Adherence to, Invasion by, and Cytokine Production in Response to Serotype VIII Group B Streptococci

Bacterial strains.We used to 20 strains of GBS, 10 of serotype III and 10 of serotype VIII, in these studies. Serotype III strain M781 was originally isolated from a newborn with meningitis (49). An invasive strain of serotype VIII, NKMC1998, was kindly provided by Kousaku Matsubara (Department of Pediatrics, Nishi-Kobe Medical Center, Kobe, Japan) (30). Vaginal isolates of serotype III strains (SMU001, SMU007, SMU016, SMU019, SMU056, SMU072, SMU075, SMU080, and SMU097) and vaginal isolates of serotype VIII strains (SMU003, SMU012, SMU014, SMU022, SMU029, SMU037, SMU044, SMU076, and SMU089) were obtained from cultures of vaginal samples from pregnant Japanese women, as described previously (21).

Cell line.The A549 cell line (ATCC CCL-185) was derived from a human lung carcinoma; it has many of the characteristics of type II alveolar epithelial cells, including surfactant production in culture, and has been used in our laboratory for invasion experiments (16). A549 cells were maintained in RPMI 1640 culture medium (GIBCO, Invitrogen, Grand Island, N.Y.) with 1% l-glutamine and 10% fetal bovine serum (GIBCO, Invitrogen) without antibiotics. The medium was replaced 1 day before the experiment. A549 cells suspended in 0.5 ml of RPMI 1640 with 10% fetal bovine serum were seeded into each well of 24-well culture plates and incubated for 4 days at 37°C with 5% CO₂. By the beginning of the assay, the cell number had increased to approximately 1.5 × 10⁶ cells per well. Cell viability was determined by 0.4% trypan blue exclusion and hemocytometer counts.

Invasion and adherence assays.The attachment and invasion assays were done essentially as described by Rubens et al. (41), and DIVAS was performed as described previously (16), with the modifications indicated below. For the conventional assays, GBS were grown to an optical density at 650 nm (OD₆₅₀) of 0.3 to 0.5 in Todd-Hewitt broth (THB) (Becton Dickinson and Co., Sparks, Md.) and inoculated onto A549 monolayers for 2 h in each well of 24-well plates at 1.5 × 10⁶ CFU/well to achieve a multiplicity of infection of 1:1. The infected monolayers were then washed three times with phosphate-buffered saline (PBS) (ICN Biochemicals, Aurora, Ohio); 1 ml of fresh RPMI 1640 medium with 10% fetal bovine serum, 5 μg of penicillin G (Sigma-Aldrich, St. Louis, Mo.) per ml, and 100 μg of gentamicin (Sigma-Aldrich) per ml was added to each well, and plates were incubated in a CO₂ incubator for 2 h. Monolayers were rinsed five times with PBS, and A549 cells were detached by addition of 0.2 ml of trypsin-EDTA (GIBCO, Invitrogen), lysed by addition of 0.8 ml of 0.025% Triton X-100 (LabChem, Pittsburgh, Pa.), transferred quantitatively to microtubes, and mixed on a Vortex mixer for 1 min. Appropriately diluted aliquots in 0.025% Triton X-100 were plated on tryptic soy-5% sheep blood agar plates. The plates were incubated at 37°C overnight, and the number of GBS CFU recovered per well was determined. Each test was done in quadruplicate.

Bacterial adherence was determined as follows. GBS grown in THB to mid-log phase (OD₆₅₀ of 0.3 to 0.5) were inoculated at 1.5 × 10⁶ CFU/well onto A549 monolayers for 2 h in each well of 24-well plates. The infected monolayers were then washed five times with PBS, detached from the wells with 0.2 ml of trypsin-EDTA, lysed with 0.8 ml of 0.025% Triton X-100, transferred quantitatively to microtubes, and vortexed vigorously for 1 min. Aliquots diluted in 0.025% Triton X-100 were plated on tryptic soy-5% sheep blood agar plates. The plates were incubated at 37°C overnight, and GBS CFU were counted. The number of attached GBS was calculated as total (attached and invaded) CFU minus invaded CFU. Each test was done in quadruplicate, and the number of CFU recovered per well was determined.

