International Immunology, Vol. 11, No. 5, 787-801,
May 1999
© 1999 Japanese Society for Immunology
Resistance of staphylococcal enterotoxin B- induced proliferation and apoptosis to the effects of dexamethasone in mouse lymphocyte cultures
Department of Experimental Pathology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA
Correspondence to: J. Tseng
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Abstract |
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Staphylococcal enterotoxin B (SEB) is a superantigen causing lymphocyte proliferation and apoptosis. Glucocorticoids are immunosuppressants and are released immediately following SEB intoxication in mice. Whether glucocorticoids affect lymphocyte proliferation and apoptosis in SEB-intoxicated mice is still unknown. To study this question, we examined the effects of dexamethasone (DEX), a synthetic glucocorticoid, on SEB-stimulated lymphocyte cultures from mouse thymus and peripheral lymphoid tissues (PLT). SEB, as well as concanavalin A (Con A), induced lymphocyte proliferation which peaked on day 4 and declined significantly on day 7. As expected, in Con A-stimulated cultures, DEX completely suppressed the proliferation of lymphocytes from both the thymus and PLT. However, in SEB-stimulated cultures, while DEX completely suppressed thymocyte proliferation, it did not suppress PLT cell proliferation even at a high concentration of 10–7 M. The proliferating cells were Vß8+ T cells of both the CD4+ and CD8+ subsets. DEX caused apoptosis. SEB also caused apoptosis, which was manifested by a maximal DNA subdiploidy on day 4 and by a maximal DNA fragmentation on day 7. Both events appeared not to be affected by DEX. The failure of DEX to affect the proliferation and apoptosis was consistent with high levels of cytokines (IL-1
, IL-2, IL-4, IL-6 and IFN-
) produced in the SEB-stimulated cultures, suggesting that the cytokines act in concert to circumvent the effects of DEX.
Keywords: cytokines, glucocorticoids, lymphocyte proliferation, superantigens, T cells
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Introduction |
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Staphylococcal enterotoxin B (SEB) is a superantigen (1,2). It binds to MHC class II molecules on antigen-presenting cells (APC) outside the groove where processed peptides of conventional antigens bind. The SEB–MHC class II complexes are presented to and engage populations of T cells that bear certain TCR Vß elements, leading to activation of both the T cells and APC. The result of this activation is the proliferation of large numbers of T cells and the production of large amounts of cytokines, including IL-2, IFN-
and tumor necrosis factor (TNF)- (3,4). Subsequently, most of the responding T cells undergo deletion and anergy (5–9). This deletion may involve apoptosis, since mice made tolerant or anergic to SEB have fewer SEB-reactive T cells and show genomic DNA fragmentation in T cells (6). The anergy and apoptosis lead to unresponsiveness to further SEB stimulation in vitro (6,8,9), while the elevation of cytokine levels appears to cause toxicosis and toxic shock (reviewed in 4,10,11).
Glucocorticoids are regulators of natural defenses in animals, including humans, acting to control immune and inflammatory reactions in the stress response (reviewed in 12). They are the final product of the hypothalamic–pituitary–adrenal (HPA), axis and their production and release are regulated in part by cytokines, such as IL-1, IL-6, IFN- and TNF-. The predominant effect of glucocorticoids on the immune and inflammatory reactions is suppressive (13). Glucocorticoids cause the apoptosis of lymphoid cells, resulting in the regression of lymphoid tissues, particularly the thymus (14). Under certain circumstances, glucocorticoids can also have immunostimulatory effects (15,16). Therefore, natural defenses are cross-regulated by the nervous and immune systems. SEB is a potent inducer of cytokines, including cytokines that are known to affect the HPA axis. This may be the reason that glucocorticoids are secreted immediately following SEB challenge in mice (17). These glucocorticoids may affect T cell proliferation and apoptosis during SEB intoxication. However, the nature of the effect of glucocorticoids on these cellular responses to SEB is not known.
A straightforward way to study the effects of glucocorticoids on SEB-stimulated T cells in vivo is to study cell proliferation and apoptosis in the presence of a glucocorticoid in SEB-stimulated lymphocyte cultures. This type of study would allow one to precisely quantify the dose and timing of exposure of the affected cells, to identify the responsive cell types, and to find possible clues as to the reasons for the effects. Surprisingly, this kind of study has not been reported in the literature. We have used dexamethasone (DEX), a potent synthetic glucocorticoid, and have tested its effects on cell proliferation and apoptosis in mouse lymphocyte cultures stimulated with SEB and have compared the results with those induced by concanavalin A (Con A). SEB, like Con A, induced cell proliferation in lymphocyte cultures of thymus and peripheral lymphoid tissues (PLT), which peaked on day 4 and declined significantly by day 7. The SEB-induced proliferating cells were in the Vß8+ T cell population. While the proliferation of SEB-stimulated thymocytes was completely suppressed by DEX, the proliferation of SEB-stimulated lymphocytes from PLT such as the spleen was not affected by DEX, although DEX may have affected the kinetics of these events. SEB also caused apoptosis of lymphocytes in culture, which was manifested by a maximal DNA subdiploidy on day 4 and maximal DNA fragmentation on day 7. Both events did not appear to have been affected by DEX. The failure of DEX to affect proliferation and apoptosis was consistent with the broad spectrum and high levels of cytokines produced in the SEB-stimulated cultures. These cytokines may have acted in concert to circumvent the effects of DEX.
