Mediator generation and signaling events in alveolar epithelial cells attacked by S. aureus alpha -toxin

Frank Rose, Gabriele Dahlem, Bernd Guthmann, Friedrich Grimminger, Ulrich Maus, Jörg Hänze, Nils Duemmer, Ulrich Grandel, Werner Seeger, and Hossein Ardeschir Ghofrani

Department of Internal Medicine, Justus-Liebig University, Giessen D-35392, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Staphylococcus aureus alpha -toxin is a pore-forming bacterial exotoxin that has been implicated as a significant virulence factor in human staphylococcal diseases. In primary cultures of rat pneumocyte type II cells and the human A549 alveolar epithelial cell line, purified alpha -toxin provoked rapid-onset phosphatidylinositol (PtdIns) hydrolysis as well as liberation of nitric oxide and the prostanoids PGE2, PGI2, and thromboxane A2. In addition, sustained upregulation of proinflammatory interleukin (IL)-8 mRNA expression and protein secretion occurred. "Priming" with low-dose IL-1beta markedly enhanced the IL-8 response to alpha -toxin, which was then accompanied by IL-6 appearance. The cytokine response was blocked by the intracellular Ca2+-chelating reagent 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, the protein kinase C inhibitor bis-indolyl maleimide I, as well as two independent inhibitors of nuclear factor-kappa B activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We conclude that alveolar epithelial cells are highly reactive target cells of staphylococcal alpha -toxin. alpha -Toxin pore-associated transmembrane Ca2+ flux and PtdIns hydrolysis-related signaling with downstream activation of protein kinase C and nuclear translocation of nuclear factor-kappa B are suggested to represent important underlying mechanisms. Such reactivity of the alveolar epithelial cells may be relevant for pathogenic sequelae in staphylococcal lung disease.

Staphylococcus aureus; sepsis; inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BACTERIAL TOXIN ATTACK on the alveolar epithelium, which represents the interface between the environment and the lung parenchyma, may contribute to lung failure under conditions of severe pneumonia and sepsis (15, 22, 40, 41). Known as adult respiratory distress syndrome, such acute lung injury is mimicked by infusion of endotoxin [a lipopolysaccharide (LPS) released from the cell walls of gram-negative bacteria] in animal models (9, 12, 18, 43). However, particularly evident in view of the increasing incidence of gram-positive sources of sepsis, there is now clear evidence that LPS is an initiator (but not the only initiator) of sepsis and septic lung failure (6, 24, 36).

In addition to LPS, pore-forming proteinaceous exotoxins may play a major role in the pathogenesis of septic organ injury, because they represent a large family of toxins that originate from both gram-positive and gram-negative bacteria and are known to exert profound effects on various target cell types (5, 10, 40, 41). The prototype of these exotoxins is the alpha -toxin of Staphylococcus aureus, which has been implicated as a significant virulence factor in various human staphylococcal diseases (29, 41). Below the threshold of causing overt cell lysis, alpha -toxin was noted to provoke a strong release response of inflammatory and vasoactive mediators in various target cells including endothelial cells and alveolar macrophages (10, 25). These cell-culture observations were well reproducible in intact organs with septic lung injury (40, 41), septic heart failure (30), and severe splanchnic malperfusion (21) being provoked by the admixture of purified LPS-free alpha -toxin to the buffer fluid of perfused lung, heart, and gut preparations, respectively. Staphylococcal alpha -toxin heptamerizes to generate hydrophilic transmembrane pores with an inner diameter of 1-2 nm in target cell membranes as the primary toxic effect (39). Extra-intracellular Ca2+ flux via these pores has been suggested as the basic mechanism that results in the rapid onset of cell-specific mediator generation (29, 35).

