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(Journal of Leukocyte Biology. 2001;70:642-648.)
© 2001 by Society for Leukocyte Biology

Involvement of p38-mitogen-activated protein kinase in staphylococcus aureus-induced neutrophil apoptosis

Helen Lundqvist-Gustafsson*, Sara Norrman*, Jessica Nilsson* and Åsa Wilsson{dagger}

Divisions of
* Pathology II and
{dagger} Medical Microbiology, Linköping University, Faculty of Health Sciences, Linköping, Sweden

Correspondence: Helen Lundqvist-Gustafsson, Division of Pathology II, Faculty of Health Sciences, S-581 85 Linköping, Sweden. E-mail: helen.lundqvist{at}pat.liu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis occurred in human neutrophils within an hour of exposure to viable serum-opsonized Staphylococcus aureus, as indicated by appearance of cells with condensed nuclei, fragmented DNA, and increased phosphatidylserine exposure. In contrast, serum-opsonized, heat-killed S. aureus did not induce apoptosis. This discrepancy could not be explained by differences in bacterial uptake or total NADPH-oxidase activity. Suppressing phagocytosis by pretreating the neutrophils with cytochalasin b or by using nonopsonized bacteria did not prevent apoptosis. A supernatant from bacteria grown for 2 h in nutrient broth had a strong proapoptotic influence that was abrogated by heat treatment. Exposure to viable S. aureus or supernatant also led to activation of p38-mitogen-activated protein kinase in the neutrophils. Inhibition of this kinase with SB203580 reduced the apoptosis-inducing capacity of both bacteria and supernatant. We conclude that S. aureus activates p38-mitogen-activated protein kinase in neutrophils and induces apoptosis, probably mediated by a bacteria-derived soluble factor(s)

Key Words: bacterial toxins • DNA-fragmentation • morphology • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Staphylococcus aureus is an important human pathogen that causes a wide variety of diseases, ranging from superficial cutaneous infections to life-threatening systemic maladies. Neutrophils constitute the primary host defense against S. aureus infection [1 ]. On invasion by S. aureus, large numbers of neutrophils are recruited from the blood and migrate into the infected tissue, where they phagocytose and kill the bacteria using reactive oxygen species (ROS), defensins, and proteolytic enzymes [2 ]. Clearance of activated neutrophils that have attacked an infection lowers the risk of tissue injury through the effects of the neutrophil-derived proteolytic enzymes and ROS, and there is now compelling evidence that such neutrophil clearance is accomplished by apoptotic cell death [3 ].

The results of studies in vitro suggest that changes in the local environment relay signals that either retard or accelerate neutrophil apoptosis. Proinflammatory mediators, such as interferon-{gamma}, granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, complement factor 5a [4 ], leukotriene B4 [5 ], and interleukin (IL)-1b [6 ], have been found to transduce survival signals to neutrophils, whereas IL-8 [7 ] and tumor necrosis factor a [8 ] induce neutrophil apoptosis. Although the signal transduction pathways involved in those effects remain largely unknown, recent findings have indicated that tyrosine phosphorylation events play a role in the signaling pathways that lead to neutrophil apoptosis [9 ]. The p38-mitogen-activated protein kinase (p38-MAPK) is a serine/threonine kinase that is stimulated by phosphorylation of tyrosine and threonine residues [10 ] during cell activation by proinflammatory cytokines, osmotic stress, and UV irradiation [11 ]. Because irradiation and other stress stimuli are known to induce apoptosis in a variety of cell types, p38-MAPK has been suggested to participate in the process leading to apoptosis in response to these stimuli [12 ]. In neutrophils, constitutive phosphorylation and activation of p38-MAPK has been suggested to participate in spontaneous apoptosis [13 ]. Another study claimed that spontaneous as well as Fas-induced apoptosis occurs independently of p38-MAPK activation, whereas stress-induced apoptosis (UV irradiation, hyperosmolarity, or sphingosine) is inhibited by the p38-MAPK inhibitor SK&F 86002 [14 ]. Together the functional significance of p38-MAPK in neutrophils and its role in apoptosis are still obscure. Perhaps divergent signals generated downstream of p38-MAPK activation can control either cell death or survival [reviewed in ref. 15 ].