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Methods |
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Mice
Male and female C3H/HeJ and BALB/cJ mice (7–12 weeks old) purchased from Jackson Laboratories (Bar Harbor, ME) were used. The mice were obtained at 5 weeks of age and allowed to acclimatize themselves to our animal facility. The mice did not seem to have been exposed to staphylococcal enterotoxin A (SEA) or SEB since quantitative ELISA of serum samples from randomly selected mice (three or four mice per 60 mice used) failed to detect anti-SEB antibodies and flow cytometric analysis of spleen cells from the mice did not show reduction or expansion of Vß8+ T cells. All animal experimental protocols were approved by our Institutional Animal Care and Use Committee, and were carried out in accordance with the Animal Welfare Act (7 USC 2131 et seq.), Army Regulation AR 70-18 and Public Law 99-198, and the Guide for the Care and Use of Laboratory Animals, NIH publ. 86-23, 1985.
Materials
RPMI 1640 medium and a DNA size ladder ranging from 100 to 2000 bp were purchased from Gibco/BRL (Gaithersburg, MD). 5-[125I]iododeoxyuridine ([125I]IUdR, sp. act. 5 mCi/mg) and methyl-[3H]thymidine ([3H]TdR, 2.0 mCi/mmol) were purchased from NEN Research Products (Boston, MA). Ficoll-Paque was purchased from Pharmacia (Piscataway, NJ). DEX, Con A and Tween 20 were purchased from Sigma (St Louis, MO). RNase A and proteinase K were purchased from Promega (Madison, WI). Agarose (NuSieve 3:1) was purchased from FMC BioProducts (Rockland, ME). SEB prepared by the method of Schantz et al. (18) was obtained in lyophilized form from the US Army Medical Research Institute of Infectious Diseases (Fort Detrick, Frederick, MD). SEA was purchased from Toxin Technology (Sarasota, FL). Monoclonal rat anti-mouse IL-2, IL-4 and IL-6 antibodies, hamster anti-mouse IL-1 and anti-IFN-, polyclonal rabbit anti-mouse IL-1 and TNF- antibodies, and recombinant mouse IL-1, IL-2, IL-4, IL-6, IFN- and TNF- were purchased from Genzyme (Cambridge, MA). Monoclonal rat anti-mouse TNF- antibody was purchased from Endogen (Boston, MA). Polyclonal rabbit anti-mouse IL-2 antibody was purchased from Collaborative Biomedicals (Bedford, MA). Polyclonal rabbit anti-mouse IFN- antibody was purchased from Biosource (Camarillo, CA). Polyclonal goat anti-mouse IL-4 and IL-6 antibodies were purchased from R & D Systems (Minneapolis, MN). Rabbit anti-mouse Ig (IgA + IgG + IgM; H + L chain specific) and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L chain specific) were purchased from Zymed (South San Francisco, CA).
Cell preparations
Mice were sacrificed, and their thymi, spleens, Peyer's patches and peripheral lymph nodes (PLN) were aseptically removed. The tissues were placed in ice-cold RPMI 1640 medium containing 50 µg/ml of gentamicin. A single-cell suspension was prepared from the lymphoid tissues by gently rubbing the tissues with a syringe plunger and passing the cells through a sterile stainless-steel mesh screen into culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 5x10–5 M 2-mercaptoethanol and 50 µg/ml gentamicin). After all cells were washed twice with Hanks' balanced salt solution (BSS), cell suspensions were treated with lysing buffer (155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA) to lyse the red blood cells. Dead cells were removed by passing the cell suspensions through a loosely packed cotton column. The cells were washed with Hanks' BSS two additional times, centrifuged and re-suspended in culture medium to a concentration of 5x106 cells/ml. Cell viability, as determined by Trypan blue exclusion, was >95%.
Panning
T cells and B cells of 4-day spleen cell cultures were separated by panning as described by Mage and et al. (19). Briefly, Petri dishes were coated with 5 µg/ml of rabbit antibodies against mouse Ig (IgA + IgG + IgM; H + L chain specific) for 4 h. After three washes, [125I]IUdR-labeled cultured cells were loaded onto the dishes and allowed to adhere for 1 h at 4°C in an ice bath. The non-adherent cells (T cell-enriched fraction) were harvested from the dishes by washing gently with medium. The adherent cells (B cell-enriched fraction) were harvested by scraping the dishes with a rubber policeman. Radioactivity was determined in a Multi Prias II -counter (Packard Instruments, Downers Grove, IL).
Lymphocyte proliferation
For lymphocyte proliferation studies, cells at a concentration of 5x106/ml were cultured in 96-well flat-bottom tissue culture plates (Falcon, Lincoln Park, NJ) in a volume of 200 µl/well and were stimulated with 10 µg/ml SEB, 2 µg/ml SEA or 2 µg/ml Con A, in the presence or absence of 10–7 M DEX (except for a titration experiment in which serial 10-fold dilutions of DEX were used). These concentrations of SEB, SEA and Con A induced maximal DNA synthesis in mouse spleen cell cultures as determined by [3H]TdR or [125I]IUdR incorporation. All cultures were performed in quadruplicate, including the control groups without mitogen. After 72 h of incubation at 37°C, with 7% CO2 in a humidified incubator, all cultures were pulsed with 0.25 µCi/well of [3H]TdR or 0.5 µCi/well of [125I]IUdR. Cells were harvested 18 h later onto glass fiber filters by using a cell harvester. Radioactivity was determined in a liquid scintillation counter (LKB, Bromma, Sweden) or in a -counter. Data in the tables are given as arithmetic means ± SEM of quadruplicate samples.