In addition to being target cells of microbial attack, recent evidence suggests that alveolar epithelial cells may actively contribute to inflammatory sequelae by liberating a variety of mediators with vasoregulatory and proinflammatory potency (17, 38). These include the cyclooxygenase products prostaglandin (PG)I2, PGE2, and thromboxane (TX)A2, the cytokines interleukin (IL)-8 and IL-6, as well as the short-lived volatile agent nitric oxide (NO) (11, 12, 15). Although homeostatic functions of these agents have been suggested, in particular for the baseline secretion of prostanoids (11, 16, 19, 40), marked upregulation of these mediators under inflammatory conditions favors a major role in the pathogenic sequelae that occurs in the diseased lung. The release of the proinflammatory multifunctional IL-6 (processing antibody production in B cells and cytotoxic T-cell differentiation) and IL-8 (a potent chemotactic factor for T lymphocytes and neutrophils) indicates a correlation with a poor prognosis and a high mortality in sepsis at the onset of multiple organ failure (8, 14, 20, 32, 33). Moreover, recent findings favor the concept that physical stress of inflamed alveolar epithelial cells such as occurs under conditions of mechanical ventilation in adult respiratory distress syndrome may cause epithelial mediator generation and thereby contribute to the appearance of ventilator-induced lung injury as well as remote organ abnormalities after entry of these agents into the systemic circulation (1, 13, 26).

Against this background, the present study investigated the influence of the alpha -toxin of Staphylococcus aureus on alveolar epithelial cells, employing both primary cultures of rat type II pneumocytes and the human alveolar epithelial A549 cell line. When recognizing that these cells are easily attacked by the staphylococcal toxin with the provocation of rapid onset and prolonged release of vasoactive and proinflammatory mediators, underlying signaling events were addressed. In essence, the findings demonstrate that alveolar epithelial cells are highly reactive target cells of Staphylococcus aureus alpha -toxin in the alveolar compartment.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Male CD18 Sprague-Dawley rats (body wt 180-200 g) were purchased from Charles River (Sulzfeld, Main, Germany). Elastase (type EC-134, sp. act. 135 U/mg protein) was purchased from Elastin Products (St. Louis, MO). DMEM was supplied by GIBCO (Karlsruhe, Germany). FCS, HEPES, Hanks' balanced salt solution, phosphate-buffered saline, trypsin-EDTA solution, and antibiotics were obtained from GIBCO. Tritiated inositol phosphates ([3H]IPx) were obtained from Amersham (Dreieich, Germany). Myo-[2-3H]inositol was purchased from New England Nuclear (Boston, MA). ELISA kits were from Coulter Immunotech (Hamburg, Germany) and Cayman Chemical (Massy Cedex, France). The lactate dehydrogenase (LDH) assay kit was purchased from Boehringer Mannheim (Mannheim, Germany). Caffeic acid phenethyl ester (CAPE) was obtained from Calbiochem (Bad Soden, Germany). Staphylococcal alpha -toxin was purified as described (3) and kindly provided by S. Bhakdi (Institute of Microbiology, Mainz, Germany). Xanthogenate tricyclodecan-9-yl (D-609) was kindly provided by M. Krönke (Köln, Germany). Tissue-culture plastic was purchased from Becton-Dickinson (Heidelberg, Germany). All other biochemicals were obtained from Merck (Munich, Germany).

Isolation of type II alveolar epithelial cells. Type II alveolar (ATII) epithelial cells were isolated as previously described in detail (42). Briefly, inflated and perfused lungs from specific pathogen-free male CD18 Sprague-Dawley rats were lavaged and filled to total lung capacity with solution containing elastase (30 U/ml) and trypsin (0.05 mg/ml). Lungs were minced and free cells were separated from lung tissue by sequential filtration through sterile gauze, 100-µm mesh, and additional 10-µm nylon mesh. "Panning" of the resultant cell suspension was performed on rat IgG-coated plates. Nonadherent ATII cells were harvested after 1 h and resuspended in DMEM containing 10% FCS. The yield was in the range of (30-50) × 106 ATII cells from each rat. The percentage of ATII cells was 94 ± 2% as assessed by modified Papanicolaou, tannic acid, and alkaline phosphatase stainings. Contaminated cells included alveolar macrophages (<2% in all experiments) and neutrophils (<2%). ATII cell viability, as assessed by 5-carboxyfluorescein diacetate (CFDA) loading and trypan blue exclusion, was persistently >95%.

