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Streptococcus pneumoniae is a major cause of pneumonia, septicemia, and meningitis. Pneumococcal infection induces host responses associated with acute inflammation. One of the key characteristics of acute inflammation is edema caused by increased vascular permeability. During pneumococcal infection, increased vascular permeability may result in vasogenic brain edema in meningitis, as well as empyema, pleural effusion, and pulmonary consolidation in pneumonia.

One of the most potent regulators of endothelial permeability is vascular endothelial growth factor (VEGF), also called vascular permeability factor. Only minutes after administration of VEGF, small capillaries and venules display endothelial fenestration and opening of tight junctions (12). VEGF has been involved in several disease states characterized by edema and increased vascular permeability, including rheumatoid arthritis, brain edema following trauma or tumor growth, allergic diseases, and parainfectious pleural effusions (1, 2, 5, 9, 13, 19). VEGF is also an endothelial mitogen which promotes embryonal vascular development and neovascularization in tumor growth (3, 6). VEGF is produced by a broad range of cell types, including macrophages, platelets, and neutrophils (4). The expression of VEGF is regulated by various stimuli, such as inflammatory cytokines (interleukin-1, tumor necrosis factor alpha, gamma interferon), hypoxia, and lipopolysaccharide (11, 18). The first line of defense against pneumococci consists predominantly of neutrophils. Since neutrophils are associated with the initial inflammatory response and can produce VEGF, we hypothesized that the increase in vascular permeability observed in pneumococcal disease may be mediated by VEGF. In this study, we assessed whether pneumococci and pneumococcal components induce secretion of VEGF by human neutrophils.

Bacterial strains, growth conditions, and pneumococcal components. The strains of S. pneumoniae tested were S3 (serotype 3; American Type Culture Collection), D39 (serotype 2), and two isogenic mutants: the pneumolysin-negative mutant PLN-A and the unencapsulated derivative R6 (a kind gift from E. Tuomanen). Bacteria were grown in Todd-Hewitt broth to mid-logarithmic phase (optical density at 660 nm of 0.5). Alternatively, bacteria were grown on tryptic soy agar containing 5% sheep blood for 16 to 18 h at 37°C in 5% CO₂. Mutant strain PLN-A was grown in the presence of erythromycin (1 µg ml¹) to maintain an environment selective for the mutant. For some experiments, bacteria were killed by heating for 10 min at 60°C. Purified cell wall was prepared from strain S3 as described previously (15). Briefly, logarithmically growing pneumococci were heat killed. Crude cell wall was extracted in 5% sodium dodecyl sulfate at 100°C for 15 min. The pellet was washed to remove detergent and then vortexed with glass beads to mechanically break any remaining intact cells. After treatment with DNase, RNase, and finally trypsin to remove associated proteins, the cell wall was reprecipitated in sodium dodecyl sulfate, washed, vacuum dried, and stored at room temperature. The cellular equivalent of 0.02 µg of cell wall approximates 10⁵ cells (17). The pneumococcal polysaccharide vaccine Pneumovax 23 was purchased from Pasteur Mérieux MSD (Brussels, Belgium). The cellular equivalent of 0.2 µg of capsular polysaccharide approximates 10⁵ cells (10).

Isolation and stimulation of neutrophils and VEGF measurement. Neutrophils were isolated by a Ficoll-Histopaque gradient from peripheral blood of healthy volunteers as described previously (14). Neutrophils were resuspended in RPMI 1640 containing 1% heat-inactivated fetal calf serum. The viability of cells exceeded 95% as determined by trypan blue exclusion. Purity was checked by optical and impedance analysis (Abbott Cell Dyn 3500) and was >98%; in the purified neutrophil fraction, platelets represented one-fifth of the cells. Neutrophils (1.25 × 10⁶ cells; 0.5 ml) were stimulated with live or heat-killed pneumococci or pneumococcal components at 37°C for 0 to 4 h in a water bath shaker. As a positive control, cells were incubated with cytochalasin B (10 µg ml¹) for 30 min, followed by addition of formylmethionylleucylphenylalanine (fMLP) at 1 µM for 30 min. As a negative control, cells were incubated with medium alone. Experiments were carried out aseptically to avoid endotoxin contamination. In addition, polymyxin B (Sigma Chemical Co., St. Louis, Mo.) was added to all test mixtures at a final concentration of 10 µg ml¹ to abolish the stimulatory effects of endotoxin (8). After incubation, the samples were centrifuged at 350 × g, and the supernatants were collected and stored at 70°C prior to VEGF assays. A commercially available human VEGF enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe, Abingdon, United Kingdom) was used to measure VEGF in culture supernatants. Measurements were done according to the manufacturer's protocol. All data are expressed as means ± standard deviations (SD).

Pneumococci induce dose- and time-dependent VEGF secretion. To determine the dose effects and kinetics of pneumococcus-induced VEGF secretion, freshly isolated neutrophils were incubated with heat-killed S. pneumoniae (R6) suspensions of several concentrations for 0 to 4 h. Heat-killed pneumococci were used to avoid differences in pneumococcal concentration during the stimulation assay due to bacterial growth and lysis. Pneumococcal suspensions at a concentration of ~10⁷ CFU ml¹ (multiplicity of infection of 4) induced VEGF secretion (Fig. 1A). Release of VEGF reached a plateau at a concentration of ~10⁸ to 10⁹ CFU ml¹ (multiplicity of infection of ~40 to 400). Though relatively high numbers of pneumococci were needed, these concentrations are not unlike those encountered in clinical disease: during meningitis, bacterial concentrations in the cerebrospinal fluid as high as 4 × 10⁹ CFU ml¹ may be reached, and in >50% of patients concentrations of ~10⁷ CFU ml¹ are found (7). Kinetics studies showed that the VEGF concentration in the supernatant was increased at 2 h and accumulated to a plateau at 4 h (Fig. 1B).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   (A) Dose-effect curve of VEGF secretion by human neutrophils (2.5 × 106 neutrophils ml1) following stimulation with different doses of heat-killed S. pneumoniae R6 at 37°C for 4 h. (B) Kinetics of VEGF secretion by human neutrophils (2.5 × 106 neutrophils ml1) following stimulation with heat-killed S. pneumoniae R6 at a concentration of 109 CFU ml1 at 37°C for 0 to 4 h. Results are means ± SD of 2 independent experiments with blood from different donors and ELISA performed in duplicate. Symbols for stimuli: , R6; , medium alone; , cytochalasin B (10 µg ml1) plus fMLP (10 µM).

Characterization of pneumococcal stimulus. Based on the dose and kinetics experiments, for all subsequent experiments 4-h incubations with bacterial suspensions of ~10⁸ to 10⁹ CFU ml¹ were used. As shown in Fig. 2, no difference was found between live and heat-killed S. pneumoniae R6. In addition, both encapsulated pneumococci (D39 and S3) and unencapsulated pneumococci (R6) induced VEGF secretion, though the unencapsulated pneumococcal strain was slightly more effective.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   VEGF secretion by neutrophils following incubation with different stimuli at 37°C for 4 h. Live pneumococci were tested at 109 CFU ml1. Polysaccharide capsule and pneumococcal cell wall were tested at a cell equivalent of 108 CFU ml1. Incubation with cytochalasin B (10 µg ml1) for 30 min followed by fMLP (10 µM) for 30 min served as a positive control; medium alone served as negative control. Data are percentages of the positive control (means ± SD [error bars]) and represent two independent experiments with blood from different donors and ELISA performed in duplicate.

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