Department of Internal Medicine, Justus-Liebig University, Giessen D-35392, Germany
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ABSTRACT |
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Staphylococcus aureus -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
-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-1
markedly
enhanced the IL-8 response to
-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-
B activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We
conclude that alveolar epithelial cells are highly reactive target
cells of staphylococcal
-toxin.
-Toxin pore-associated transmembrane Ca2+ flux and PtdIns hydrolysis-related
signaling with downstream activation of protein kinase C and nuclear
translocation of nuclear factor-
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
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INTRODUCTION |
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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 -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,
-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
-toxin to the
buffer fluid of perfused lung, heart, and gut preparations,
respectively. Staphylococcal
-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 -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
-toxin in the alveolar compartment.
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MATERIALS AND METHODS |
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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 -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-PGF1, 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
-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
-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-1, the epithelial cells were preincubated with
50 U/ml for 3 h. With respect to IL-1
(50 U/ml), tumor necrosis
factor-
(TNF-
, 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
-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.
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RESULTS |
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Influence of -Toxin on Inositol Phosphates in ATII Cells
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Impact of -Toxin on Inflammatory Mediator Release from ATII and
A549 Cells
PG and NO release.
Incubation of ATII cells with -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,
-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
-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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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
-toxin for several hours caused a significant accumulation of IL-8,
which did not, however, approach the effect of TNF-
stimulation
(Fig. 4A). No significant
effect on the release of IL-6 and GM-CSF was observed. In contrast,
IL-1
induced the release of IL-6, IL-8, and GM-CSF. Interestingly,
after pretreatment of A549 cells with a subthreshold dose of IL-1
(50 U/ml, 4 h), the
-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
-toxin revealed a marked upregulation in the presence and absence of
IL-1
pretreatment (Fig. 5).
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Impact of Different Inhibitors on IL-8 Release in A549 Cells
Exposed to -Toxin
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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 ![]() |
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
-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-1
. All
effects occurred below the threshold of overt
-toxin-induced cell
damage. Analysis of intracellular signaling events in concert with
previously documented
-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-
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 -toxin-attacked endothelial cells by Ca2+-sensitive fluorochromes (35).
Third, in line with the preceding studies in other
-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
-toxin.
Interestingly, and again in line with previous studies on
-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-1 followed by
-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-1
-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
-toxin attack of other cell types. The signaling events underlying
the cytokine upregulation are apparently more complex than those
suggested for
-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-
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-
B, which is operative via the degradation of the NF-
B
inhibitor I-
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-
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
-toxin challenge (10), and time-
and dose-dependent appearance of IPx in
-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
-toxin was
drastically suppressed by two inhibitors of NF-
B activation, PDTC
and CAPE, which directly interfere with the phosphorylation and
translocation of NF-
B to the nucleus (23).
Notwithstanding, these data strongly support the view that the
suggested sequence of signaling events underlies cytokine synthesis in
-toxin-exposed epithelial cells. The cooperativity of low-dose IL-1
priming and
-toxin exposure in eliciting IL-8 and IL-6 synthesis, although being well imaginable as both events merge in the
pathway of NF-
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
-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-1
, 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
-toxin is the prototype of a large family of
pore-forming proteinaceous exotoxins generated by both gram-positive
and -negative bacteria (4).
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ACKNOWLEDGEMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547, Cardiopulmonary Vascular System).
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Acute Respiratory Distress Syndrome Network.
Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome.
N Engl J Med
342:
1301-1308,
2000
1a.
Bearz, A,
Tell G,
Colombatti A,
Formisano S,
and
Pucillo C.
Fibronectin binding promotes a PKC-dependent modulation of NF-B in human T cells.
Biochem Biophys Res Commun
243:
732-737,
1998[ISI][Medline].
2.
Berridge, MJ,
Dawson RMC,
Downes CP,
Heslop JP,
and
Irvine RF.
Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.
Biochem J
212:
473-482,
1983[ISI][Medline].
3.
Bhakdi, S,
Bayley H,
Valeva A,
Walev I,
Walker B,
Weller U,
Kehoe M,
and
Palmer M.
Staphylococcal -toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.
Arch Microbiol
165:
73-79,
1996[ISI][Medline].
4.
Bhakdi, S,
Muhly M,
Mannhardt U,
Hugo F,
Klapettek K,
Mueller-Eckhardt C,
and
Roka L.
Staphylococcal -toxin promotes blood coagulation via attack on human platelets.
J Exp Med
168:
527,
1988
5.
Bhakdi, S,
and
Tranum-Jensen J.
-Toxin of Staphylococcus aureus.
Microbiol Rev
55:
733-751,
1991[ISI].
6.
Bone, RC.
Gram-positive organismus and sepsis.
Arch Intern Med
154:
26,
1994[Abstract].