A549 cells were obtained by American Type Culture Collection (CCL-185). This transformed human cell line, which is established from explanted lung carcinoma, reveals typical characteristics of ATII cells.

Phosphoinositide metabolism. The phosphatidylinositol (PtdIns) turnover of stimulated ATII cells was investigated by measuring the accumulation of IPx according to Berridge and colleagues (2). Cells were cultured on 35-mm dishes at a density of 3 × 106 cells/well. Cellular phospholipid pools were enriched with myo-[3H]inositol (10 µCi/well) in DMEM containing 2% FCS plus 40 mM HEPES buffer (pH 7.4) and were incubated at 37°C for 12 h. Before experimental use, cells were washed twice in Hanks' balanced salt solution containing 20 mM HEPES and 10 mM LiCl. At different times after stimulus application, samples were quenched with trichloracetic acid (final concentration, 7.5%), kept on ice for 15 min, and extracted four times with diethylether. The aqueous phase was neutralized with sodium tetraborate to pH 8.0 and processed to separate IPx on Dowex anion-exchange columns as described by Berridge and colleagues (2). The column was eluted sequentially as follows: with water (for free [3H]inositol), 5 mM sodium tetraborate-60 mM sodium formate (for glycerophospho-[3H]inositol), 0.1 M formic acid-0.2 M ammonium formate (for [3H]IP1), 0.1 M formic acid-0.5 M ammonium formate (for [3H]IP2), and 0.1 M formic acid-1.0 M ammonium formate (for [3H]IP3); samples were then processed for liquid scintillation counting and collectively depicted as IPx.

Immunoassays. IL-8, IL-6, and granulocyte-monocyte colony-stimulating factor (GM-CSF) were analyzed using ELISA (Coulter-Immunotech). The detection limit of these ELISAs was <8 pg/ml. TXB2, 6-keto-PGF1alpha , and PGE2 were analyzed using an ELISA technique from Cayman Chemical. The detection limit of these assays was <20 pg/ml.

Isolation of total cellular RNA and reverse transcription. Total cellular RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform method as previously described (7). The constituent mRNA was reverse transcribed according to the instructions of the manufacturer (StrataScript RT-PCR kit; Stratagene, Heidelberg, Germany) in a final volume of 25 µl. The synthesis of complementary DNA was carried out in a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CT) for 50 min at 37°C, and enzyme inactivation was achieved by heating the reaction to 94°C for 7 min. Subsequently, the reaction mixture was diluted with RNAse-free water to 60 µl and stored at -85°C until used.

Amplification of IL-8 cDNA. The PCR was performed in 1× PCR buffer (Perkin-Elmer), 1 mM each of dNTP (dATP, dCTP, dGTP, and dTTP), 1 µM of intron-spanning cytokine-specific primer (Stratagene), 0.75 U AmpliTaq DNA polymerase (Perkin-Elmer), and 2 µl of first-strand cDNA, respectively, in a total volume of 25 µl. PCR profiles consisted of initial denaturation at 94°C (for 1.5 min) followed by 35 cycles of denaturation (94°C for 50 s), primer annealing (60°C for 60 s), and primer extension (72°C for 60 s) in a GeneAmp PCR System 2400. The final extension was performed at 72°C for 7 min. Aliquots of PCR products were electrophoresed through 1.8% (wt/vol) NuSieve-agarose gels stained with ethidium bromide for ~2 h at 75 V. Negative controls were routinely performed by running PCR without a cDNA template to exclude false-positive amplification products. Positive controls were performed using cDNA preparations obtained from LPS-stimulated (100 ng/ml for 6 h) alveolar macrophages. To verify the specificity of PCR amplifications obtained from the above-mentioned procedure, automated DNA sequencing was carried out on the purified cDNA samples according to the instructions of the manufacturer (model 373A; Applied Biosystems, Darmstadt, Germany). By comparing the resulting cDNA sequences with the corresponding published sequences, we identified PCR products as expected segments of spliced cytokine or beta -actin mRNA species. With PCR conditions optimized for primer and magnesium concentrations and cycle numbers, amplification of cDNA samples was verified to be in the exponential phase of PCR by comparing the amount of input RNA equivalents with the yield of the respective cytokine and beta -actin PCR products.

