial monolayer. Neither strain elicits translocation of NF-{kappa}B to the nucleus, nor is there upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin. The role of pneumolysin in pneumococcus-induced neutrophil transmigration appears to require a complex interaction between the bacteria, the neutrophil, and the endothelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human PMN purification. Human PMNs were isolated according to standard techniques from heparin anticoagulated venous blood from healthy consenting adults in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. PMN were isolated using dextran sedimentation and Hypaque-Ficoll density-gradient separation, followed by hypotonic lysis of erythrocytes as previously described (4). Purified PMNs were resuspended in 0.9% saline before use in migration experiments.

Endothelial cell culture. Pulmonary microvascular endothelial cells (PMVEC) were purchased from Clonetics (San Diego, CA) and cultured on collagen-coated flasks (type VI, human placental collagen; Sigma, St. Louis, MO) using endothelial growth medium-2 (Clonetics) with added bovine brain extract, VEGF, EGF, gentamicin, and hydrocortisone according to manufacturer's specifications. Cells were received from Clonetics at passage 5 and used between passages 5 and 9. Cells were passed from T-75 flasks into experimental plates when ~70–80% confluent. PMVEC were detached using trypsin-EDTA and cultured on collagen-coated Transwell (Costar, Cambridge, MA) 12-mm filters (transmigration experiments), Transwell 24-mm filters (immunofluorescence microscopy), or 96-well plates (whole-cell ELISA). Cell monolayers on Transwell filters were monitored by measuring resistance changes across the endothelial cell monolayer using an End Ohm epithelial voltohmeter (World Precision Instruments, Sarasota, FL). The microvascular endothelial cell monolayers reached an average resistance of 34–37 {Omega}/cm2 across the monolayer within 5–6 days. Medium was changed on the monolayer every 48 h.

Bacterial cell cultures. S. pneumoniae serotype-2 strain D39 and its isogenic pneumolysin-negative mutant plnA– (gifts from Elaine Tuomanen, St. Jude Children's Research Hospital) were stored at –80°C, and an aliquot was streaked on blood agar plates 16 h before use. The serotype 2 D39 pneumolysin-negative mutant complemented with reinsertion of the pneumolysin gene (Pn+) and the purified pneumolysin toxin were gifts of Dr. Timothy Mitchell (University of Glasgow, Glasgow, Scotland). Bacteria were resuspended in endothelial basal medium (EBM), and bacterial concentration was determined by measuring optical diameter at 520 nm. Bacteria were added to the Transwell lower chambers at known optical diameter, and samples from time 0 and at 4 h (just before addition of the PMNs) were plated for colony-forming unit (CFU) determination to determine the growth of each bacterium during the initial 4-h bacteria/endothelial cell incubation.

In certain experiments, bacteria were EtOH fixed or heat killed before use in transmigration studies. EtOH fixation was performed by placing an aliquot of the strain in 70% EtOH on ice for 30 min, and organisms were heat killed by incubation at 65°C for 20 min. Plating of the samples on blood agar plates confirmed that EtOH and heat treatment eliminated all viable bacteria.

PMN transendothelial migration. We performed migration assays across PMVEC monolayers using a Transwell system (Fig. 1) as previously described. (16) The endothelial cells were not activated with serum or cytokines before the transmigration assay. Transwell filters with attached endothelial cell monolayers were transferred to clean 12-well plates and washed twice with HBSS. After removal of the HBSS, 400 µl of EBM (Clonetics) was added to the Transwell filter compartment. Intact bacteria were added to the lower compartment in a total volume of 1 ml and incubated with the endothelial cells for 4 h at 37°C (except in time course studies, where timing is specified in RESULTS). At the end of this 4-h incubation, 10-µl samples were removed from the lower chamber for quantitation of the bacterial CFU, and 2 x 106 PMNs were added to the Transwell (upper chamber) in a volume of 100 µl. PMNs were allowed to migrate over a 3-h period at 37°C. At the conclusion of the incubation, migrated cells were collected from the lower chamber for counting in a hemacytometer in triplicate. In preliminary experiments, PMNs recovered from above and below the endothelial monolayer accounted for ~90% of the initial number added, indicating that relatively few PMN were trapped within or in transit across the endothelium at any time examined. Therefore, percent PMN transmigration was calculated simply by dividing the number of PMN recovered from the lower chamber by the number of PMN initially added.


Figure 1
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  		Fig. 1. In vitro transmigration model. Pulmonary microvascular endothelial cells (PMVECs) were grown on a Transwell filter until peak electrical resistance signaling formation of a monolayer. Bacteria were then added to the lower chamber (abluminal surface) and incubated at 37°C, 5% CO2, and 95% relative humidity for 4 h; this was followed by addition of polymorphonuclear leukocytes (PMNs) to the upper chamber (luminal surface).