Flow cytometry
Cells stained with mAb reagents or propidium iodide were identified and analyzed in a FACScan flow cytometer using the Lysys II program (Becton Dickinson Immunocytometry Systems, San Jose, CA). By light-scatter analysis, three populations of cells were identified: apoptotic cells, non-proliferating cells and proliferating cells. Apoptotic cells were identified by the low forward light scatter of the dead cell population (20) or by low fluorescence intensity (subdiploid DNA content) with propidium iodide (21). Non-proliferating cells were mostly small lymphocytes, while proliferating cells were blast cells. All three cell populations were gated and further analyzed for the membrane markers CD4, CD8, Vß8 and Vß6. Double staining was usually performed with anti-CD4 or anti-CD8 reagents in combination with either anti-Vß8 or anti-Vß6 mAb reagents with the contrasting colors of FITC or phycoerythrin. Ten thousand cells were analyzed for each test.
Agarose gel electrophoresis of DNA
DNA from aliquots of cell cultures was extracted by a slight modification of the method of Sambrook et al. (22). Unlike the published procedure, phenol and formalin were not used in the extraction. Briefly, 107 cells were pelleted by centrifugation, and the pellets were resuspended, lysed and digested in 100 µl of Tris–EDTA extraction cocktail (10 mM Tris, 10 mM EDTA, 10 µg/ml RNAse A, 0.1% SDS, and 100 µg/ml proteinase K). This DNA extraction mixture was incubated at 56°C overnight. The absorbance of the DNA samples was then measured at 260 and 280 nm with a UV-1201 spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) and the DNA concentrations were calculated. Approximately 1.5 µg of DNA was applied to each slot of a flat-bed agarose gel (1.5%; NuSieve 3:1) and electrophoresed in 0.5xTris–borate–EDTA (TBE) buffer (0.045 M Tris–borate and 0.001 M EDTA) at room temperature. A DNA ladder of 100–2000 bp was used as size markers. After electrophoresis, DNA stained with ethidium bromide in the gel was examined under a UV light. A photograph was then taken of the gel.
Propidium iodide staining
The procedures of Nicoletti et al. for quantifying apoptosis (DNA subdiploidy) (21) and of Sgonc and Wick (23) for quantifying cells in various stages of the cell cycle were followed. Briefly, 1 ml of cell suspension (1–3x106 cells/ml) was pipetted into a 12x75 mm centrifuge tube (Falcon 2054) and cells were centrifuged for 10 min at 400 g at 4°C. The supernatant was discarded and the pellet was resuspended in 1.5 ml hypotonic fluorochrome solution (50 µg/ml propidium iodide in 0.1% sodium citrate and 0.1% Triton X-100). The tube was placed at 4°C in the dark overnight and analyzed by flow cytometry within 24 h in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Ten thousand cells were analyzed for each test. The data were analyzed using the Lysys II program (Becton Dickinson).
Cytokine quantification
IL-1, IL-2, IL-4, IL-6, TNF- and IFN- levels in culture supernatants were assayed by ELISA. Briefly, 96-well U-bottom Immulon II microtiter plates (Dynatech, Chantilly, VA) were coated with monoclonal anti-mouse cytokine antibody (2 µg/ml) in 0.05 M bicarbonate buffer, pH 9.6, at room temperature overnight. After three washes with 0.05% Tween 20 in PBS, the coated plates were blocked with 2% BSA in PBS (BSA/PBS) at 37°C for 1 h. After three washes, culture supernatants were placed into the plates without dilution. Cytokine standards (recombinant cytokines) were prepared at this step at various concentrations by serial dilution. Each sample or standard was determined in triplicate. The plates were incubated at 4°C overnight. After three washes, polyclonal anti-mouse cytokine antibody was placed into the plates which were subsequently incubated at 37°C for 2 h. HRP-goat anti-rabbit IgG (H + L) was applied at a pre-titrated dilution (0.375 mg/ml) to the plates after three washes. After a 2 h incubation at 37°C the plates were washed 3 times. Color was developed by adding 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (Kirkegaard & Perry, Gaithersburg, MD) and hydrogen peroxide, and measured in an ELISA reader (Biotek ELISA kinetics reader; Biotek Instruments, Winooski, VT) at 405 nm with background subtraction at 630 nm. Results are expressed in ng/ml, pg/ml or Biological Response Modifiers Program units (BRMP)/ml, as noted in the table, based on standard curves made with recombinant cytokine standards.
Statistical analyses
Data between experimental groups were compared and analyzed using Student's t-test. Data from time course studies were compared and analyzed by one-way ANOVA (24).
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Results |
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Dose response of DEX
The effect of glucocorticoids on SEB-induced proliferation in lymphocyte cultures has not been reported. DEX was chosen for our studies because it is a stable and potent synthetic glucocorticoid, is easily obtainable, and is widely used clinically as an immunosuppressive and anti-inflammatory drug. In the first experiments, we determined the dose–response curve of DEX on SEB-stimulated lymphocyte proliferation in spleen cell cultures, i.e. the effects of different concentrations and times of addition of DEX to the cultures were assessed. Cultures were done for 4 and 7 days, corresponding respectively to the peak of the increase in SEB-responsive T cells in vivo and the end of a period of decline in the numbers of T cells due to apoptosis (6). Spleen cell cultures were stimulated with SEB plus DEX and DNA synthesis (as an indicator of cell proliferation) was measured by [3H]thymidine incorporation. The results are shown in Table 1
and Fig. 1. SEB (10 µg/ml) induced proliferation of lymphocytes that peaked on day 4 and subsided significantly by day 7. When different concentrations of DEX were added at the establishment of the cultures (Table 1), a high concentration (10–6 M) of DEX, as expected, suppressed SEB-induced proliferation in both the 4- and 7-day cell cultures to levels 2- to 4-fold lower than those of cultures stimulated with SEB only. However, a complete resistance to suppression and sometimes an enhancement of proliferation were seen in 4-day cultures when the concentration of DEX was lowered to 10–7 and 10–8 M. The suppressive effect of DEX was only partial in 7-day cultures. When 10–7 M DEX was added at different times after cell cultures were established (Fig. 1), a maximal suppression of proliferation (down from ~30,000 to <10,000 c.p.m. of [125I]IUdR incorporation) was seen at 24 h. However, if DEX was added at time 0 or 72 h after the establishment of the cultures, the level of IUdR incorporation was as high as or higher than with SEB alone. Thus, depending on the concentration of DEX and its time of addition to spleen cell cultures, SEB-induced proliferation can be resistant to or be suppressed by DEX.