Intracellular production of ROS induces neutrophil apoptosis [16 17 18 ], whereas the presence of antioxidants prolongs cell survival [19 ]. Phagocytosis of Escherichia coli induces neutrophil apoptosis through an oxygen-dependent mechanism [20 ], suggesting activation-induced regulation of the life span of neutrophils.

The aim of the work reported here was to study the effects of viable and heat-killed S. aureus on neutrophil survival by assaying phagocytosis, NADPH oxidase activity, and activation of p38-MAPK.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The reagents and their sources were as follows: dextran and Ficoll-Paque (Pharmacia, Uppsala, Sweden); nutrient broth no. 2 (Oxoid, London, United Kingdom); p-hydroxyphenylacetic acid (pHPA), propidium iodide (PI), Triton X-100, 5-aminophthalhydrazide (luminol), diphenyleneiodonium (DPI), and fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO); sodium azide (NaN3) and gentian violet (Merck, Darmstadt, Germany); horseradish peroxidase (HRP), superoxide dismutase (SOD), catalase, annexin V-FLUOS, phenylmethanesulfonyl fluoride (PMSF), aprotinin, pepstatin, leupeptin, Nonidet P40 (NP-40), bovine serum albumin (BSA), and sodium deoxycholate (Roche, Mannheim, Germany); SB203580 (Calbiochem-Behring, La Jolla, CA); sodium orthovanadate (Na3VO4; Janssen Chimica, Geel, Belgium); and an enhanced chemiluminescence detection system (Amersham, Cardiff, United Kingdom). The antibodies used were rabbit polyclonal anti-phospho-p38-MAPK (Thr180/Tyr182) and anti-p38-MAPK (New England Biolabs, Inc., Beverly, MA); and HRP-conjugated goat anti-rabbit (Dakopatts, Copenhagen, Denmark).

Isolation of human neutrophils
Human neutrophils were isolated from freshly drawn heparinized blood obtained from the blood bank at Linköping University Hospital. Erythrocytes were removed by dextran sedimentation followed by hypotonic lysis. The lysate was centrifuged on a Ficoll-Paque gradient [21 ], and the extracted granulocyte fraction was washed twice and resuspended in Krebs-Ringer glucose buffer (KRG, containing 10 mM glucose, 1.5 mM Mg2+, and 1 mM Ca2+, pH 7.3). The suspended cells were placed on ice and used within 2 h of preparation. Microscopic examination revealed that at least 97% of the cells obtained in this way were neutrophils.

Cultivation and opsonization of S. aureus
S. aureus bacteria (strain WOOD 46; catalase positive, protein-A free) were stored at -70°C. Cultures were grown for 18 h in liquid growth medium and then transferred to fresh medium and cultured for another 2 h. Thereafter, the bacteria were washed and opsonized in 20% normal human serum (37°C, 20 min), washed once, and subsequently resuspended at 108/mL in KRG and kept on ice. Portions of the suspension were boiled for 15 min (100% of the bacteria killed) before opsonization, and other portions were not opsonized.

In some experiments, broth from the above-mentioned 2-h cultivation period was centrifuged and sterile filtered and then either added directly to neutrophils or boiled for 15 min and then added to neutrophils.

FITC-labeling of bacteria
Bacteria (109/mL) were labeled with FITC (0.25 mg/mL) in carbonate buffer [3.35% Na2CO3, 1.54% NaHCO3 (v/w), pH 10.0) for 30 min at 37°C. After being washed four times in PBS, cells were resuspended in KRG and stored on ice.

Phagocytosis
Neutrophils (106 cells) were mixed with FITC-labeled opsonized bacteria (107) in KRG and incubated for 60 min. At predetermined intervals, one drop of the reaction mixture was mixed with two drops of crystal violet (0.5 mg/mL in KRG) and immediately analyzed by fluorescence microscopy. Bacteria that were not ingested were stained with the dye, and their fluorescence was quenched, whereas ingested S. aureus remained fluorescent [22 ].

Assessment of apoptosis
Neutrophils (106) in KRG were prewarmed at 37°C for 5 min, and S. aureus bacteria (106–108) in suspension were subsequently added to a total volume of 1 mL. The samples were analyzed after 60 min to assess apoptosis.