7.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
8.
Fujishima, S,
Sasaki J,
Shinozawa Y,
Takuma K,
Kimura H,
Suzuki M,
Kanazawa M,
Hori S,
and
Aikawa N.
Serum MIP-1 and IL-8 in septic patients.
Intensive Care Med
11:
1169-1175,
1996.
9.
Ghosh, S,
Latimer RD,
Gray BM,
Harwood RJ,
and
Oduro A.
Endotoxin-induced organ injury.
Am J Respir Crit Care Med
21 Suppl 2:
19-24,
1993.
10.
Grimminger, F,
Rose F,
Sibelius U,
Meinhardt M,
Potzsch B,
Spriestersbach R,
Bhakdi S,
Suttorp N,
and
Seeger W.
Human endothelial cell activation and mediator release in response to the bacterial exotoxins Escherichia coli hemolysin and staphylococcal -toxin.
J Immunol
159:
1909-1916,
1997[Abstract].
11.
Grimminger, F,
von Kurten I,
Walmrath D,
and
Seeger W.
Type II alveolar epithelial eicosanoid metabolism: predominance of cyclooxygenase pathways and transcellular lipoxygenase metabolism in co-culture with neutrophils.
Am J Respir Cell Mol Biol
6:
9-16,
1992[ISI][Medline].
12.
Gutierrez, HH,
Pitt BR,
Schwarz M,
Watkins SC,
Lowenstein C,
Caniggia I,
Chumley P,
and
Freeman BA.
Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators.
Am J Physiol Lung Cell Mol Physiol
268:
L501-L508,
1995
13.
Haitsma, JJ,
Uhlig S,
Goggel R,
Verbrugge SJ,
Lachmann U,
and
Lachmann B.
Ventilator-induced lung injury leads to loss of alveolar and systemic compartmentalization of tumor necrosis factor-.
Intensive Care Med
26:
1515-1522,
2000[ISI][Medline].
14.
Hamano, K,
Gohra H,
Noda H,
Katoh T,
Fujimura Y,
Zempo N,
and
Esato K.
Increased serum interleukin-8: correlation with poor prognosis in patients with postoperative multiple organ failure.
World J Surg
22:
1077-1081,
1998[ISI][Medline].
15.
Larsson, BM,
Larsson K,
Malmberg P,
and
Palmberg L.
Gram positive bacteria induce IL-6 and IL-8 production in human alveolar macrophages and epithelial cells.
Inflammation
23:
217-230,
1999[ISI][Medline].
16.
Lassus, P,
Wolff H,
and
Andersson S.
Cyclooxygenase-2 in human perinatal lung.
Pediatr Res
47:
602-605,
2000
17.
Lesur, O,
Berthiaume Y,
Blaise G,
Damas P,
Deland E,
Guimond JG,
and
Michel RP.
Acute respiratory distress syndrome: 30 years later.
Can Respir J
6:
71-86,
1999[Medline].
18.
Li, XY,
Donaldson K,
and
MacNee W.
Lipopolysaccharide-induced alveolar epithelial permeability: the role of nitric oxide.
Am J Respir Crit Care Med
157:
1027-1033,
1998
19.
Lin, CH,
Sheu SY,
Lee HM,
Ho YS,
Lee WS,
Ko WC,
and
Sheu JR.
Involvement of protein kinase C- in IL-1
-induced cyclooxygenase-2 expression in human pulmonary epithelial cells.
Mol Pharmacol
57:
36-43,
2000
20.
Lin, KJ,
Lin J,
Hanasawa K,
Tani T,
and
Kodama M.
Interleukin-8 as a predictor of the severity of bacteremia and infectious disease.
Shock
14:
95-100,
2000[ISI][Medline].
21.
Mayer, K,
Temmesfeld-Wollbruck B,
Friedland A,
Olschewski H,
Reich M,
Seeger W,
and
Grimminger AF.
Severe microcirculatory abnormalities elicited by E coli hemolysin in the rabbit ileum mucosa.
Am J Respir Crit Care Med
160:
1171-1178,
1999
22.
McElroy, MC,
Harty HR,
Hosford GE,
Boylan GM,
Pittet JF,
and
Foster TJ.
-Toxin damages the air-blood barrier of the lung in a rat model of Staphylococcus aureus-induced pneumonia.
Infect Immunt
67:
5541-5544,
1999
23.
Munoz, C,
Salcedo DP,
del Carmen Castellanos M,
Alfranca A,
Aragones J,
Vara A,
Redondo JM,
and
de Landazuri MO.
Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-B and AP-1 transcription factors activity.
Blood
88:
3482-3490,
1996
24.
Opal, SM,
and
Cohen J.
Clinical gram-positive sepsis: does it fundamentally differ from gram-negative bacterial sepsis?