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), medium only (•) and DEX only (
) served as controls. Vertical lines represent SEM of the mean. The data are representative of three repeated experiments, which yielded similar results.Differential suppression by DEX of SEB- and Con A-stimulated lymphocyte cultures
To determine whether the resistance of SEB-induced lymphocyte proliferation to suppression by DEX was also a property of lymphocyte cultures from other PLT and if the resistance was different from that induced by Con A, lymphocytes from various lymphoid tissues were cultured with SEB plus DEX (10–7 M) and the resulting DNA synthesis (cell proliferation) was compared with that induced by Con A plus the same concentration of DEX (10–7 M). The result is shown in Table 2
. Either SEB plus DEX or Con A plus DEX was added at the establishment of the cultures. Proliferation was subsequently measured in the 4-day cultures by [125I]IUdR incorporation. In the cultures of thymocytes, DEX completely suppressed the proliferation induced by either SEB or Con A. However, in lymphocyte cultures of PLT such as the spleen, Peyer's patches and PLN, SEB-induced proliferation was essentially not suppressed by the concentration of DEX and occasionally an enhancement of proliferation was seen in spleen cell cultures. Furthermore, this resistance to DEX was in sharp contrast to the results in similar lymphocyte cultures stimulated with Con A plus DEX, in which DEX completely suppressed proliferation. These results indicate that, unlike SEB-induced proliferation, Con A-induced proliferation of lymphocytes from PLT is not resistant to the inhibitory effect of glucocorticoids.
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Resistance to DEX was also manifested by SEA-stimulated cultures
To investigate if the proliferation induced by other staphylococcal enterotoxins showed similar resistance to inhibition by DEX, SEA was tested in the presence of DEX. The result is shown in Table 3
. SEA-induced lymphocyte proliferation was similar to that induced by SEB. Proliferation of spleen cells from both BALB/cJ and C3H/HeJ mice was resistant to DEX when stimulated with either SEB or SEA. In contrast, the proliferation of thymocytes of both strains of mice was sensitive to DEX. Both SEA- and SEB-induced proliferation in thymocyte cultures were suppressed by DEX. These results suggest that resistance of SEB-induced proliferation to DEX is a general phenomenon in lymphocyte cultures of PLT of the mouse and that resistance to DEX may be a common characteristic of lymphocyte proliferation induced by staphylococcal enterotoxins.
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The resistant cells were in the T cell population
SEB is a superantigen. It therefore causes a large proportion of T cells to proliferate. To determine if DEX-resistant proliferation occurred in the T cell population, T and B cells labeled with [125I]IUdR from cultures stimulated with SEB or SEB plus DEX were separated by panning and both the B and T cell fractions were counted in a
-counter. The result is shown in Table 4. The proliferating cells identified by [125I]IUdR incorporation were virtually all in the T cell population; the B cell population showed virtually no proliferation. Thus, T cells responsive to SEB were resistant to DEX suppressive effects.
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Proliferating and non-proliferating Vß T cell subsets
To identify and further delineate the T cell populations that are resistant to the suppressive effect of DEX, the cells were analyzed by flow cytometry for proliferating and non-proliferating cell populations, and Vß T cell subsets were identified in these populations. Spleen cells were cultured with DEX, SEB and SEB plus DEX. At various times after the initiation of the cultures, cells were removed and proliferating and non-proliferating cells as well as T cells with different TCR Vß elements were identified in a FACScan flow cytometer by double staining with antibody reagents to T cells and Vß chains. Vß6+ T cells, which are non-responsive to SEB, and Vß8+ T cells, which are SEB responders, were then enumerated in the proliferating and non-proliferating cell populations. The results are shown in Figs 2–5
. In SEB-stimulated cultures, non-proliferating T cells which are SEB-reactive (CD4+Vß8+ cells in Fig. 2B) decreased slowly from ~2.7 to 1% in 7 days. The non-reactive (CD4+Vß6+) T cells (Fig. 2F) showed a slight increase (from ~1 to 1.5%) at day 2 and subsided dramatically to ~0.5% in days 4–7. In contrast, when DEX was added to the SEB-stimulated cultures, the frequencies of both the SEB-reactive (CD4+Vß8+) (Fig. 2B) and non-reactive (CD4+Vß6+) (Fig. 2F) T cells declined dramatically to the same low levels of those in cultures stimulated with DEX only. In these cultures, the low frequencies (0.1–0.5%) of these cells persisted from days 4 to 7. These patterns of decrease were consistent with the changing patterns of total CD4+ cells, Vß8+, Vß6+ and total non-proliferating cells (Fig. 2A, C–E, G and 2H). Similar patterns were seen for CD8+Vß8+ and CD8+Vß6+ non-proliferating cells (Fig. 3B, F and others in Fig. 3). Thus, all non-proliferating T cells (SEB-reactive and non-reactive) were sensitive to the suppressive effect of DEX.