Morphology
Neutrophils stained with Türck’s reagent (0.02% gentian violet in 6% acetic acid) were evaluated by light microscopy, and the portion of cells with condensed nuclei was determined. In each sample, 200–400 cells were counted.

Exposure of phosphatidylserine
Neutrophils were stained with a combination of annexin V-FLUOS and PI according to the instructions of the manufacturer. Cells (104 per sample) were analyzed in a flow cytometer (Becton Dickinson, Heidelberg, Germany) using Lysys II software. Cells with an increased FITC fluorescence, corresponding to an increased exposure of phosphatidylserine, were considered apoptotic [23 ], whereas cells that exhibited both increased FITC and PI fluorescence were considered necrotic. Cell debris was excluded by raising the forward scatter threshold.

Fragmented DNA
Fragmented DNA was analyzed by flow cytometry performed on PI-stained, permeabilized neutrophils [24 ]. Briefly described, pelleted cells were resuspended in 1.5 mL of a hypotonic solution containing 50 mg/mL of PI, 0.1% sodium citrate, and 0.1% Triton X-100 and were then incubated overnight. The fluorescence of DNA was detected in a flow cytometer (Becton Dickinson; 105 events per sample) using Lysys II software. The broad, low-fluorescence intensity peaks from apoptotic neutrophil DNA were easily distinguished from the narrow, high-intensity peak of intact DNA. The percentage of low-fluorescence (fragmented) DNA was analyzed with CellQuest software.

Production of reactive oxygen species
Hydrogen peroxide production
Cellular H2O2 production was assayed as an increase in oxidized pHPA, using a modified version of the method published by Hyslop and Sklar [25 ], as earlier described [26 ]. Neutrophils (106) were suspended in KRG (0.9 mL) supplemented with 0.5 mg of pHPA, 4 U of HRP, and 1 mM NaN3 to block the intracellular H2O2-consuming enzymes myeloperoxidase and catalase [27 ]. Thereafter, the cells were prewarmed at 37°C for 5 min and challenged with S. aureus (100 µL containing 107 bacteria). The cells were subsequently removed by centrifugation (10 s at 13,000 g), and the oxidized pHPA was detected in a Shimadzu spectrofluorimeter (RF-540; {lambda}ex, 317 nm; {lambda}em, 400 nm). Fluorescence values were converted to nanomoles of H2O2 by comparison with a standard curve obtained using known amounts of H2O2.

Chemiluminescence
Chemiluminescence (CL) was measured as previously described [28 ]. Neutrophils (106) were mixed in polypropylene tubes with KRG supplemented with either 50 µM luminol and 4 U of HRP (total CL) or 50 µM luminol, 2000 U of catalase, and 200 U of SOD (intracellular CL), prewarmed at 37°C for 5 min, and then challenged with S. aureus (107). Light emission was recorded continuously at 37°C in a Biolumat LB 9509 (Berthold, Wildbad, Germany).

Preparation of cell lysates and immunoblotting
Neutrophils (107) were challenged for 5 min with viable or heat-killed serum-opsonized S. aureus (108), after which 1 mL of ice-cold PBS (pH 7.3) supplemented with 1 mM Na3VO4 was added. Cells were pelleted and resuspended in 0.3 mL of lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) pH 7.4), supplemented with 1 mM Na3VO4, 10 µg/mL of aprotinin, 10 µg/mL of leupeptin, 1.4 µg/mL of pepstatin, and 1 mM PMSF). After 15 min at 4°C, the lysate was collected by centrifuging for 10 min at 4°C and 15,800 g, heated in sample buffer at 95°C for 5 min [29 ], and then centrifuged for 2 min at 15,800 g. Samples of the prepared lysates were subjected to SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred onto a nitrocellulose membrane using transfer buffer. The membranes were subsequently blocked for 60 min in TBS-BSA-Tween buffer (200 mM Tris and 137 mM NaCl [pH 7.6], supplemented with 0.1% Tween-20 and 5% BSA) and then incubated for 60 min with anti-phospho-p38-MAPK antibody (diluted 1:1,000 in TBS-BSA-Tween) which recognizes only p38-MAPK when it is activated by dual phosphorylation at Thr-180 and Tyr-182. The membranes were then washed and incubated for 60 min with an HRP-conjugated goat anti-rabbit antibody (diluted 1:1,700 in TBS-BSA-Tween) and thereafter washed and developed using the enhanced CL detection system. To confirm the identity of the protein and to ensure that each lane was loaded with the same amount of protein, the blot was stripped and reprobed with an anti-p38-MAPK antibody (diluted 1:1,000) that recognized both phosphorylated and nonphosphorylated p38-MAPK.