 
Bacterial translocation across the endothelial monolayer. Transwell filters with attached endothelial cell monolayers were transferred to clean 12-well plates and washed twice with HBSS. After removal of the HBSS, 400 µl of EBM (Clonetics) was added to the Transwell filter upper compartment. Bacteria (D39 or plnA–; 3 x 106) were added to the lower compartment in a total volume of 1 ml and incubated with the endothelial cells at 37°C. No bacteria were added to the upper chamber of the Transwell. Aliquots (50 µl) were removed from the upper and lower chambers of the Transwell at 1, 2, 4, and 6 h after the initial inoculum was added to the lower chamber and used for enumeration of CFU. The number of bacteria translocating to the upper chamber was expressed as a percentage of the initial inoculum in the lower chamber, and the CFU per milliliter present in the lower chamber over the time course studied is given for comparison to account for bacterial growth during the course of the assay.

Analysis of chemokine expression. Supernatants from the both the upper and lower chambers of the Transwell system during transmigration experiments were collected after PMN migration and filtered to remove bacteria and neutrophils before storage at –20°C. Levels of IL-8, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T-expressed and presumably secreted (RANTES), IL-6, growth-related oncogene (GRO)-{alpha}, and TNF-{alpha} were assayed in the supernatant using standard ELISA techniques. Briefly, 96-well plates were coated with a monoclonal antibody to the protein of interest (R&D Systems). Samples (50 µl) from the collected supernatant were added to the plate followed by a biotinylated detection antibody. HRP-conjugated streptavidin (Pierce Chemicals) was then added to the plate, followed by a color substrate TMB (Sigma). Standard curves were made using recombinant protein for each of the proteins of interest (R&D Systems). Absorbance at 450 nm was read, and protein concentrations of unknown samples were extrapolated based on the standard curve.

Assays of surface expression of endothelial cell adhesion molecules. To assess endothelial cell surface expression of adhesion molecules, monolayers were grown on 6.5-mm Transwell filters to peak resistance as previously described. Whole bacteria or TNF-{alpha} (10 ng/ml) was added to the lower chamber of the Transwell apparatus and incubated for 6 h. At the completion of the incubation, cells were washed twice with HBSS and then blocked for 30 min with HBSS containing 0.5% BSA. In ELISA for E-selectin, cells were then fixed with 0.5% glutaraldehyde at 4°C for 30 min, before addition of the primary antibody. In ELISA for ICAM-1 and VCAM-1, primary antibody was added immediately after the blocking step. Primary antibodies were added 100 µl/well, diluted in PBS + 0.1% BSA, and incubated for 1 h at room temperature on a rotating shaker. The primary antibodies used were E-selectin (2 µg/ml) (BD Pharmingen, San Diego, CA), ICAM-1 (0.375 µg/ml) (Clone 6.5B5, Dako, Glostrup, Denmark), and VCAM-1 (0.5 µg/ml) (BD Pharmingen). After this incubation, cells were washed five times with HBSS/0.1% BSA and incubated with peroxidase-conjugated goat anti-mouse secondary antibody (dilution of 1:1,000) for 1 h at room temperature. After the secondary antibody, cells were washed an additional five times, and liquid TMB substrate (Sigma) was added. Optical density at 650 nm was read after 30 min. For each ELISA, an isotype-matched control Ab was used in place of the primary antibody in three wells, and this background was subtracted from the signal.

Assessment of nuclear translocation of NF-{kappa}B by microscopy. PMVEC were grown on Transwell 24-mm filters (Costar) to peak resistance. Filters with attached endothelial cells were rinsed twice with HBSS. After removal of the HBSS, 1,500 µl of EBM (Clonetics) was added to the Transwell filter compartment. Bacteria suspended at the described concentrations in EBM or TNF-{alpha} (10 ng/ml) (positive control) were added to the lower compartment in a total volume of 2.5 ml and coincubated for 1–6 h at 37°C. After the incubation, filters were washed twice with PBS, fixed with 2% paraformaldehyde at room temperature for 20 min, permeabilized with 0.1% Triton for 10 min at room temperature, and blocked overnight with PBS + 0.5% BSA. Primary antibody (anti-NF-{kappa}B p65, 1:25) was applied to the luminal surface of the endothelial cells on the filter for 1 h at room temperature (Santa Cruz Biotechnology). After cells were washed three times with PBS + 0.5% BSA, Texas red labeled goat anti-mouse Ab (Molecular Probes, Eugene, OR) was applied (1:1,000 dilution) for 1 h at room temperature. Specificity of staining was assessed by use of isotype control mouse antibodies (Sigma). Filters were washed and mounted on glass slides. Samples were viewed using a Zeiss Axioplan2 photomicroscope (Carl Zeiss, Thornwood, NY), and digital images were obtained using a Zeiss AxioCam and AxioVision 3.1 software.