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) served as controls. On days 0, 2, 4 and 7 (shown on the x-axis), cells were harvested and double stained with mAb reagents against CD4 and Vß8 or Vß6 markers and analyzed in a FACScan flow cytometer. Viable non-proliferating cells were gated and stained cells were examined. Percentages are expressed relative to the total (10,000) cells examined. The top panel represents double staining with anti-CD4 and anti-Vß8: (A) total CD4+ cells, (B) CD4+Vß8+ cells, (C) CD4–Vß8+ cells and (D) gated cells (viable non-proliferating cells). The bottom panel represents double staining with anti-CD4 and anti-Vß6: (E) total CD4+ cells, (F) CD4+Vß6+ cells, (G) CD4–Vß6+ cells and (H) gated cells (viable non-proliferating cells). The data are representative of four repeated experiments, which showed similar results.
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However, when proliferating cells were examined, different patterns were seen for Vß8+ and Vß6+ T cells (Figs 4 and 5
). For CD4+Vß8+ proliferating cells (Fig. 4B), there was an increase (from 0.5 to 10.7%) in the cell frequency that peaked on day 4 for cultures stimulated with SEB plus DEX. For cultures stimulated with SEB only, there was a smaller increase, from 0.5 to 7.2%, a frequency that lasted until day 7. These patterns of increase were similar to the pattern of percent increase of total CD4+ cells, Vß8+ cells and total proliferating cells (Fig. 4A, C and D). In contrast to the increases of CD4+Vß8+ proliferating cells, the CD4+Vß6+ proliferating cells showed a percent decrease that was similar to the pattern of percent decrease of total Vß6+ cells (Fig. 4F and G). This decrease of Vß6+ proliferating cells seemed to be due to the proportional increase of Vß8+ proliferating cells in the culture. When CD8+Vß8+ and CD8+Vß6+ proliferating cells were examined (Fig. 5B and F), they showed similar patterns to the CD4+Vß8+ and the CD4+Vß6+ proliferating cells respectively (Fig. 4B and F). All these results strongly suggest that Vß8+ T cells induced by SEB to proliferate are resistant to the suppressive effect of DEX.
DNA fragmentation
SEB causes lymphocyte proliferation and apoptosis of Vß8+ T cells (6,8). Glucocorticoids arrest lymphocytes at the G0/G1 phase of the cell cycle and kill them through the process of apoptosis (reviewed in 14,25). To understand the reason why proliferation was resistant to DEX in our cultures, and whether the apoptosis caused by SEB and by DEX were related events, it was therefore necessary to determine when apoptosis was occurring and whether DEX would affect SEB-induced apoptosis in our cultures. Since DNA fragmentation is a clear indication of apoptosis, DNA fragmentation and its relationship to the changes of the dead cell population in our cultures were examined. Figure 6(A and B) shows representative results of an aliquot of the lymphocyte cultures in the experiments described above (the flow cytometric analyses shown in Figs 2–5). The DNA fragmentation patterns (Fig. 6B) were compared with the population changes of apoptotic cells as defined by forward light scatter in a flow cytometer (Fig. 6A). Based on the fluorescence intensity of DNA stained by ethidium bromide (Fig. 6B), on day 2, cultures stimulated with DEX only or with SEB plus DEX both showed more intense staining of small DNA ladder fragments (multiples of 200 bp) than did cultures with medium only or with SEB only. However, on day 4, cultures with SEB and with SEB plus DEX showed the same intensity of fluorescent staining of small DNA ladder fragments, which was the lowest staining intensity of any cultures examined on that day. Cultures stimulated with DEX only continued to show the most intense pattern of DNA fragmentation from days 4 to 7. On day 7, cultures stimulated with SEB alone showed clear but less intense DNA fragmentation than did the cultures stimulated with SEB plus DEX. This difference may be due to the apoptotic effects of DEX on SEB-reactive T cells that have completed the cell cycle and have reached the G1 phase. Both DNA ladders of the cultures stimulated with SEB and with SEB plus DEX were more intense than those of the corresponding cultures on day 4; however, the intensity was still less than that of the DEX-stimulated cultures. Overall, the DNA fragmentation patterns (Fig. 6B) were consistent with the proportional increases and decreases of apoptotic cells in the cultures (Fig. 6A). They were also consistent with the proportional increases and decreases of SEB-reactive and non-reactive Vß T cells (Figs 2–5). These results suggest that most of the dead cells in all the cultures were apoptotic cells with DNA fragmentation. The apoptosis induced by SEB may not be affected by DEX.
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Apoptosis and proliferation assessed by propidium iodide staining
To further characterize and understand the effects of DEX on SEB-induced proliferation and apoptosis in our cultures, cells from spleen cell cultures stimulated with SEB or SEB plus DEX were stained with propidium iodide and analyzed by flow cytometry (21,23). The fluorescence histograms, proportions of apoptotic cells defined by DNA subdiploidy and cells at different phases of the cell cycle were then compared to those from cultures stimulated with Con A or Con A plus DEX. The fluorescence histograms are shown in Fig. 7
and the data are summarized in Table 5. From days 2 to 7, cultures stimulated with SEB only and with SEB plus DEX showed essentially a similar pattern of apoptotic cells and cells at different phases of the cell cycle, while cultures stimulated with DEX and with Con A plus DEX showed an another pattern (Fig. 7), suggesting again a differential effect of DEX on SEB- and Con A-stimulated cultures. Cultures stimulated with SEB and with SEB plus DEX generally had similar proportions of cells in apoptosis, the G0/G1 phases and the mitotic (MI) phase (Table 5). For viable cells, these cultures had a higher proportion of cells in MI than in the G0/G1 phases (Fig. 7) and the ranges of ratios of cells in these two phases (MI:G0/G1 ratios) were 0.6–3.5 and 1.1–4.4 for SEB and SEB plus DEX respectively (Table 5). Since these ranges overlapped, there was no significant difference in the results of the two treatments. In contrast, these ratios were different from those of cells stimulated with DEX or with Con A plus DEX, among which there were more viable cells in the G0/G1 phases than in mitosis (the MI:G0/G1 ratios were 0.5–0.7 and 0.4–0.6). These results further strengthen the conclusion that SEB-induced proliferation is resistant to DEX while that induced by Con A is sensitive to DEX.