Statistics
The Mann-Whitney U test was used to evaluate differences between groups of cells. P values were considered significant when <0.05, and they are distinguished by a system of asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Exposure to viable S. aureus induces apoptosis in human neutrophils
Initially we investigated interactions of S. aureus with neutrophils. Human neutrophils were prepared from fresh blood and then exposed to different numbers of viable or heat-killed (100°C for 15 min), serum-opsonized S. aureus for 60 min. Thereafter, the neutrophils were permeabilized, the nuclei were stained with PI, and the pattern of DNA fluorescence was recorded by flow cytometry. The results indicated that apoptosis was induced by viable but not by heat-killed S. aureus (Fig. 1 ), and there was a strong positive correlation between the number of bacteria used and induction of apoptosis. Therefore, further experiments were performed to determine the proapoptotic effect of S. aureus at a neutrophil-to-bacteria ratio of 1:10. Neutrophils were examined for changes in nuclear morphology after staining with Türck’s reagent, as well as for exposure of phosphatidylserine in the outer leaflet of the plasma membrane. In support of our initial results, use of this approach showed that many cells interacting with viable S. aureus displayed apoptotic morphology as well as an increase in phosphatidylserine exposure (Fig. 2 ). We then turned to a study of the mechanisms underlying S. aureus-induced apoptosis.



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  		Figure 1. Correlation between number of S. aureus and neutrophil apoptosis. Neutrophils were challenged with different numbers of viable ({blacksquare}) or heat-killed ({square}) S. aureus for 60 min. Thereafter, the neutrophils were permeabilized and then PI stained overnight, and fragmented DNA was subsequently analyzed by flow cytometry (104 events/sample). Apoptosis is presented as percent fragmented DNA of total. Data are means ± SD (n=3).

 


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  		Figure 2. Apoptotic morphology and exposure of phosphatidylserine in neutrophils exposed to viable S. aureus. Neutrophils were challenged with viable or heat-killed S. aureus at a ratio of 1:10 for 60 min. (A) The neutrophils were subsequently stained with gentian violet and analyzed by light microscopy. Cells with condensed nuclei characteristic of apoptosis are shown in the micrograph (arrowheads), and the data in the diagram represent means ± SD (n=5; *, P<0.05). (B) Alternatively, neutrophils were washed in PBS and stained with annexin V-FLUOS in combination with PI and then analyzed by flow cytometry. Cells exhibiting enhanced fluorescence in both the FL1 and the FL3 channel (i.e., necrotic cells) were gated away, and data on the rest of the neutrophils were plotted as histograms. The results are representative of three separate experiments.

 
Role of NADPH oxidase activity in S. aureus-induced apoptosis
In a previous study [16 ], we discovered that apoptosis is accelerated by H2O2 generated by NADPH oxidase; thus we analyzed the formation of H2O2, using the pHPA technique, and the level of luminol-amplified CL in neutrophils interacting with S. aureus. The oxidative activity elicited by S. aureus from 106 neutrophils was monitored for 60 min, and no differences were seen in total NADPH-oxidase activity comparing neutrophils exposed to viable versus dead bacteria (P>0.1). Production of H2O2 was 35.75 ± 9.18 nmol for viable and 37.57 ± 19.18 nmol for heat-killed bacteria (mean±SD, n=4), and the CL response (the integral) was 5.36 x 109 ± 0.94 x 109 cpm for viable and 5.33 x 109 ± 0.79 x 109 cpm for heat-killed bacteria (mean±SD, n=6). However, qualitative differences were observed; viable bacteria caused release of ROS that was quenched by SOD and catalase, whereas only an intracellular response was induced by heat-killed S. aureus (Fig. 3 ). To further examine the role of NADPH oxidase in S. aureus-induced apoptosis, before challenging with bacteria, neutrophils were pretreated for 10 min with a concentration of 10 µM of the NADPH oxidase inhibitor DPI [30 31 ]. Such treatment totally abrogated the respiratory burst (data not shown), but it did not significantly inhibit S. aureus-induced apoptosis (P>0.1). Flow cytometry of PI-stained nuclei revealed that the apoptosis in response to S. aureus was 28 ± 15% in DPI-pretreated cells and 37 ± 11% in untreated cells (mean±SD; n=6).