Determination of NO by a chemiluminescence technique. NO was detected as previously described by our group (27). Briefly, NO is rapidly converted to nitrite and nitrate, summarized as NOx, in oxygen-containing solutions such as the perfusate of isolated perfused organs. To monitor accumulating NOx, samples from the recirculating perfusate were transferred into a reaction vessel containing 80 ml of 0.1 mol/l vanadium (III) chloride in 2.0 mol/l HCl at 98°C. This solution quantitatively reduced NOx back to NO. Arising NO was then removed from the reaction vessel by inert oxygen-free nitrogen continuously flushed through the liquid (160 ml/min), which after passage of a liquid trap and an acidic vapor trap entered a chemiluminescence detector (model UPK-300; UPK, Bad Nauheim, Germany). Calibration was performed with buffer fluids that contained known concentrations of nitrite and nitrate.

Experimental protocols. Freshly isolated ATII cells were seeded at a density of 0.9 × 106 cells/cm2 on 12-well culture dishes. The plating efficiency was typically ~70%. For experiments with measurement of epithelial phosphoinositide generation (extraction of cells and cell supernatant), PG, NO, cytokine release (measurement in extracted-cell supernatant), and mRNA expression (extraction of cells), confluent monolayers of ATII and A549 cells were used within 36 h. For experiments with D609 (5 µg/ml), pyrrolidine dithiocarbamate (PDTC; 150 µM), CAPE (30 µg/ml), 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; 5 mM), and bis-indolyl maleimide I (1 µM), the epithelial cells were preincubated for 1.0 h. For priming experiments with IL-1beta , the epithelial cells were preincubated with 50 U/ml for 3 h. With respect to IL-1beta (50 U/ml), tumor necrosis factor-alpha (TNF-alpha , 10 ng/ml), and A-23187 (10 µM), these concentrations address the dose range usually reported for the use of these agents in in vitro studies. The maximum doses of alpha -toxin were chosen to stay below the threshold of overt cell lysis (see the data on LDH release).

Measurement of LDH. LDH release, a marker for overt cytotoxicity, was quantified by standard colorimetric technique. Enzyme release was expressed as a percentage of total enzyme activity liberated in the presence of 100 µg/ml mellitin.

Statistical analysis. The data are given as means ± SE. ANOVA with Scheffé's post test was used to test for significant differences between the different groups; P < 0.05 was considered to indicate statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Influence of alpha -Toxin on Inositol Phosphates in ATII Cells

Incubation of ATII cells with alpha -toxin provoked a strong time- and dose-dependent PtdIns hydrolysis with accumulation of IPx (Fig. 1). The maximal response elicited by alpha -toxin (10 µg/ml) approached ~80% of that provoked by ATP at optimum concentration (50 µg/ml) and even surpassed the response to optimum concentrations of the calcium ionophore A-23187. Data with primary type II cells have substantially been reproduced in experiments analyzing the phosphoinositide metabolism of A549 cells (data not given).


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Fig. 1.   Time course and dose dependency of inositol phosphate (IP) generation in response to alpha -toxin. Type II alveolar (ATII) epithelial cells prelabeled with [3H]inositol were incubated with alpha -toxin (1 and 10 µg/ml), A-23187 (10 µM; A23), or ATP (50 µM) for different time periods. Extracted [3H]IPx were separated by anion-exchange chromatography. [3H]IP3, [3H]IP2, and [3H]IP1 are collectively depicted as [3H]IPx. Means ± SE of 4 independent experiments each are given; #P < 0.05, significantly different from control; *P < 0.05, significantly different from A-23187; and +P < 0.05, significantly different from 1 µg/ml alpha -toxin.

