Pneumococcal Infections in Humans Are Associated with Increased Apoptosis and Trafficking of Type 1 Cytokine-Producing T Cells

Patients.Twenty-two consecutive patients (10 men, 12 women) with infections caused by S. pneumoniae in a department of infectious diseases were included. The median age was 68 years (range, 37 to 91 years). None of the patients was HIV positive or had cancer. Eleven patients had bacteremia, including 6 patients with meningitis, but none had multiple organ failure. The remaining patients had noninvasive pneumonia. A microbiologic diagnosis of S. pneumoniae infection was obtained in 19 patients. The remaining three patients were included because they had radiologically verified lobar pneumonia, a high level of C-reactive protein (CRP) at admission, and an immediate clinical response to treatment with penicillin G. The median length of hospitalization was 13 days (range, 2 to 38 days). Blood samples were collected during the first 24 h after admission (day 0) and after 7 days. Twenty-two healthy, sex- and age-matched subjects served as a control group.

Isolation of mononuclear cells.Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep (Nyegaard, Oslo, Norway) density centrifugation. The cells were frozen and stored in liquid nitrogen. Before use, the cells were rapidly thawed and washed. The viability of the cells was ascertained by trypan blue exclusion. Previous studies have shown that freezing does not significantly change the expression of the surface markers used, intracellular cytokine expression, or phytohemagglutinin (PHA)-induced apoptosis (13). Blood samples were collected during the same time period and treated in the exact same way for patients and controls. For each donor, samples from days 0 and 7 were analyzed on the same day together with samples from the healthy age- and sex-matched control in all of the cellular immune assays in order to eliminate the influence of day-to-day variation.

Clinical chemistry tests.Standard laboratory procedures were used for clinical chemistry tests. The concentration of leukocyte subsets was determined with a cell counter (Technic H.1; Miles, Tarrytown, N.Y.).

Cultivation of PBMC.PBMC were resuspended in RPMI 1640, supplemented with 15% heat-inactivated pooled human serum, 20 IU of penicillin per ml, and 20 μg of streptomycin per ml (Gibco, Paisley, United Kingdom) and seeded into 24-well plates (Nunc, Roskilde, Denmark). Each well contained 10⁶ PBMC in 1 ml of medium. The cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. For intracellular cytokine detection, the cells were incubated with 1.5 μM monensin (Sigma, St. Louis, Mo.), 1 μM ionomycin, and 50 ng of phorbol myristate acetate per ml for 4 h. For detection of apoptosis, the cells were incubated with PHA in a final concentration of 40 μg/ml for 24 h.

Flow cytometry.For surface staining, three-color flow cytometry was applied by using a monoclonal antibody (MAb) conjugated to either fluorescein isothiocyanate (FITC), phycoerythrin, or Cy5 and specific for the determinants listed: CD3 (UCHT1; DAKO, Glostrup, Denmark) CD4 (MT310; DAKO) CD8 (DK25; DAKO), CD19 (HD37; DAKO), CD45R0 (F0800; DAKO), CD45RA (4KB5;DAKO), CD62L (FMC46, DAKO), CD56 (MOC-1; DAKO), and CD95 (M019751; Pharmingen, San Diego, Calif.). All samples were analyzed on a flow cytometer (Epics XL-MCL; Coulter, Hialeah, Fla.). Data were analyzed with Winlist software (Verity, Topsham, Maine). The absolute number of the lymphocyte subset was found by multiplying the lymphocyte counts by the proportion of lymphocytes.

Intracellular cytokine staining.The method used for intracellular cytokine staining was based on other studies (15). In brief, following surface staining, cells were washed twice in staining buffer, fixed in staining buffer containing 2% formaldehyde (Sigma), and washed twice in a freshly made saponin buffer (phosphate-buffered saline [PBS], bovine serum albumin [BSA], NaN₃ containing 0.1% [wt/vol] saponin Σ), and incubated with anticytokine antibody (IL-2, tumor necrosis factor alpha [TNF-α], IFN-γ, or IL-4 from Pharmingen) for 30 min in the dark (40°C). Following intracellular labeling, the cells were washed twice in saponin buffer and twice in staining buffer, resuspended in the same buffer, and analyzed.

Analysis of apoptosis by annexin V and 7AAD staining.PBMC were labeled with annexin V (M046701; Pharmingen) and 7-aminoactinomycin D (7AAD) (Sigma) in binding buffer (Pharmingen) according to the manufacturer's directions and analyzed by flow cytometry.

Levels of sFas ligand in plasma.Levels of sFasL in plasma were measured by enzyme-linked immunosorbent assay (ELISA) (Naka-ku, Nagoya, Japan).

Statistical analysis.Statistical calculations were performed with Sigma Stat software. Data are presented as medians and quartiles, because initial analysis revealed that the lymphocyte data were not normally distributed. Parameters obtained on day 1 were compared to data obtained on day 7 by Wilcoxon's signed-rank test for paired data. Furthermore, data from patients on day 1 or day 7 were compared to those from controls by Mann-Whitney rank-sum test for independent groups. P values of <0.05 were considered significant. Preliminary data analysis revealed that there were no significant differences between patients with pneumonia and patients with bacteremia or meningitis according to the parameters investigated. Consequently, patient data were pooled in the final analyses.