Statistical analysis. Differences between experimental groups with paired comparisons composed of normally distributed data were analyzed for statistical significance using Student's t-test. Differences among groups of three or more data points were analyzed by ANOVA. Nonparametric evaluation of data sets was performed using the Mann-Whitney rank sum test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Pneumolysin-dependent transmigration of PMN across PMVECs. PMN migration across intact PMVEC monolayers was assessed for resting PMVEC monolayers and in response to several concentrations of intact, live S. pneumoniae or purified pneumolysin. In the absence of added bacteria or other stimulus, there was minimal migration of PMNs across the intact endothelial monolayer (<10,000 cells or <0.5% of the added PMNs). Live S. pneumoniae (serotype 2, D39) added at the abluminal surface of an intact endothelial monolayer potently elicited neutrophil migration, in a dose-dependent manner, across the monolayer in a luminal to abluminal direction. In contrast, an isogenic pneumolysin-deficient mutant (plnA–) was markedly less potent in inducing PMN transmigration. Complementation of the mutant strain by reinsertion of the pneumolysin gene (Pn+) restored the ability of the organism to elicit PMN transmigration (Fig. 2). Time course studies were performed to determine whether the reduced migration in response to the pneumolysin-deficient mutant simply reflected a delay in the onset of migration. After addition of 5 x 106 CFU of each strain to PMVEC monolayers, there was reduced PMN migration in response to plnA– at all time points (Fig. 3), indicating that the decrease in the number of PMNs transmigrating in response to the mutant organism did not reflect simply a delay in onset of the migratory response.


Figure 2
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  		Fig. 2. Migration of human neutrophils across a PMVEC monolayer in response to intact, live Streptococcus pneumoniae: encapsulated, serotype 2 D39 vs. isogenic pneumolysin-deficient mutant, plnA– vs. the pneumolysin-deficient strain complemented with reinsertion of the pneumolysin gene Pn+. X-axis represents bacterial colony-forming units (CFUs) present in lower chamber of the Transwell system at the time PMNs were added to the assay, after 4-h bacterial coincubation with the endothelial monolayer. The parent strain D39 potently elicited PMN transendothelial migration in a dose-dependent manner to a much greater extent than plnA–. Complementation of the deficient strain with reinsertion of Pn+ restored migration to levels similar to the those for the parent strain. *P ≤ 0.05 compared with D39; n = 6 experiments.

 

Figure 3
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  		Fig. 3. Kinetic analysis of transendothelial migration of neutrophils in response to intact live D39 vs. plnA–. D39 (5 x 106 CFU/ml) elicited greater PMN migration across the endothelial monolayer at all time points between 1 and 4 h after addition of PMNs to the monolayer than did plnA–. *P ≤ 0.05 compared with D39; n = 4 experiments.

 
To provide further evidence of the role of pneumolysin in neutrophil migration elicited by pneumococci, we investigated migration in response to EtOH-fixed or heat-killed organisms. Purified pneumolysin is unaffected by EtOH treatment but is heat labile (28). The difference in neutrophil migration in response to D39 vs. plnA– was also seen in ethanol-fixed bacteria, but overall transmigration of PMN was substantially decreased. Very few neutrophils migrated in response to either of the strains after heat killing (Fig. 4). These findings are consistent with an important role of pneumolysin in pneumococcal-induced PMN transmigration and also suggest a need for intact, live pneumococci to induce the greatest response.


Figure 4
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  		Fig. 4. Neutrophil transmigration across PMVEC monolayer in response to live, EtOH-fixed (70% EtOH on ice for 30 min) or heat-killed (65°C for 20 min) D39 or plnA–. Initial inoculum for both strains before described treatment was 3 x 106 CFU/ml. Both live and EtOH-killed D39 elicited substantially greater PMN migration than an equivalent number of pneumolysin-deficient organisms. Heat-killed bacteria of either strain elicited very little PMN migration. *P ≤ 0.05 compared with D39; n = 3 experiments, with 12 Transwells/condition.

 
Role of bacterial products in eliciting migration. To determine whether pneumolysin acts independently as a secreted/released bacterial product, we tested the ability of purified pneumolysin to elicit neutrophil transmigration in the absence of intact bacteria. Purified pneumolysin at 30 ng/ml (pneumolysin equivalent to 104 CFU of D39) (32) elicited neutrophil migration of 4.3% of the added PMNs. There was an increase in the number of PMNs migrating in response to higher concentrations of the purified toxin to a maximum migration of 29% of added PMNs in response to 3 µg/ml of pneumolysin, with a decrease in migration at higher doses of pneumolysin (Fig. 5). Inspection of the monolayer by immunofluorescence microscopy after incubation with concentrations of purified pneumolysin of 0.3–30 µg/ml demonstrated significant dose-dependent destruction of the architecture of the endothelial monolayer that was not seen in response to any of the concentrations of intact organisms studied (data not shown). In addition, electrical resistance across the monolayer declined to <10% of the starting value after incubation with these doses of pneumolysin. These data suggest that the action of pneumolysin in pneumococcus-induced PMN transmigration does not occur independently of the bacterial cell and support the contention that host cell responses to intact bacteria may change quantitatively, and perhaps qualitatively, from that induced, even by highly potent, isolated products of the bacteria.


Figure 5
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  		Fig. 5. Neutrophil transendothelial migration in response to purified pneumolysin incubated in the abluminal compartment of the Transwell system. There was a dose-dependent induction of PMN transendothelial migration in response to concentrations of pneumolysin from 30 ng/ml to 3 µg/ml but a decline in migration at higher concentrations of the toxin; n = 6 experiments.