DIVAS.DIVAS was developed to study microbe-host cell interactions with bacteria held at steady-state growth in a chemostat under defined environmental and nutritional conditions (16, 25). Bacteria held at the steady state of growth are perfused slowly over an established monolayer of tissue culture cells as described below. In brief, a 1-liter chemostat vessel (Applikon, Foster City, Calif.) with a working volume of 0.5 liter was used to grow GBS in chemically defined medium with glucose limitation at a mass doubling time (t_d) of 1.8 h (fast growth). The temperature (37°C), pH (pH 7.3), and mixing rate (400 rpm) were maintained at constant levels throughout the experiments. Levels of dissolved oxygen (DO₂) were monitored with a polarographic DO₂ probe and controlled by the addition of a mixture of compressed nitrogen and oxygen. All growth parameters (pH, temperature, mixing rate, media inflow rate, and DO₂) were computer controlled with a BioController (model ADI 1030; Applikon).

Attachment and invasion experiments were performed with A549 respiratory epithelial cells grown in 60-mm-diameter culture dishes. Confluent cells were washed twice with 3.0 ml of PBS prior to use in DIVAS. GBS from the chemostat were perfused over the A549 cells by using a plate holder apparatus with inlet and outlet ports to allow for perfusion of bacteria (25). The apparatus was placed on a rotary shaker at 20 rpm in a 37°C incubator, and perfusion of GBS proceeded for 2 h, after which the plates were disconnected from the outflow line of the chemostat. Cell monolayers were washed with PBS, extracellular GBS were killed with antibiotics, eukaryotic cells were disrupted, and internalized GBS were quantified by standard plate counts as described previously (16, 25).

SMU007 (serotype III) and SMU029 (serotype VIII) GBS were used for DIVAS assay. Invasion and adherence with DIVAS were performed with GBS held at a t_d of 1.8 h and 12% DO₂, conditions that result in high levels of invasion (16). Cytokine assays were also performed twice on filtered outflow supernatant fluid samples obtained at 0, 1, and 2 h after GBS passed over the A549 cell monolayer.

Induction of cytokines from human lung epithelial cells.The following GBS strains were used in the experiments involving cytokine production by human A549 lung epithelial cells: serotype III M781, SMU007, SMU016, SMU001, SMU056, and SMU019 and serotype VIII NKMC1998, SMU014, SMU037, SMU029, SMU003, and SMU044.

Sampling method for cytokine assays.GBS were grown to an OD₆₅₀ of 0.3 to 0.5 in THB. GBS were inoculated at 1.5 × 10⁶ CFU/well onto A549 monolayers in each well of 24-well plates at an MOI of 1:1. The supernatant fluid was removed after 2 h of contact with the bacteria. The supernatant fluids were clarified by centrifugation and were filtered (0.22-μm-pore-size filter) for clarification.

Cytokine assay.Cytokines secreted into the culture supernatant were detected with enzyme-linked immunosorbent assays. Human TNF-α or human TNF-α US (ultrasensitive), human IL-8/NAP-1, human IL-10 or human IL-10 US, and human IL-12 US immunoassay kits (all from BioSource International, Camarillo, Calif.) were used to detect TNF-α, IL-8, IL-10, and IL-12, respectively. The quantitative limits of detection were 15.6 to 1,000, 0.5 to 32, 15.6 to 1,000, 7.8 to 500, 0.78 to 50, and 1.56 to 100 pg/ml for the TNF-α, TNF-α US, IL-8/NAP-1, IL-10, IL-10 US, and IL-12 US kits, respectively. All assays were conducted according to the manufacture's instructions in duplicate.

Effect of stimulation with GBS CPS on TNF-α production.TNF-α production by A549 cells stimulated with serotype III and VIII GBS CPS at 2, 5, and 10 μg/ml was investigated. GBS serotype III and serotype VIII CPS were purified as previously described (47, 48). Each test was done in triplicate, and cytokine assays were conducted twice in duplicate. Because of the potential for the presence of trace amounts of endotoxin in the purified CPS, each CPS was exposed to polymyxin B (2, 5, and 10 μg/ml) (Sigma-Aldrich) at stimulation. Each test was done in quadruplicate, and cytokine assays were conducted twice in duplicate.

Statistical analyses.The significance of GBS invasion for two different variables was determined by the unpaired t test with the Welch correction. These analyses were performed with Instat version 3.0a (GraphPad, San Diego, Calif.). P values of <0.05 were considered significant.

Adherence and invasion.Adherence to and invasion into A549 cells by serotype VIII GBS was significantly higher than those by serotype III GBS (Table 1) both for the invasive isolate (P < 0.0001) and for vaginal isolates (P < 0.0001). For serotype VIII, a significant difference (P < 0.005) between the invasion into A549 cells by invasive isolate NKMC1998 and vaginal isolates was seen, whereas for serotype III, no significant difference (P > 0.05) between invasive and vaginal isolates was noted. For both serotypes, there was no significant difference (P > 0.05) in the adherence of invasive and vaginal isolates to A549 cells.