Impact of alpha -Toxin on Inflammatory Mediator Release from ATII and A549 Cells

PG and NO release. Incubation of ATII cells with alpha -toxin caused rapid-onset liberation of substantial quantities of the short-lived vasodilatory agent NO as assessed by chemiluminescence technique (Fig. 2). This increase was comparable to that in response to A-23187 (1 µM). In parallel, alpha -toxin induced the liberation of substantial quantities of the vasodilatory agents PGI2 and PGE2 as well as the potent vasoconstrictor TXA2 in a dose-dependent manner in both ATII and A549 cells (Fig. 3). The toxin-evoked PGI2 formation was blocked to <15% in the presence of 250 µM acetylsalicylic acid. ATII-cell stimulation with 1 and 10 µg/ml alpha -toxin in the absence of intracellular Ca2+ suppressed the PGI2 synthesis to <10% of the respective controls (n = 4 experiments each).


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Fig. 2.   Nitric oxide (NO) formation in response to alpha -toxin stimulation. ATII cells were incubated with 0.1 µg/ml alpha -toxin or 1 µM A-23187 for 30 min. NO release into the supernatant was quantified by chemiluminescence technique as described in MATERIALS AND METHODS. Means ± SE of 3 independent experiments each are shown; #P < 0.05, significantly different from control.



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Fig. 3.   Dose dependency of PGI2 (given as accumulation of stable product PGF1alpha ), PGE2, and thromboxane (TX)A2 (given as accumulation of stable product TXB2) formation in reponse to alpha -toxin challenge. ATII (A) and A549 (B) cells were incubated for 20 min with alpha -toxin (1 and 10 µg/ml) or with A-23187 (10 µM). Means ± SE of 4 independent experiments each are given; #P < 0.05, significantly different from control.

IL-8, IL-6, and GM-CSF. For measurement of cytokines, human A549 cells have been used with the aim of analyzing the liberation of inflammatory cytokines relevant to infection and sepsis in humans. Incubation of A549 cells with purified alpha -toxin for several hours caused a significant accumulation of IL-8, which did not, however, approach the effect of TNF-alpha stimulation (Fig. 4A). No significant effect on the release of IL-6 and GM-CSF was observed. In contrast, IL-1beta induced the release of IL-6, IL-8, and GM-CSF. Interestingly, after pretreatment of A549 cells with a subthreshold dose of IL-1beta (50 U/ml, 4 h), the alpha -toxin-induced release of IL-8 and IL-6 in these epithelial cells was markedly increased up to threefold (Fig. 4, B and C). Total amounts of these mediators were then in the range of ~8 ng/ml (IL-8), ~500 pg/ml (IL-6), and ~100 pg/ml (GM-CSF). In parallel, the mRNA expression of IL-8 in response to alpha -toxin revealed a marked upregulation in the presence and absence of IL-1beta pretreatment (Fig. 5).


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Fig. 4.   Time course and dose dependency of cytokine release in response to alpha -toxin (0.1 µg/ml) and tumor necrosis factor-alpha (TNF-alpha ; 10 ng/ml) without (A) and with (B and C) preceding interleukin (IL)-1beta priming (50 U/ml for 4 h). Release of IL-8, IL-6, and granulocyte-monocyte colony-stimulating factor (GM-CSF) into the supernatant was quantified by ELISA. Means ± SE of 4 independent experiments each are given; #P < 0.05, significantly different from control; *P < 0.05, significantly different from IL-1.



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Fig. 5.   Time course of IL-8 mRNA expression in response to alpha -toxin (0.1 µg/ml) without (A) and with (B) preceding IL-1beta priming (50 U/ml for 4 h). Data are presented by the photography of the ethidium bromide-stained gel electrophoresis.

Impact of Different Inhibitors on IL-8 Release in A549 Cells Exposed to alpha -Toxin

When A549 cells were preincubated with the NF-kappa B inhibitors PDTC and CAPE, the cells failed to liberate substantial amounts of IL-8 in response to alpha -toxin. A549 cells preincubated with BAPTA (an inhibitor of epithelial Ca2+ release) and bis-indolyl maleimide I [a highly specific inhibitor of epithelial protein kinase C (PKC)] also liberated far less quantities of IL-8 in response to alpha -toxin. All inhibitory capacities were demonstrated for epithelial cells with and without preceding IL-1beta priming (Fig. 6).