 
Although pneumolysin is a cytosolic protein, it acts on host cells and therefore presumably depends on release of pneumolysin from the intact bacterial cell (e.g., lysis of pneumococci). We also tested the ability of conditioned media from each of the two strains (D39 and plnA–) to induce neutrophil transmigration. The conditioned media were obtained by culturing live bacteria for 6 h in the endothelial medium used for transmigration studies, followed by filtration (0.2 µM, low-protein binding) to remove intact bacteria before addition of the media to the lower chamber (abluminal surface) of the Transwell apparatus. In addition, intact plnA– were supplemented with conditioned medium from D39 to determine whether soluble/secreted products released from the wild-type strain could enhance the PMN transmigration response induced by intact pneumolysin-deficient mutants. Neither the conditioned medium from D39 nor that from plnA– elicited PMN migration to the extent induced by the intact bacteria (Fig. 6). Incubation of the monolayer with live plnA– combined with conditioned medium from D39 also did not elicit migration of PMN at a greater level than that seen with the mutant bacteria alone (Fig. 6). These results suggest that the effects of pneumolysin require a more complex interaction between pneumococci and the endothelium to elicit PMN transmigration.


Figure 6
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  		Fig. 6. Neutrophil transendothelial migration in response to intact, live bacteria or conditioned medium (CM) from each of the strains. CM was obtained by 6-h culture of the live bacteria in endothelial basal medium at 37°C, followed by filtration to remove intact bacteria. CM from both strains elicited substantially less PMN migration than intact organisms. CM from D39 coincubated with intact, live plnA– did not increase neutrophil migration above that seen with plnA– alone. *P ≤ 0.05 compared with D39; n = 4 experiments.

 
Bacterial translocation across the monolayer. One possible reason for enhanced PMN transmigration-inducing effects of live pneumococci is the need for bacteria to infect and translocate across the endothelium to have their greatest effect. To begin to address this possibility, we first examined growth and translocation of wild-type and mutant pneumococci across the endothelial monolayer in an abluminal to luminal direction. The upper and lower chambers of the Transwell system were sampled at 1, 2, 4, and 6 h after addition of bacteria to the lower chamber at time 0. Both D39 and plnA– were cultured from the upper chambers of their respective Transwells within 1 h of addition of the bacteria to the lower chamber, and the number in the upper chamber steadily increased over several hours. At 6 h, the number of D39 translocated to the upper chamber had further increased to 24.1 ± 1.6% of CFU count in the lower chamber; however, translocation of the pneumolysin-deficient mutant appeared to have plateaued (Fig. 7). These data indicate that not only was there direct interaction of the S. pneumoniae at the abluminal surface of the endothelial monolayer, but bacterial translocation was occurring throughout our migration assays.


Figure 7
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  		Fig. 7. Bacterial translocation across an intact monolayer of PMVEC. D39 and plnA– both translocated in the abluminal to luminal direction across an intact endothelial monolayer within 1 h of addition to the lower chamber of the Transwell. There was a relative increase in the number of bacteria translocated to the upper chamber at each time point from 1 to 4 h for both D39 and plnA–. At 6 h after the initial inoculum was added, the number of D39 in the upper chamber had further increased to 24% of the CFU count in the lower chamber, whereas the translocation of plnA– appeared to have plateaued. Results for number of translocated bacteria (upper chamber) are expressed as a percentage of the CFU count in the lower chamber at the same time point. *P ≤ 0.05 compared with plnA–; n = 4 experiments.

 
The integrity of the monolayer was assessed during many of these studies by measurement of the electrical resistance across the monolayer before and after incubation with the bacterial stimulus. The measured resistance across the monolayer after a 4-h incubation with 1 x 106 live D39 declined by 23% and by 21% after incubation with heat-killed S. pneumoniae but remained significantly elevated over the resistance across the filter alone. The similar decrement in resistance after exposure to live vs. heat-killed D39 despite significantly greater PMN migration in response to the live organisms indicate that the PMN migration elicited is not simply a result of destruction of the monolayer. These data taken together with the studies of bacterial translocation suggest an active role of the endothelial monolayer in the interaction with the live bacteria and in eliciting PMN transmigration.

Role of chemokines in eliciting transmigration. Although bacterial translocation appears to occur during pneumococcal-endothelial cell interaction in vitro, it did not require pneumolysin and thus is not an explanation for the disparity in neutrophil migration. We investigated the role of several chemokines that are expressed and secreted by the endothelium in response to inflammatory stimuli to determine whether differential expression was elicited by the two strains of bacteria. Both strains of bacteria potently induced IL-8 secretion at both the luminal (Fig. 8A) and abluminal (Fig. 8B) surfaces of the endothelial monolayer in a dose-dependent manner. When normalized for CFU count, the magnitude of IL-8 secretion was very similar in response to D39 and plnA–. In addition, there was greater secretion of IL-8 into the luminal compartment than into the abluminal compartment for several of the concentrations of bacteria studied. Together, these data suggest that IL-8 is not the primary mediator responsible for eliciting neutrophil migration under the conditions studied. There were not detectable levels of MCP-1, IL-6, or GRO-{alpha} elicited in response to either strain of S. pneumoniae. RANTES was produced in response to both D39 and plnA– at similar levels (data not shown). In addition, the cytokine TNF-{alpha} was measured as a potential neutrophil product that might be generated in response to neutrophil-bacterial interactions and perpetuate the migratory response. However, levels of TNF-{alpha} were undetectable in both the luminal and abluminal compartments in response to both strains of S. pneumoniae.