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  		Figure 3. Chemiluminescence in neutrophils interacting with S. aureus. Neutrophils (106) were mixed with S. aureus at a 1:10 ratio in KRG buffer supplemented with 50 µM luminol and 4 U of HRP [total CL (a and c)] or with 50 µM luminol, 2000 U of catalase, and 200 U of SOD [intracellular CL (b and d)]. Results from one representative experiment are shown.

 
S. aureus-induced apoptosis is not dependent on phagocytosis
Another possible explanation for the difference we observed between neutrophil responses to viable and heat-killed S. aureus could be a less efficient uptake of the dead bacteria. Therefore, we assessed phagocytosis by using FITC-stained S. aureus in the presence of crystal violet; with that approach, bacteria that are phagocytosed can be distinguished from those that are simply attached to the surface of a neutrophil [22 ]. No difference in regard to phagocytosis was discerned between viable and heat-killed S. aureus (data not shown).

We noted that unopsonized bacteria, which were not as readily taken up by neutrophils as opsonized bacteria (47% phagocytosing cells with 12±7 bacteria/phagocyte for nonopsonized bacteria compared with 94% phagocytosing cells with 12±4 bacteria/phagocyte for opsonized bacteria), were also capable of inducing apoptosis, although they did so less effectively than the opsonized bacteria. Pretreatment of neutrophils with 5 µM cytochalasin b for 10 min inhibited phagocytosis (48% phagocytosing cells with 3±2 bacteria/phagocyte) but did not abrogate the capacity of viable bacteria to induce apoptosis (Fig. 4 ). Cytochalasin b alone did not provoke apoptosis (data not shown). These findings show that phagocytosis of the bacteria is not a prerequisite of apoptosis.



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  		Figure 4. S. aureus-induced neutrophil apoptosis is not dependent on phagocytosis. Neutrophils were exposed to S. aureus for 60 min under different conditions. Flow cytometry was performed on PI-stained nuclei, and the amount of fragmented DNA was determined. Apoptosis is presented as percent fragmented DNA of total. The results are shown as means ± SD. Significant differences from control cells (open bar) are marked with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

 
Apoptosis is induced by a soluble factor
In light of our findings, we speculated that neutrophil apoptosis could be elicited by a certain factor or factors released by viable S. aureus. To test this hypothesis, we treated neutrophils for 60 min with sterile-filtered supernatant from the 2-h cultivation of bacteria in nutrient broth. Such treatment readily induced apoptosis, but the effect was lost after boiling the supernatant for 15 min (Fig. 5 ). In contrast, nutrient broth alone or the supernatant from viable S. aureus incubated in KRG buffer at 37°C for 60 min did not induce neutrophil apoptosis (data not shown).



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  		Figure 5. S. aureus-induced neutrophil apoptosis is activated by a soluble factor. Neutrophils were exposed for 60 min to fresh or boiled (15 min, 100°C) sterile filtered supernatant from S. aureus cultivated in nutrient broth for 2 h. Flow cytometry was performed on PI-stained nuclei, and the amount of fragmented DNA was determined. Apoptosis is presented as percent fragmented DNA of total. The results are shown as means ± SD (n=3; **, P<0.01).

 
Kinetics of S. aureus-induced apoptosis
From our findings to this point, we could not exclude the possibility that the heat-killed bacteria induce apoptosis too, but that the signal might be weaker or delayed compared with that provoked by viable bacteria and supernatant. Therefore, we performed time studies of apoptosis after challenge with the different stimuli. The degree of apoptosis induced by viable bacteria or supernatant steadily increased over time (Fig. 6 ). Heat-killed bacteria evoked a much weaker response and never reached the same values as viable bacteria or supernatant. After 18 h, spontaneous apoptosis in control cells was substantial, and there were no differences between samples.