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Fig. 6.   Influence of 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; 5 mM), bis-indolyl maleimide I (bis; 1 µM), pyrrolidine dithiocarbamate (PDTC; 100 µM), and caffeic acid phenethyl ester (CAPE; 50 µg/ml) on epithelial cytokine release. A549 cells were pretreated with each inhibitor for 30 min and exposed to alpha -toxin (0.1 µg/ml for 6 h). All experiments were performed in cells both with and without preceding IL-1beta priming (50 U/ml for 4 h). Release of IL-8 into the supernatant was quantified by ELISA. Response in the absence of inhibitor was set to 100%. Means ± SE of 4 independent experiments each are given; #P < 0.05, significantly different from alpha -toxin.

Control Experiments

ATII cells loaded with CFDA as a marker of vitality exhibited fluorochromasia within minutes and showed no decrease in fluorescence activity after application of alpha -toxin in the highest concentration range used. LDH release from the epithelial cells as a marker of overt cell lysis was <2% of total enzyme activity in the absence of alpha -toxin (control). In the concentration range used, alpha -toxin incubation evoked only moderate protracted LDH release: ~4-5% of total cellular LDH was liberated within 60 min in response to 1.0 µg/ml alpha -toxin, and 8-10% was released in the presence of 10 µg/ml alpha -toxin. During the incubation periods used, 0.1 µg of alpha -toxin did not induce substantial LDH release (<2% of total enzyme activity). IL-1beta (used in concentrations up to 50 U/ml) was ineffective with respect to LDH release.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In the present study, which was performed in both primary rat ATII cells and the human alveolar epithelial A549 cell line, staphylococcal alpha -toxin was noted to provoke rapid-onset liberation of NO and various prostanoids. Moreover, prolonged upregulation of proinflammatory cytokine synthesis (predominantly IL-8) was observed in response to the toxin, and this was markedly enhanced when the epithelial cells had previously been "primed" by low-dose incubation with IL-1beta . All effects occurred below the threshold of overt alpha -toxin-induced cell damage. Analysis of intracellular signaling events in concert with previously documented alpha -toxin effects on different target cells suggest that an initial extra-intracellular Ca2+ shift as well as the PtdIns response, PKC activation, and nuclear translocation of NF-kappa B are important mechanisms that underlie the rapid and prolonged secretory responses.

Transmembrane Ca2+ flux appears to be the major event underlying pronounced early onset of NO synthesis and PG liberation including both vasodilatory prostanoids (PGI2 and PGE2) and the vasoconstrictor agent TXA2. First, the toxin of Staphylococcus aureus is well known to oligomerize on target cell membranes and form hydrophilic transmembrane pores of ~1-2 nm in inner diameter, which allows bivalent cation flux across the membrane alongside the large extra-intracellular gradient (39). Second, such transmembrane Ca2+ flux has been directly demonstrated in alpha -toxin-attacked endothelial cells by Ca2+-sensitive fluorochromes (35). Third, in line with the preceding studies in other alpha -toxin-sensitive cell types, both the prostanoid release and the NO formation were virtually fully blocked when the alveolar epithelial cells were exposed to the staphylococcal toxin in the absence of extracellular Ca2+ (10). Fourth, an increase in intracellular Ca2+ is known to be a prerequisite of and sufficient for induction of prostanoid formation (via activation of phospholipolytic activities and liberation of free arachidonic acid) and (NO-synthase related) NO formation, both events which are currently observed in the alveolar epithelial cells exposed to the alpha -toxin. Interestingly, and again in line with previous studies on alpha -toxin-elicited cellular responses, sublethal doses presented as the optimum toxin concentrations, which indicates that rather than overt cell lysis, discrete pore formation in still-viable cells represents the precondition for maximum mediator provocation. We are aware of the fact that contamination of the freshly isolated ATII cells with macrophages may interfere with the interpretation of the mediator response. To overcome this issue, data gained with ATII cells were reproduced with A549 cells (see Fig. 3B).