Figure 8
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  		Fig. 8. Endothelial IL-8 expression induced by incubation of the monolayer with D39 vs. plnA–. There was a dose-dependent increase in IL-8 secreted into the supernatant at the luminal surface (A; upper chamber of the Transwell) and the abluminal surface (B; lower chamber) supernatant in response to both D39 and plnA–. There were no significant differences in the amount of IL-8 secretion elicited by D39 vs. plnA–; n = 8 experiments.

 
Expression of endothelial cell adhesion molecules. Neutrophil migration into the lung in response to S. pneumoniae utilizes both CD18-dependent and -independent pathways. We investigated the surface expression of several endothelial cell adhesion molecules that might participate in neutrophil adhesion and transmigration and might be differentially upregulated in the presence or absence of the bacterial virulence factor pneumolysin. There was constitutive surface expression of ICAM-1 but no increase in surface levels after incubation of the monolayer with either D39 or plnA– (Fig. 9A). Similarly, there was no upregulation of VCAM-1 or E-selectin by either strain (Fig. 9, B and C), similar to our previous findings using a type 3 pneumococcal strain.(16) TNF-{alpha} (10 ng/ml) was used as a positive control and elicited significant upregulation of ICAM-1, VCAM-1, and E-selectin consistent with previously published data (20).


Figure 9
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  		Fig. 9. PMVEC surface expression of adhesion molecules. A: ICAM-1 was constitutively expressed by the endothelial monolayer and upregulated by exposure to TNF-{alpha}, but there was no effect on surface expression in response to coincubation with D39 or plnA– (5 x 106 CFU/ml) for 6 h. There was no expression of VCAM-1 (B) or E-selectin (C) by endothelial cells in the unstimulated monolayer, and there was no upregulation by exposure to D39 or plnA– at the abluminal surface. TNF-{alpha} elicited significantly increased surface expression of both molecules. *P < 0.05 compared with baseline expression; n = 4–6 experiments.

 
Assessment of nuclear translocation of NF-{kappa}B in response to S. pneumoniae. In view of the lack of upregulation of any of these adhesion molecules by S. pneumoniae, we explored whether NF-{kappa}B had any role in pneumococcal activation of the endothelial monolayer. Although it has been previously demonstrated that certain strains of S. pneumoniae may elicit activation of NF-{kappa}B (1, 18), this activation process by pneumococci appears to be restricted to certain host cell types (27). PMVEC monolayers were incubated with D39 or plnA– for time points ranging from 1 to 6 h followed by fixation, permeabilization, and immunostaining for NF-{kappa}B. There was no evidence of translocation of NF-{kappa}B p65 to the nucleus in any of the monolayers exposed to S. pneumoniae (using CFU counts ranging from 1 x 106/ml to 5 x 107/ml) (data not shown). TNF-{alpha} (10 ng/ml) was used as a positive control and elicited nuclear translocation of p65 in >90% of the cells of the monolayer at 1, 2, 4, and 6 h of stimulation, consistent with previously reports (22).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The intensely inflammatory neutrophilic infiltrate that accompanies pneumococcal pneumonia is responsible for significant host tissue damage, which contributes to morbidity. The role of pneumolysin in pathogenicity has primarily been studied with animal models with intranasal infection, instillation of purified pneumolysin (24), or intratracheal administration of live bacteria. The requirement for pneumolysin in the pathogenesis of bronchopneumonia is well established (10, 11). With the use of endotracheal instillation of bacteria, the pneumolysin-deficient mutant has been demonstrated to have reduced capacity to separate tight junctions of the alveolar epithelium and thus an inability to penetrate the alveolar capillary barrier and invade into the interstitial space (26). These data suggest that the reduced virulence associated with pneumolysin deficiency may be attributable to an altered interaction at the alveolar epithelium.

Interactions between intact pneumococci and host cells occurring after penetration of the alveolar epithelium are also highly relevant to the pathogenesis of pneumococcal pneumonia, as this organism has been demonstrated to move across the respiratory epithelium by transcytosis (17) and invade the bloodstream, causing bacteremia (6, 29). In the present investigation, the pneumolysin-deficient mutants are presented at the abluminal surface of the endothelium, as would occur when an organism has already successfully traversed the epithelium and interstitium. In this study, we demonstrate the potent induction of neutrophil transendothelial migration across a pulmonary microvascular endothelial monolayer in response to intact S. pneumoniae (serotype 2, D39) and a marked reduction in neutrophil migration elicited in response to the pneumolysin-deficient mutant plnA–. The PMN migratory response occurred in a dose-dependent manner, with 105 wild-type bacteria eliciting significant migration and a plateau in the response with 5 x 106 bacteria. The peak migration to the parent strain D39 included >55% of the PMNs, but only one-quarter of the PMNs migrated to a similar inoculum size of plnA–. Our results demonstrate both a shift in the dose response to the pneumolysin-deficient mutant and a diminished maximal response.