Crit Care Med
27:
1608-1616,
1999[ISI][Medline].
25.
Rose, F,
Kiss L,
Grimminger F,
Mayer K,
Grandel U,
Seeger W,
Bieniek E,
and
Sibelius U.
E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid.
Am J Physiol Lung Cell Mol Physiol
279:
L100-L109,
2000
26.
dos Santos, CC,
and
Slutsky AS.
Mechanotransduction, ventilator-induced lung injury and multiple organ dysfunction syndrome.
Intensive Care Med
26:
638-642,
2000[ISI][Medline].
27.
Schütte, H,
Mayer K,
Gessler T,
Ruhl M,
Schlaudraff J,
Burger H,
Seeger W,
and
Grimminger F.
Nitric oxide biosynthesis in an exotoxin-induced septic lung model: role of cNOS and impact on pulmonary hemodynamics.
Am J Resp Crit Care Med
157:
498-504,
1998
28.
Schütze, S,
Potthoff K,
Machleidt T,
Berkovic D,
Wiegmann K,
and
Krönke M.
TNF activates NF-B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown.
Cell
71:
765-776,
1992[ISI][Medline].
29.
Seeger, W,
Bauer M,
and
Bhakdi S.
Staphylococcal -toxin elicits hypertension in isolated rabbit lungs: evidence for thromboxane formation and the role of extracellular calcium.
J Clin Invest
74:
849,
1984[ISI][Medline].
30.
Sibelius, U,
Grandel U,
Buerke M,
Mueller D,
Kiss L,
Kraemer HJ,
Braun-Dullaeus R,
Haberbosch W,
Seeger W,
and
Grimminger F.
Staphylococcal -toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts: role of thromboxane generation.
Circulation
101:
78-85,
2000
31.
Soderquist, B,
Kallman J,
Holmberg H,
Vikerfors T,
and
Kihlstrom E.
Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins.
APMIS
106:
1157-1164,
1998[ISI][Medline].
32.
Spittler, A,
Razenberger M,
Kupper H,
Kaul M,
Hackl W,
Boltz-Nitulescu G,
Fugger R,
and
Roth E.
Relationship between interleukin-6 plasma concentration in patients with sepsis, monocyte phenotype, monocyte phagocytic properties, and cytokine production.
Clin Infect Dis
31:
1338-1342,
2000[ISI][Medline].
33.
Splawski, JB,
McAnally LM,
and
Lipsky PE.
IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2).
J Immunol
144:
562-569,
1990
34.
Sugiyama, S,
Kugiyama K,
Ogata N,
Doi H,
Ota Y,
Ohgushi M,
Matsumura T,
Oka H,
and
Yasue H.
Biphasic regulation of transcription factor nuclear factor-B activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway.
Arterioscler Thromb Vasc Biol
18:
568-576,
1998
35.
Suttorp, N,
and
Habben E.
Effect of staphylococcal -toxin on intracellular Ca2+ in poly-morpho-nuclear leukocytes.
Infect Immun
56:
2228,
1988[ISI][Medline].
36.
Tanowitz, HB,
and
Chan J.
Gram-positive sepsis.
Crit Care Med
28:
3081-3082,
2000[ISI][Medline].
38.
Tomashefski, JF, Jr.
Pulmonary pathology of acute respiratory distress syndrome.
Clin Chest Med
21:
435-466,
2000[ISI][Medline].
39.
Valeva, A,
Palmer M,
and
Bhakdi S.
Staphylococcal -toxin: formation of the heptameric pore is partially cooperative and proceeds through multiple intermediate stages.
Biochemistry
36:
13298-13304,
1997[ISI][Medline].
40.
Walmrath, D,
Ghofrani HA,
Grimminger F,
and
Seeger W.
Synergism of alveolar endotoxin "priming" and intravascular exotoxin challenge in lung injury.
Am J Respir Crit Care Med
154:
460-468,
1996[Abstract].
41.
Walmrath, D,
Griebner M,
Kolb B,
Grimminger F,
Galanos C,
Schade U,
and
Seeger W.
Endotoxin primes perfused rabbit lungs for enhanced vasoconstrictor response to staphylococcal -toxin.
Am Rev Respir Dis
148:
1179-1186,
1993[ISI][Medline].
42.
Wirtz, HRW,
and
Dobbs LG.
Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells.
Science
250:
1266-1269,
1990[ISI][Medline].
43.
Xavier, AM,
Isowa N,
Cai L,
Dziak E,
Opas M,
McRitchie DI,
Slutsky AS,
Keshavjee SH,
and
Liu M.
Tumor necrosis factor- mediates lipopolysaccharide-induced macrophage inflammatory protein-2 release from alveolar epithelial cells. Autoregulation in host defense.
Am J Respir Cell Mol Biol
21:
510-520,
1999