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When apoptotic cells (cells with subdiploid DNA content) were examined (Fig. 7
and Table 5), a different effect of DEX on SEB- and Con A-stimulated cultures was seen. Cultures stimulated with DEX consistently showed a broad spectrum of propidium iodide staining intensity and high proportions (38.2–62.8%) of apoptotic cells from days 2 to 7. A similar broad spectrum of staining intensity and high proportions (32.1–60.4%) of apoptotic cells were also seen in cultures stimulated with Con A plus DEX. However, although cultures stimulated with SEB plus DEX on day 2 also showed a similar spectrum of staining intensity and pattern, a smaller proportion (25.1%) of apoptotic cells was seen. At the same time (day 2), SEB-stimulated cultures showed a distinct population but a similarly small proportion (21.3%) of apoptotic cells. On day 4, both cultures stimulated with SEB only and with SEB plus DEX showed the same broad spectrum of staining intensity and two large and distinct populations of apoptotic cells (Fig. 7; 67.7 and 57.9% respectively in Table 5), one with more DNA than the other. Interestingly, this apoptosis appeared at the same time as the peak of cell proliferation and could be the result of cell proliferation (Figs 3–5). The pattern of two populations of apoptotic cells was the most prominent feature among all the cultures; however, this pattern disappeared by day 7 (Fig. 7). On day 7, cultures stimulated with SEB plus DEX had half the proportion of apoptotic cells as did the cultures stimulated with SEB only (12.4 versus 24.3% in Table 5). Comparing these results to the results of apoptosis as defined by DNA fragmentation and apoptotic cells as defined by light scatter (Fig. 6A and B), it appears that a large proportion of the apoptotic cells detected by propidium iodide staining on days 4 and 7 in the cultures stimulated with SEB or with SEB plus DEX did not yet have extensive DNA fragmentation. The apoptosis seen on day 4 was apparently caused by SEB rather than by DEX since cultures stimulated with SEB only and those stimulated with SEB plus DEX showed virtually the same pattern of DNA subdiploidy and DNA fragmentation. In contrast, DEX appears to consistently cause apoptosis in cultures stimulated with Con A (Con A plus DEX cultures) since these cultures showed virtually the same patterns and proportions of subdiploid cells and cells at different phases of the cell cycle as the cultures stimulated only with DEX.
Cytokine profiles induced in lymphocyte cultures by SEB, SEA, and Con A
Staphylococcal enterotoxins are superantigens. They are powerful stimulants for APC and T cells. They could have induced the expression of several genes including cytokine genes in our cultures. Since the main function of DEX is to suppress cytokine production, the resistance to DEX of lymphocyte proliferation in our SEB- and SEA-stimulated cultures could be due to the failure of DEX induced-transcription factors to suppress the transcription of cytokine genes and/or due to the action of expressed cytokines which overcame the suppressive effect of DEX. In both cases, cytokines are the indicators in our cultures. Thus, cytokines from cultures stimulated with Con A, SEB or SEA with and without DEX were quantified and the results are summarized in Table 6. On day 4, compared to the level of cytokines in cultures with medium only, both SEB- and SEA-stimulated cultures showed a similar broad spectrum of cytokines: 2- to 8-fold elevation of IL-1, IL-2, IL-4, IL-6, TNF- and IFN-. The levels of all these cytokines, except for TNF-, were essentially not reduced at all by DEX in the SEB plus DEX or SEA plus DEX cultures. In contrast, in the Con A-stimulated cultures, the cytokine pattern was narrowed to IL-1 and IL-2, both of which were highly elevated. However, these cytokines, particularly IL-2, were dramatically reduced when DEX was added to the cultures. These results suggest that SEB and SEA strongly induce the expression of various cytokine genes, which are resistant to suppression by DEX. Alternatively, the wide spectrum of cytokines and their combined effects in SEB- and SEA-stimulated cultures might have overcome the suppressive effects of DEX. The proliferation of the SEB-, SEA- and Con A-stimulated cultures seems to have been driven mainly by IL-2.
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When cytokines from day 7 cultures were quantified, SEB- and SEA-stimulated cultures maintained a wide spectrum of cytokines; however, the levels, particularly those of IL-2, were dramatically reduced. IL-1
levels, in contrast, were elevated. Cultures stimulated with SEB plus DEX or with SEA plus DEX also showed the same wide spectrum of cytokines, but the cytokine levels were generally reduced to levels lower than those of cultures stimulated with SEB or SEA only. These higher levels of cytokines may account for the persistence of Vß8+ T cells in the cultures stimulated with SEB or with SEA only (Fig. 4). In cultures stimulated with Con A, the IL-2 level was reduced almost to the same baseline level as the cultures with medium only. A greater reduction of IL-2 levels was seen in cultures stimulated with Con A plus DEX. Again, these results suggest that IL-2 may be the driving factor for proliferation in SEB-, SEA- and Con A-stimulated cultures, and that the expression of cytokine genes is resistant to DEX and the concerted effects of cytokines in cultures stimulated with SEB plus DEX or SEA plus DEX may overcome the suppressive effects of DEX. |
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We have found in the present study that SEB-induced proliferation in lymphocyte cultures from PLT, compared to those from the thymus, was relatively resistant to suppression by DEX. The resistance to DEX was found in the proliferating T cell population, which consists mainly of SEB-reactive Vß8+ T cells of both the CD4+ and CD8+ T cell subsets. This DEX resistance was also seen in SEA-stimulated cultures, suggesting that the resistance may be a general characteristic of responses to staphylococcal enterotoxins. In contrast to SEB-stimulated cultures, proliferation in Con A-stimulated cultures was completely suppressed by DEX. DEX persistently caused the apoptosis of lymphocytes stimulated by DEX and by Con A but not the lymphocytes stimulated by SEB. However, SEB by itself also caused apoptosis, which did not appear to be affected by DEX. The resistance of SEB-induced proliferation and apoptosis to DEX was consistent with the broad spectrum of high levels of cytokines produced in the cultures, which seems to suggest that SEB-induced cytokine gene expression is resistant to DEX and its effects, and that the cytokines produced in the cultures have acted in concert to circumvent the effects of DEX.