The marked difference between the wild-type and pneumolysin-deficient mutants suggests that changes in pneumococcal-endothelial cell interactions somehow affected by pneumolysin are also critical for recruitment of PMN and hence the intensity of the inflammatory response. It is not completely clear how the expression of pneumolysin affects pneumococcal-induced neutrophil transendothelial migration. However, our findings strongly suggest that pneumolysin does not act alone: 1) live bacteria (D39) have much greater potency than conditioned media (D39 or plnA–) or than EtOH-fixed bacteria, and 2) conditioned media from the wild-type D39 combined with the pneumolysin-deficient organisms did not enhance migration. These data indicate that the role of pneumolysin in pneumococcal-endothelial cell interactions in our model system is not simply a result of secretion of the toxin during autolysis of the bacteria. In addition, in contrast to observations from a murine bronchopneumonia model where purified pneumolysin coinstilled into the trachea with the pneumolysin-deficient mutant restored the pattern of bacterial replication and invasion seen with the wild-type bacteria (26), purified pneumolysin was highly toxic to the endothelium and elicited reduced levels of neutrophil migration. Together, these findings suggest a more complex interaction between the pneumococcus, the endothelial cell, and the PMN and suggest that the greater ability of the wild-type strain to elicit neutrophil migration might result from direct interaction of the bacteria with the endothelium and local delivery of specific pneumococcal products (e.g., pneumolysin).

Pneumolysin has been demonstrated to be required for normal adherence to respiratory epithelium using both an intact animal model and an organ culture model (21, 26). The requirement for pneumolysin in pneumococcal attachment/invasion of the pulmonary endothelium has not been studied and could potentially explain the reduced ability of the pneumolysin-deficient mutant to activate the endothelium for neutrophil migration. In our analysis of bacterial translocation across the endothelium, both strains moved from the luminal to abluminal surface within 1 h of infection at similar levels, and the only apparent difference in translocation occurred at the 6-h time point. Our data do not exclude differences in invasion of the cells of the monolayer as the monolayer was not lysed during this assay; however, it appears that the pneumolysin-deficient mutant is able to attach to and move across the microvascular endothelial cell monolayer. Although our data suggest that pneumolysin is not required for attachment to the endothelial monolayer, there may be significant alterations in the receptors engaged and/or the signaling initiated in the absence of pneumolysin. In addition, our model system was designed to specifically explore a role for the pulmonary endothelium in pneumococcus-induced transmigration; hence, the epithelium is not present. This reduction in complexity is also a limitation of our in vitro model, as secreted products from the alveolar epithelium and interstitium would certainly be modifiers of the endothelial cell response to S. pneumoniae.

The molecular participants involved in pneumococcal-endothelial cell interactions are likely highly complex and may employ multiple diverse endothelial cell surface receptors Recently, Toll-like receptor 4 (TLR4) was implicated as a receptor recognizing pneumolysin and TLR4–/– macrophages demonstrated to be hyporesponsive to purified pneumolysin. (13) Although these studies focused predominantly on the role of macrophage TLR4, the data suggest that epithelial or endothelial TLR4 might also function in the recognition of S. pneumoniae. In addition, a specific role for endothelial TLR4 has been demonstrated for pulmonary neutrophil sequestration in response to LPS instillation (2). We postulated that recognition of S. pneumoniae by the microvascular endothelial cell and subsequent activation of the endothelium might require two or more distinct receptors that act sequentially with pneumolysin signaling through TLR4 as one step in this process. We explored IL-8 secretion by the endothelium as one proinflammatory marker of TLR4-dependent cell activation and a source of neutrophil chemoattractant and hypothesized that IL-8 secretion would be markedly diminished in the supernatant of monolayers exposed to the pneumolysin-deficient mutant. Interestingly, the wild-type and pneumolysin-deficient mutants dose dependently elicited very similar levels of IL-8 production by the endothelium. These data not only suggest that pneumolysin-mediated TLR4 activation is not the primary effector of neutrophil migration but also that neutrophil migration does not occur based on a chemoattractant gradient of IL-8.

The primary endothelial receptors interacting with the intact pneumococcus in the pulmonary capillary bed have not been defined and may require pneumolysin-sufficient organisms for full activation. Interaction with the airway epithelium is mediated by pneumococcal cell surface phosphorylcholine acting as a direct ligand for the PAFR (7), which is upregulated on the epithelium in response to inflammatory stimuli. The PAFR is also widely expressed on the vascular endothelium, and in vitro studies demonstrate that virulent pneumococci adhere to brain endothelium in a PAFR-dependent manner and appear to enter these cells by receptor-mediated endocytosis (7, 25). This receptor-ligand pairing does not require pneumolysin. Interactions between S. pneumoniae and the vascular endothelium have predominantly been studied in the context of pneumococcal meningitis. The microvascular endothelium of the brain is distinct from all other endothelium in terms of barrier function, and the pneumococcus interacts at the luminal surface. In contrast, during the onset of pneumococcal pneumonia, the organism enters the lower respiratory tract from the upper respiratory tract, and interactions between the pneumococcus and the pulmonary microvascular endothelium occur exclusively at the abluminal surface. Further investigations of pneumococcal interactions with the pulmonary microvascular endothelium, including the role of the PAFR, are needed.