In addition to the early NO and PG responses, a sustained and dose-dependent liberation of the proinflammatory cytokine IL-8 was observed in the alveolar epithelial cells. Local synthesis of IL-8 induces recruitment and activation of immunogenic and inflammatory cells (in particular, neutrophils and macrophages), which are major contributors to both pulmonary host defense and inflammatory tissue injury. Priming of A549 cells with low doses of IL-1beta followed by alpha -toxin treatment caused an even more impressive upregulation of IL-8 and in addition, IL-6, on both the message and protein levels, whereas the IL-1beta -induced GM-CSF release was not influenced by the staphylococcal toxin. Such prolonged upregulation of cytokine synthesis has hitherto not been reported for Staphylococcus aureus alpha -toxin attack of other cell types. The signaling events underlying the cytokine upregulation are apparently more complex than those suggested for alpha -toxin-elicited early NO and prostanoid generation. The current data favor a significant role of the PtdIns hydrolysis-related signal transduction pathway with phospholipase C (PLC)-dependent IP and diacylglycerole (DAG) formation, downstream PKC activation (and putatively ceramide appearance), and nuclear translocation of NF-kappa B for transcriptional activation of the cytokine genes. Besides increasing PKC activity, DAG has been demonstrated to activate acidic sphingomyelinase with liberation of ceramide (28). Both PKC- and ceramide-induced nuclear translocation of NF-kappa B, which is operative via the degradation of the NF-kappa B inhibitor I-kappa B, is well established for many cell types (1a, 28, 34). Upregulation of the genes encoding for the cytokines IL-8 and IL-6 requires NF-kappa B binding to positive regulatory domains in the respective promoter regions (23). The suggested sequence of signaling events is supported by the following findings: 1) the coappearance of DAG and IPx, arising due to PtdIns-PLC activity, was previously reported for human endothelial cells in response to alpha -toxin challenge (10), and time- and dose-dependent appearance of IPx in alpha -toxin-exposed epithelial cells was demonstrated in the present investigation; 2) IL-8 generation was markedly reduced after pretreatment of the epithelial cells with the PKC inhibitor bis-indolyl maleimide I; and 3) cytokine synthesis in response to alpha -toxin was drastically suppressed by two inhibitors of NF-kappa B activation, PDTC and CAPE, which directly interfere with the phosphorylation and translocation of NF-kappa B to the nucleus (23).

Notwithstanding, these data strongly support the view that the suggested sequence of signaling events underlies cytokine synthesis in alpha -toxin-exposed epithelial cells. The cooperativity of low-dose IL-1beta priming and alpha -toxin exposure in eliciting IL-8 and IL-6 synthesis, although being well imaginable as both events merge in the pathway of NF-kappa B-related cytokine upregulation, remains to be clarified on a molecular basis.

In conclusion, the present study forwarded interesting new findings that may be relevant for inflammatory events in the alveolar compartment under conditions of Staphylococcus aureus pneumonia (primary or secondary) and sepsis. The alveolar epithelial cells turned out to be sensitive target cells for staphylococcal alpha -toxin attack, which resulted in immediate and strong NO release and prostanoid generation and prolonged upregulation of proinflammatory cytokine synthesis. Interestingly, marked enhancement of cytokine formation was noted upon alveolar epithelial priming with low doses of IL-1beta , which mimicked inflammatory conditions in the alveolar compartment. These findings lend further credit to the concept that alveolar epithelial cells are not only targets of but active contributors to inflammatory sequelae in the lung parenchyma, and they may similarly hold true for further bacterial toxins given the fact that the staphylococcal alpha -toxin is the prototype of a large family of pore-forming proteinaceous exotoxins generated by both gram-positive and -negative bacteria (4).


    ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547, Cardiopulmonary Vascular System).


    FOOTNOTES

Address for reprint requests and other correspondence: F. Rose, Dept. of Internal Medicine, Klinikstraße 36, D-35392 Giessen, Germany (E-mail: Frank.Rose{at}innere.med.uni-giessen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00156.2001

Received 10 May 2001; accepted in final form 12 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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