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  		Figure 6. Kinetics of S. aureus-induced apoptosis. Neutrophils were exposed to viable or heat-killed S. aureus or sterile filtered supernatant for different periods of time. Flow cytometry was performed on PI-stained nuclei, and the amount of fragmented DNA was determined. Apoptosis is presented as percent fragmented DNA of total. The results are shown as means ± SD (n=3).

 
Role of p38-MAPK in S. aureus-induced apoptosis
Activation of p38-MAPK has been shown to take place during stress-induced apoptosis in human neutrophils [14 ]. To determine whether p38-MAPK is activated in neutrophils during interaction with S. aureus, we performed Western blotting, using an anti-phospho-p38-MAPK antibody to detect protein phosphorylation. We found that viable but not heat-killed S. aureus strongly activated p38-MAPK, as did bacteria-derived supernatant (Fig. 7a ). Reprobing the same blots with anti-total p38-MAPK antibody confirmed the identity of the protein and revealed that similar amounts of proteins had been loaded in each lane. To ascertain whether signaling through p38-MAPK is also involved in S. aureus-induced apoptosis, we pretreated neutrophils with the specific inhibitor SB203580. Treatment with different concentrations (1–10 µM) for 15 min on ice and for 10 min at 37°C before challenging with viable bacteria, revealed that SB203580 reduced S. aureus-induced apoptosis by ~50% at the concentration 10 µM (Fig. 7b) . Further experiments with this concentration showed that SB203580 significantly reduced S. aureus-induced apoptosis (P>0.05) as well as supernatant-induced apoptosis (P>0.05) (Fig. 7c) . SB203580 alone or the carrier dimethyl sulfoxide had no effect on apoptosis (data not shown).



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  		Figure 7. Viable S. aureus and S. aureus-derived supernatant induce phosphorylation of p38-MAPK. (A) Phosphorylation of p38-MAPK in neutrophils stimulated with S. aureus was analyzed by Western blotting with an anti-phospho-p38-MAPK antibody. (B) Concentration inhibition curve of the p38-MAPK inhibitor SB203580. Neutrophils were pretreated with SB203580 (1–10 µM) or with buffer alone for 15 min on ice and then for 10 min at 37°C, and they were subsequently incubated with viable S. aureus for 60 min. Flow cytometry was performed on PI-stained nuclei, and the amount of fragmented DNA was determined. The results are representative of two separate experiments. (C) Neutrophils were or were not pretreated with 10 µM SB203580 and subsequently incubated with viable S. aureus or with S. aureus-derived supernatant for 60 min. Apoptosis was analyzed as described in B. The results are shown as means ± SD (n=3; *, P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we show that viable S. aureus could rapidly induce apoptosis in human neutrophils. Examining the extent of apoptosis, we found good correlation between the results of the morphological evaluations and analysis of DNA, as well as phosphatidylserine exposure assessed by flow cytometry. We also found that heat-killed bacteria had only a weak proapoptotic effect that was seen much later than for viable bacteria (4 h compared with 1 h). This result indicated that S. aureus must be viable to generate reliable signals that induce apoptosis. These findings are compatible with data obtained in studies of endothelial cells, showing that the apoptosis-inducing capacity of S. aureus was abrogated by UV irradiation [32 ], which might apply to other bacteria as well, because Oishi and Machida [33 ] reported that neutrophil apoptosis is provoked by viable but not by heat-killed E. coli. On the other hand, Baran et al. [34 ] recently observed that degradation of DNA was delayed in neutrophils exposed to viable S. aureus. The discrepancy between our results and those of Baran and coworkers might be explained by, among other things, differences in the following aspects of experimental design: strain variability (they used the Cowan ATCC 25923 strain, and we used the WOOD 46 strain), the ratio of neutrophils to bacteria (they used a ratio of 1:20, and we used 1:10), and exposure time (they assessed neutrophil apoptosis 12–24 h after addition of bacteria, whereas we did so after 1 h). However, in our systems, the rate of apoptosis induced by viable S. aureus increased over time, well beyond the basal level of spontaneous apoptosis in control cells for <=12 h. After 18 h, there was no longer any difference between samples due to substantial spontaneous apoptosis in the control population.