The molecular determinants of neutrophil-endothelial cell interactions in response to S. pneumoniae in the lung are also not well defined. We have previously shown that a type 3 pneumococcal strain did not enhance surface expression of ICAM-1, ICAM-2, VCAM-1, or E-selectin on pulmonary microvascular endothelium. Similarly, in the present investigation, neither the wild-type (type 2 strain) D39 nor the pneumolysin-deficient mutant elicited increased expression of ICAM-1, VCAM-1, or E-selectin on the pulmonary microvascular endothelium. These data are in agreement with the published literature, which suggest that 1) ICAM-1 has no role in S. pneumoniae-induced neutrophil recruitment to the lung in a murine model (5), 2) VCAM-1 may have a minor role in movement of PMN from the vascular space to the lung, but this role likely occurs beyond the endothelium (i.e., at the epithelium or interstitium) (23, 30), and 3) pneumococcal pneumonia induces E-selectin-independent migration of neutrophils (15). Together with our finding that endothelial cell NF-{kappa}B was not activated by S. pneumoniae, these data suggest that the molecular determinants of neutrophil transendothelial migration elicited by the pneumococcus may be unique from those defined for other gram-positive bacteria and gram-negative bacteria.

The present study demonstrates that pneumolysin is necessary to elicit neutrophil transendothelial migration and suggests that direct interaction of the pneumolysin-containing organism may be necessary in this process. Whatever precise mechanism(s), our findings indicate a novel role for pneumolysin in pneumococcus-induced migration of PMN across the pulmonary microvascular endothelium. The in vitro model described in this study should help, in the future, to better define the molecular basis of pneumococci, endothelial cells, and PMN interactions needed to induce maximal recruitment of PMN from the vascular space to the lung.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Environmental Health Sciences Grant ES-00378 and American Lung Association Grant RG-127-N (to J. G. Moreland).