Patients are characterized by low numbers of lymphocytes.Patients had elevated levels of CRP, increased numbers of neutrophils, and low hemoglobin levels at admission to the hospital compared to the control group (Table 1). These parameters were partly normalized after the first week of treatment, although they were still significantly different from those of the controls on day 7. Thrombocytes were within the range of the controls on day 1, but elevated on day 7, whereas the monocyte count was constantly increased on days 1 and 7. In contrast, the number of lymphocytes was decreased at admission, but increased during the first week of hospitalization, although it did not reach the levels in the controls on day 7. Accordingly, the acute phase of the infection was associated with lymphopenia and a subsequent release of lymphocytes to the peripheral blood in response to chemotherapy.

Activated or memory T lymphocytes, but not naive cells, disappear from circulation.In order to further characterize the distribution of lymphocytes during infection, we looked at changes in percentages of different subsets. On day 1, percentages of CD8⁺ cells were severely decreased, whereas those of CD4⁺ cells were unaltered. After 1 week, the percentage of CD4⁺ cells was elevated compared to that at day 1 and to that of controls, whereas the percentage of CD8⁺ cells was increased compared to that at day 1, but did not reach the ranges observed in controls (Table 2). When the absolute numbers in the blood were calculated, it was revealed that the numbers of both CD4⁺ and CD8⁺ cells were decreased on day 1. On day 7, the number of CD4⁺ cells was normalized: whereas the number of CD8⁺ cells was elevated compared with that at day 1, it was still decreased compared with the number in controls (Fig. 1A and B). No significant differences in percentages of naive cells (CD62L⁺ CD45RA⁺) and activated or memory cells (CD45R0⁺ and CD95⁺) among CD4⁺ and CD8⁺ cells were observed (Table 2). However, calculations of the absolute numbers revealed that the infection-induced decrease in counts of CD4⁺ and CD8⁺ cells in the circulation was due to a disappearance of activated cells, whereas the number of naive cells was unaffected (Fig. 1A and B). The percentage of NK cells was decreased at admission, but not after 1 week when compared to that of the control, whereas the percentage of B cells was unaffected (Table 2).

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FIG. 1.

Absolute numbers of CD4⁺ subsets (A), CD8⁺ subsets (B), CD4⁺ cytokine-producing cells (C), and CD8⁺ cytokine-producing cells (D). Results (medians and quartiles) from patients with pneumococcal infections at day 1 (black bars) and day 7 (white bars) after the start of treatment and in a control group (gray bars) are shown. P < 0.05 for significant difference from day 1 (_∗) or from day 7 (#).

T lymphocytes with a type 1 cytokine profile reappear in the circulation in relation to treatment.The percentage of IL-2-producing CD4⁺ cells was lower at day 1 when compared to patients at day 7, and lower percentages of IFN-γ-producing CD4⁺ cells were observed in patients on days 1 and 7 when compared to those in the control group (Fig. 2A). Within the CD8⁺ subset, more IFN-γ-producing cells were found at day 7 than at day 1 (Fig. 2B). When the absolute number of T lymphocytes with potential cytokine production was calculated, it became clear that CD4⁺ cells and CD8⁺ cells producing IL-2, TNF-α, and IFN-γ disappeared from the circulation in relation to the acute phase of the infection, but had partly returned on day 7. The percentage or absolute number of CD4⁺ T lymphocytes expressing IL-4 was not affected during the infection (Fig. 1C and D).

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FIG. 2.

Percentage of cytokine-producing CD4⁺ (A) or CD8⁺ (B) cells from patients with pneumococcal infections at day 1 (black bars) and day 7 (white bars) after the start of treatment and in a control group (gray bars). Medians and quartiles are shown. P < 0.05 for significant difference from day 1 (_∗) or from day 7 (#).

Increased apoptosis among lymphocytes and elevated levels of sFasL in serum.Annexin V-positive, 7AAD-negative cells following PHA stimulation were identified as markers of cells primed for apoptosis, whereas 7AAD-positive cells marked dead cells (Fig. 3). The percentages of annexin V-positive, 7AAD-negative cells were significantly elevated at day 1 both compared to the levels at day 7 and in the control group. There was no difference between the levels in day 7 patients and the controls. Furthermore, there was no difference in 7AAD-positive cells in relation to the disease (data not shown). Finally, levels of sFasL in serum were significantly higher in patients at admission compared to those in controls, whereas the difference between day 1 and day 7 did not reach significance (Fig. 4).

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FIG. 3.

PHA-induced apoptosis (Annexin V⁺7AAD⁻) of lymphocytes from patients with pneumococcal infections (black bar) and 7 days after initiation of chemotherapy (white bar), as well as age-matched, healthy control donors (gray bars). Medians and quartiles are shown. P < 0.05 for significant difference from day 1 (_∗).

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FIG. 4.

Levels of sFasL in serum obtained from patients with pneumococcal infections at admission (black bar) or 7 days after admission (white bar) and from age-matched, healthy control donors (gray bar). Medians and quartiles are shown. P < 0.05 for significant difference from day 1 (_∗).