To determine the invasiveness of GBS held at a steady state of growth and perfused over A549 cells, we performed DIVAS with two selected strains of GBS: serotype III SMU007 and serotype VIII SMU029. A mean (± standard deviation) of 17 ± 2.8 GBS serotype III SMU007 cells adhered per A549 cell, compared with 24 ± 0.7 GBS serotype VIII SMU029 cells (P < 0.05) (Fig. 1).

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FIG. 1.

Adherence of GBS to (top) and invasion of GBS into (bottom) A549 cells during fast growth (t_d of 1.8 h) with 12% DO₂ in DIVAS. Error bars indicate standard deviations.

GBS serotype VIII SMU029 grown in continuous culture invaded A549 cells more readily than did GBS serotype III SMU007 (Fig. 1). The CFU of GBS serotypes III and VIII grown at a t_d of 1.8 h with 12% DO₂ that invaded A549 cells were 672 ± 56 and 7,900 ± 1,800, respectively (P < 0.0001).

Cytokine production.Secretion of TNF-α, IL-8, and IL-10 by A549 cells in response to GBS infection was significantly increased compared with secretion by the untreated control group. There was a significant difference between TNF-α production by serotypes III and VIII for both invasive and vaginal isolates (P < 0.0001 and P < 0.0001 for invasive and vaginal isolates, respectively) (Fig. 2). There was a significant difference in IL-8 production between serotypes III and VIII for both invasive and vaginal isolates (P < 0.001 and P < 0.001 for invasive and vaginal isolates, respectively) (Fig. 2). IL-10 production by A549 cells was similar to that of TNF-α. There was a significant difference in IL-10 production in response to serotypes III and VIII for both invasive (P < 0.05) and vaginal (P < 0.01) isolates (Fig. 2). IL-12 production by A549 cells in response to GBS infection was observed for serotype III, while it was not observed for serotype VIII (Fig. 2).

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FIG. 2.

TNF-α, IL-8, IL-10, and IL-12 production by A549 cells after 2 h of stimulation with GBS serotype III and serotype VIII strains. The dotted line shows the average of each cytokine value. The means of TNF-α production following stimulation with serotypes III and VIII are 75.1 and 46.1 pg/ml, respectively. The means of IL-8 production for serotypes III and VIII are 541.2 and 677.4 pg/ml, respectively. The means of IL-10 production for serotypes III and VIII are 11.9 and 9.7 pg/ml, respectively. The mean of IL-12 production for serotype III is 1.74 pg/ml. Error bars indicate standard deviations.

Since the flow of chemically defined medium in the DIVAS assay is continuous, cytokines do not accumulate but are diluted in this open-flow system over time; thus, the level of cytokine production was relatively low in all samples. Nevertheless, TNF-α production in response to serotypes III and VIII differed significantly (P < 0.0001 and P < 0.0001 at 1 and 2 h, respectively) (Fig. 3). Also, there was a significant difference in IL-8 production in response to serotypes III and VIII (P < 0.0005 and P < 0.0001 at 1 and 2 h, respectively) (Fig. 3). On the other hand, IL-10 production could be detected only for serotype III GBS at 2 h and was below the detection limit for serotype VIII (Fig. 3).

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FIG. 3.

TNF-α, IL-8, and IL-10 production by A549 cells in DIVAS following stimulation with GBS serotype III and VIII strains. Error bars indicate standard deviations.

Effect of stimulation with GBS CPS on TNF-α production.TNF-α production by A549 cells increased in a dose-dependent manner with both serotypes after stimulation with CPS (Fig. 4). There was a significant difference in TNF-α production in response to CPS of serotypes III and VIII at all concentrations tested (P < 0.005, P < 0.0001, and P < 0.0001 at CPS concentrations of 2, 5, and 10 μg/ml, respectively) (Fig. 4). TNF-α production by A549 cells was inhibited by only 26.2% (serotype III CPS) and 21.5% (serotype VIII CPS) following stimulation of A549 cells by CPS with polymyxin B. TNF-α production by A549 cells differed significantly (P < 0.0005) following stimulation by either serotype III CPS or serotype VIII CPS.

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FIG. 4.

TNF-α production by A549 cells after stimulation by GBS serotype III and VIII CPS. (Top panel) Induction of TNF-α by purified CPS with and without addition of polymyxin B. (Bottom panel) THF-α production as a function of CPS concentration. Error bars indicate standard deviations.