   ACKNOWLEDGMENTS
 
The authors thank Jerrold P. Weiss for assistance with critical review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. G. Moreland, Division of Pediatric Critical Care, Dept. of Pediatrics/2JCP, The Univ. of Iowa, Iowa City, IA 52242 (e-mail: jessica-moreland{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amory-Rivier CF, Mohler J, Bedos JP, Azoulay-Dupuis E, Henin D, Muffat-Joly M, Carbon C, and Moine P. Nuclear factor-{kappa}B activation in mouse lung lavage cells in response to Streptococcus pneumoniae pulmonary infection. Crit Care Med 28: 3249–3256, 2000.[CrossRef][ISI][Medline]
  2. Andonegui G, Bonder CS, Green F, Mullaly SC, Zbytnuik L, Raharjo E, and Kubes P. Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs. J Clin Invest 111: 1011–1020, 2003.[CrossRef][ISI][Medline]
  3. Berry A, Yother J, Briles D, Hansman D, and Paton J. Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae. Infect Immun 57: 2037–2042, 1989.[Abstract/Free Full Text]
  4. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. J Clin Lab Invest 21: 77–89, 1968.[ISI][Medline]
  5. Bullard DC, Qin L, Lorenzo I, Quinlin WM, Doyle NA, Bosse R, Vestweber D, Doerschuk CM, and Beaudet AL. P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli. J Clin Invest 95: 1782–1788, 1995.[ISI][Medline]
  6. Catterall J. Streptococcus pneumoniae. Thorax 54: 929–937, 1999.
  7. Cundell D, Gerard N, Gerard C, Idanpaan-Heikkla I, and Tuomanen E. Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377: 435–438, 1995.[CrossRef][Medline]
  8. Gilbert R, Jimenez J, Chen S, Tickle I, Rossjohn J, Parker M, Andrew P, and Saibil H. Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae. Cell 97: 647–655, 1999.[CrossRef][ISI][Medline]
  9. Ishizuka S, Yamaya M, Suzuki T, Nakayama K, Kamanaka M, Ida S, Sekizawa K, and Sasaki H. Acid exposure stimulates the adherence of Streptococcus pneumoniae to cultured human airway epithelial cells: effects on platelet-activating factor receptor expression. Am J Respir Cell Mol Biol 24: 459–468, 2001.[Abstract/Free Full Text]
  10. Jounblat R, Kadioglu A, Mitchell TJ, and Andrew PW. Pneumococcal behavior and host responses during bronchopneumonia are affected differently by the cytolytic and complement-activating activities of pneumolysin. Infect Immun 71: 1813–1819, 2003.[Abstract/Free Full Text]
  11. Kadioglu A, Gingles NA, Grattan K, Kerr A, Mitchell TJ, and Andrew PW. Host cellular immune response to pneumococcal lung infection in mice. Infect Immun 68: 492–501, 2000.[Abstract/Free Full Text]
  12. Laterre PF, Garber G, Levy H, Wunderink R, Kinasewitz GT, Sollet JP, Maki DG, Bates B, Yan SC, and Dhainaut JF. Severe community-acquired pneumonia as a cause of severe sepsis: data from the PROWESS study. Crit Care Med 33: 952–961, 2005.[CrossRef][ISI][Medline]
  13. Malley R, Henneke P, Morse SC, Cieslewicz MJ, Lipsitch M, Thompson CM, Kurt-Jones E, Paton JC, Wessels MR, and Golenbock DT. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infections. Proc Natl Acad Sci USA 100: 1966–1971, 2003.[Abstract/Free Full Text]
  14. Mitchell T, Andrew P, Saunders F, Smith A, and Boulnois G. Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute-phase protein. Mol Microbiol 5: 1883–1888, 1991.[ISI][Medline]
  15. Mizgerd JP, Meek BB, Kutkoski GJ, Bullard DC, Beaudet AL, and Doerschuk CM. Selectins and neutrophil traffic: margination and Streptococcus pneumoniae-induced emigration in murine lungs. J Exp Med 184: 639–645, 1996.[Abstract/Free Full Text]
  16. Moreland J, Bailey G, Nauseef WM, and Weiss JP. Organism-specific neutrophil-endothelial cell interactions in response to E. coli, S pneumoniae, and S aureus. J Immunol 172: 476–432, 2004.
  17. Novak R and Tuomanen E. Pathogenesis of pneumococcal pneumonia. Semin Respir Infect 14: 209–217, 1999.[Medline]
  18. Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, and Hippenstiel S. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae. J Biol Chem 279: 36426–36432, 2004.[Abstract/Free Full Text]
  19. Paton J. The contribution of pneumolysin to the pathogenicity of Streptococcus pneumoniae. Trends Microbiol 4: 103–106, 1996.[CrossRef][ISI][Medline]
  20. Pober JS. Effects of tumour necrosis factor and related cytokines on vascular endothelial cells. Ciba Found Symp 131: 170–184, 1987.[Medline]
  21. Rayner C, Jackson A, Rutman A, Dewar A, Mitchell T, Andrew P, Cole P, and Wilson R. Interaction of pneumolysin-sufficient and -deficient isogenic variants of Streptococcus pneumoniae with human respiratory mucosa. Infect Immun 63: 442–447, 1995.[Abstract]
  22. Read MA, Whitley MZ, Williams AJ, and Collins T. NF-{kappa}B and I{kappa}B{alpha}: an inducible regulatory system in endothelial activation. J Exp Med 179: 503–512, 1994.[Abstract/Free Full Text]
  23. Ridger V, Wagner B, Wallace W, and Hellewell P. Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation. J Immunol 166: 3484–3490, 2001.[Abstract/Free Full Text]
  24. Rijneveld AW, van den Dobbelsteen GP, Florquin S, Standiford TJ, Speelman P, van Alphen L, and van der Poll T. Roles of interleukin-6 and macrophage inflammatory protein-2 in pneumolysin-induced lung inflammation in mice. J Infect Dis 185: 123–126, 2002.[CrossRef][ISI][Medline]
  25. Ring A, Weiser JN, and Tuomanen EI. Pneumococcal trafficking across the blood-brain barrier. J Clin Invest 102: 347–360, 1998.[ISI][Medline]
  26. Rubins J, Charboneau D, Paton J, Mitchell T, and Andrew P. Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia. J Clin Invest 95: 142–150, 1995.[ISI][Medline]
  27. Spellerberg B, Rosenow C, Sha W, and Tuomanen EI. Pneumococcal cell wall activates NF-{kappa}B in human monocytes: aspects distinct from endotoxin. Microb Pathog 20: 309–317, 1996.[CrossRef][ISI][Medline]
  28. Steinfort C, Wilson R, Mitchell T, Feldman C, Rutman A, Todd H, Syke D, Walker J, Saunders K, and Andrew PW. Effects of Streptococcus pneumoniae on human respiratory epithelium in vitro. Infect Immun 57: 2006–2013, 1989.[Abstract/Free Full Text]
  29. Tan T. Update on pneumococcal infections of the respiratory tract. Semin Respir Infect 17: 3–9, 2002.[CrossRef][Medline]
  30. Tasaka S, Richer SE, Mizgerd JP, and Doerschuk CM. Very late antigen-4 in CD18-independent neutrophil emigration during acute bacterial pneumonia in mice. Am J Respir Crit Care Med 166: 53–60, 2002.[Abstract/Free Full Text]
  31. Weiss K, Low DE, Cortes L, Beaupre A, Gauthier R, Gregoire P, Legare M, Nepveu F, Thibert D, Tremblay C, and Tremblay J. Clinical characteristics at initial presentation and impact of dual therapy on the outcome of bacteremic Streptococcus pneumoniae pneumonia in adults. Can Respir J 11: 589–593, 2004.[Medline]
  32. Zysk G, Schneider-Wald B, Hwang J, Bejo L, Kim K, Mitchell T, Hakenbeck R, and Heinz HP. Pneumolysin is the main inducer of cytotoxicity to brain microvascular endothelial cells caused by Streptococcus pneumoniae. Infect Immun 69: 845–852, 2001.[Abstract/Free Full Text]




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