When mice are injected with SEB or their peripheral T cells are stimulated with SEB in culture, the Vß8+ T cell population shows a strong pattern of proliferation followed by apoptosis and anergy (5–9,26,27). The apoptosis of Vß8+ T cells in vivo seems to occur in two phases, one on day 1 and the other on days 3–5 following SEB injection (5,6,26,27). Before the apoptosis occurs on days 3–5, the cells proliferate on days 2–3 (26,27). Although it remains to be proven, the decrease in Vß8+ T cell numbers on days 3–5 is believed to be due to this apoptosis (6,26,27). This sequence of events, characterized by early apoptosis followed by proliferation and late apoptosis, was also seen in our cultures stimulated with SEB and with SEB plus DEX. However, it should be noted that the early apoptosis seen in our cultures seemed to be caused by the suppressive effects of DEX on non-proliferating cells (Figs 2 and 3), while the proliferation and late apoptosis were mainly driven by SEB (Figs 4–7 and Table 5). When all the cells from the cultures were stained with propidium iodide and were analyzed by flow cytometry (Fig. 7 and Table 5), apoptosis defined by DNA subdiploidy was maximally seen on day 4, suggesting that apoptosis in another cell population had already been occurring and reached a peak on day 4 before DNA fragmentation and cell death occurred on day 7. The apoptosis in this cell population could have started immediately following proliferation of the Vß8+ T cell population. Based on the close similarity in the profile and proportion of propidium iodide-stained cells, the apoptosis seemed to have been driven by SEB and was DEX resistant (Fig. 7 and Table 5).
The differential suppressive effects of DEX on the proliferation of peripheral lymphocytes in SEB-stimulated and Con A-stimulated cultures may simply be due to the difference in the effectiveness of stimulation by Con A and by SEB. It is known that Con A binds and stimulates T cells via the carbohydrate moiety of the TCR; APC may be brought into contact with T cells by Con A via the carbohydrate moiety (28). Although this non-specific cell-cell contact between APC and T cells appears to be analogous to that brought about by SEB, the cell-cell interaction does not seem to result in gene transcription as strong and diverse as that induced by SEB (see below). SEB is a superantigen; it can effectively bring APC and T cells into close contact by bridging MHC class II molecules and Vß chains of the TCR (1–4). This close cell contact may be further strengthened by the complementary interactions between various adhesion molecules, accessory molecules and their corresponding receptors on APC and T cells, resulting in strong, combined signal transduction via various pathways, which quickly leads to strong transcription of various genes. This is conceivable since stimulation of T cells through the TCR complex with the involvement of T cell adhesion molecules, accessory molecules and their complementary receptors results in a strong transcription and stabilization of mRNA messages of various cytokine and chemokine genes (29–34). Supporting this explanation in the present study is the wide spectrum of high levels of cytokines produced in the SEB-stimulated cultures (Table 6). These high levels and the wide spectrum of cytokines were also seen in cultures stimulated with SEB plus DEX, suggesting that transcription of cytokine genes in SEB-stimulated cells is resistant to DEX and/or to the inhibitory effects of DEX-induced transcription factors.
Another possible mechanism for the differential effects of DEX on Con A- and SEB- stimulated cultures may be the concerted action of cytokines produced in the cultures. In the Con A-stimulated cultures, the cytokine spectrum was restricted and was characterized mainly by dramatic increases of IL-1 and IL-2. In SEB-stimulated cultures, however, the cytokine spectrum was relatively broad and showed a dramatic increase not only in IL-1 and IL-2 but also in IL-4, IL-6, TNF- and IFN-. Since IL-2 is the predominant cytokine in both Con A- and SEB-stimulated cultures, it is possible that proliferation in these cultures was driven by IL-2 (Table 6). DEX greatly suppressed IL-1 and IL-2 production in Con A-stimulated cultures. However, though it suppressed the production of TNF, DEX did not seem to affect the production of IL-1, IL-2, IL-4, IL-6 and IFN- in cultures stimulated with SEB plus DEX. Therefore, it is likely that the broad spectrum of cytokines, including those such as macrophage migration inhibitory factor (MIF) that was not measured, and their high levels in the cultures may be the main reason that SEB-stimulated cultures are resistant to DEX. Support for this explanation can also be seen in published reports on the rescue by cytokines of DEX-treated lymphocytes in cultures and of SEB-driven apoptosis of T cells in mice. For example, IL-2 and IL-4 can rescue T cells from apoptosis induced by DEX (35–37), IL-6 can rescue resting T cells from apoptosis (38), TNF- induced by LPS following SEB challenge in mice can prevent T cell death (39), and IL-1, IL-6 and IFN- can abrogate the DEX-mediated inhibition of T cell proliferation (40). Based on these findings, it should be possible to rescue lymphocytes in our cultures stimulated with Con A plus DEX or SEB plus DEX by adding cytokines, especially IL-1 plus IL-2.