It is not yet known how S. aureus causes neutrophil apoptosis. The strong positive correlation between the number of viable bacteria and the extent of apoptotic neutrophils could indicate that apoptosis is initiated by phagocytosis-induced cellular activation (e.g., degranulation and NADPH oxidase activation). In a previous study of human neutrophils [16 ], we noted that activation of the plasma membrane-bound oxidase by N-formyl-methionyl-leucyl-phenylalanine did not induce apoptosis, whereas apoptosis was induced by intracellular formation of H2O2 during stimulation of NADPH oxidase with phorbol myristate acetate or ionomycin. It is interesting that only the viable S. aureus caused extracellular release of ROS. However, we found no difference between the levels of NADPH oxidase activity that occurred in response to viable and heat-killed bacteria in quantitative terms. Thus, we concluded that ROS derived from NADPH oxidase are not major signals in S. aureus-induced apoptosis. This is further supported by the fact that apoptosis was not blocked in neutrophils pretreated with the NADPH oxidase inhibitor DPI in concentrations that completely abrogated the respiratory burst.

Several of our observations indicated that S. aureus can induce neutrophil apoptosis, even if the bacteria are not ingested. Apoptosis is not blocked by pretreatment with a known inhibitor of phagocytosis (cytochalasin b) or by exposure to unopsonized bacteria, which are not readily taken up; thus, it is unlikely that phagocytosis per se initiates proapoptotic signaling. It is interesting that the supernatant from the 2-h cultivation of S. aureus also had a strong apoptosis-inducing effect. Notably, that effect was lost when the supernatant was boiled for 15 min, similar to what was seen on heat inactivation of the bacteria. Thus, S. aureus-induced apoptosis in neutrophils seemed to be largely caused by a soluble, bacteria-derived factor. Other investigators have studied the role of S. aureus toxins in regulating apoptosis in various cells involved in the immune response. Staphylococcal enterotoxin B has been observed to induce apoptosis in T lymphocytes in patients suffering from atopic dermatitis and might thereby dictate the pathogenicity of the disease [35 ]. Furthermore, S. aureus-derived {alpha}-toxin induces apoptosis in T lymphocytes caused by formation of small transmembrane pores [36 ].

A number of recent studies have suggested that tyrosine phosphorylation is involved in signaling pathways that often lead to neutrophil apoptosis. It has been reported that apoptosis induced by stress (i.e., sphingosine, UV irradiation, or hyperosmolarity) initiates activation of p38-MAPK [14 15 ], but data on the role of p38-MAPK in spontaneous apoptosis are conflicting [13 14 ]. We found an interesting difference in signaling exhibited by neutrophils; phosphorylation of p38-MAPK occurred in neutrophils exposed to viable bacteria or bacteria-derived supernatant, but in those treated with heat-killed bacteria either no phosphorylation or a very modest one took place. Moreover, inhibition of p38-MAPK by SB203580 significantly reduced the apoptosis-inducing capacity of these stimuli. At least one S. aureus-derived toxin, namely the staphylococcal superantigen toxic shock syndrome toxin-1, has been implicated in activation of protein tyrosine kinases in target cells [37 ]. Our data suggest a similar scenario for S. aureus-induced apoptosis, although the activating factor is not yet known.

Together, our results show that interaction of human neutrophils with viable S. aureus rapidly induced apoptosis, a process that involves p38-MAPK. The causative factor(s) appeared to be released from the bacteria. Additional studies are needed to identify this factor or factors and to further investigate p38-MAPK regarding its role in apoptosis signal transduction in neutrophils and its involvement in the proapoptotic machinery. Based on our findings, we speculate that the ability of viable S. aureus to induce apoptosis may represent an active strategy to avoid being killed by cells of the immune system.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Medical Research Council (no. 5968); the Faculty of Health Sciences, Linköping University; the King Gustaf V 80-Year Foundation; the Swedish Society of Medicine; the Tore Nilsson Foundation; and the Lars Hierta Memorial Foundation. We thank Prof. Olle Stendahl for valuable discussions, Tina Andersson and Maria Sisell for skillful technical assistance, and Patty Ödman for linguistic revision of the manuscript.

Received July 5, 2000; revised April 23, 2001; accepted April 24, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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