However, it should be noted that the action of cytokines in cultures stimulated with SEB plus DEX involves a complicated mechanism. Adding cytokines to the cultures did not have a simple outcome. In a preliminary study in which we added two arbitrary amounts (1 and 10 µg/ml) of several individual cytokines or combinations of two or three cytokines to cultures stimulated with SEB and SEB plus DEX, the results of proliferation ([125I]IUdR incorporation) varied irregularly among the cultures and were difficult to interpret (C.-F. Weng, unpublished results). This may have been due to the complicated action network and effects on the functional balance of at least six cytokines (IL-1, IL-2, IL-4, IL-6, TNF- and IFN-) produced in the cultures, of which the levels and functions were altered by added exogenous cytokines. However, when a certain concentration (10 µg/ml) of mAb to IL-1, IL-2, IL-4, IL-6 and IFN- was added separately to the spleen cell cultures stimulated with SEB or with SEB plus DEX, anti-IL-1 enhanced proliferation while anti-IL-2, anti-IL-4 and anti-IL-6 each partially inhibited proliferation (C.-F. Weng, unpublished results). This approach of inhibiting the actions of cytokines in cultures will be continued in the future to further delineate the network of cytokines and their functional balances in our cultures.
It has been well demonstrated that the addition of either anti-CD3 or DEX to the culture of T cell hybridomas or T cell clones specific for pigeon cytochrome c results in cell death while the combined addition of anti-CD3 and DEX to the T cell cultures prevents cell death (41). The death and survival of the T cells are associated with the expression of the death receptor, Fas, and its ligand (FasL) (42). More recently, it has been further demonstrated that DEX induces the expression of a gene of the leucine zipper protein family, which appears to prevent cell death caused by activation through the TCR–CD3 complex but not through other proapoptotic stimuli (43). Interestingly, the leucine zipper protein gene is expressed in lymphocytes freshly isolated from thymus, spleen and lymph nodes, and DEX strongly induces its expression in lymphocyte cultures of thymus and lymph nodes (43). The leucine zipper protein may be a candidate transcription factor involved in the regulation of T cell apoptosis. The pattern of expression and action of the leucine zipper protein suggests an attractive hypothesis to explain the mechanism of DEX resistance in our cultures. However, it should be noted that there are three major differences between our culture system and the anti-CD3-stimulated culture system described above. First, in our culture system, both APC and T cells are stimulated by SEB while, in the other, only T cells (T cell hybridomas and T cell clones) are stimulated by anti-CD3. Second, in our culture system, SEB induces cell proliferation which may immediately be followed by apoptosis while, in the other, anti-CD3 causes immediate apoptosis. Third, DEX is unable to suppress cell proliferation and apoptosis in our SEB-stimulated cultures while, in the other, it is able to prevent apoptosis induced by anti-CD3. Whether and how the leucine zipper protein regulates cell proliferation and apoptosis in our cultures remain to be studied.
It is generally accepted that the production of glucocorticoids in response to inflammation and immune stimulation is a powerful natural defense mechanism that hosts use to cope with diseases (reviewed in 44). In cultures stimulated with SEB plus DEX, we have found that the proliferation and apoptotic events appear to have been driven mainly by SEB and are resistant to the effects of 10–7 M DEX. At 10–7 M DEX in mice is functionally equivalent to 3 times the peak concentration of corticosterone released into the blood circulation immediately following SEB challenge (17,45). However, this finding does not imply that glucocorticoids produced or introduced in SEB-challenged hosts are not effective at all. As shown in our initial culture experiments (Table 1 and Fig. 1), the time of addition and the concentration of DEX (and presumably other glucocorticoids) determine whether it will have a suppressive effect. These time and concentration effects have also been seen in in vivo animal studies. It has been demonstrated that adrenalectomy of mice as well as parenteral injection of a glucocorticoid antagonist (RU486) before SEB challenge renders mice more sensitive to lethal SEB intoxication (17). An injection of DEX applied 2 h before SEB challenge or up to 2 h after challenge prevents the death of galactosamine-primed mice (17). Cortisone enhances the apoptosis of SEB-reactive Vß8+ T cells when it is administered later in mice with SEB intoxication (46). Temporary depression of cortisol levels in the blood circulation is related to the outcome of toxic shock in monkeys immediately following SEB aerosol challenge (47). Because of the resistance to the effects of DEX displayed by SEB-stimulated lymphocytes, high concentrations of glucocorticoids and their production and administration before or immediately following SEB challenge may be necessary to have protective effects. Unfortunately, the production and release of corticosterone following SEB challenge in some animals is brief and at relatively low levels (17,45), but understanding the dose and time requirements for the administration of exogenous glucocorticoids may make therapeutic intervention possible.
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Note |
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The views and opinions expressed are solely those of the authors and do not necessarily reflect those of the Department of Defense or the US Army.
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Abbreviations |
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| APC | antigen-presenting cell |
| BRMP | Biological Response Modifiers Program units |
| BSS | balanced salt solution |
| Con A | concanavalin A |
| DEX | dexamethasone |
| HRP | horseradish peroxidase |
| HPA | hypothalamic–pituitary–adrenal |
| IUdR | iododeoxyuridine |
| MI | mitosis |
| PLN | peripheral lymph node |
| PLT | peripheral lymphoid tissue |
| SEA | staphylococcal enterotoxin A |
| SEB | staphylococcal enterotoxin B |
| TBE | Tris–borate–EDTA |
| TdR | thymidine |
| TNF | tumor necrosis factor |
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Notes |
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1 Present address: Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan
Transmitting editor: K. Knight
Received 6 March 1998, accepted 2 February 1999.
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