INFECTION AND IMMUNITY, Nov. 1990, p. 3796-3801
Vol. 58, No. 11
0019-9567/90/113796-06$02.00/0
Effects of Escherichia coli Hemolysin
on
Endothelial Cell Function
NORBERT SUTTORP,l* BARBARA FLOER,1 HANS SCHNITTLER,2 WERNER SEEGER,' AND SUCHARIT BHAKDI3 Department of Internal MedicinelDivision of Clinical Pathophysiology and Experimental Medicine' and Department of Medical Microbiology,3 Justus Liebig University, D-6300 Giessen, and Department of Anatomy and Cell Biology, Philipps University, D-3550 Marburg,2 Federal Republic of Germany Received 2 March 1990/Accepted 30 August 1990
Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (-0.05 hemolytic units/ml; .5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2' but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initihting different Ca2+dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin. Several lines of evidence suggest that Escherichia coli hemolysin (Hly) is an important virulence factor in extraintestinal E. coli infections. In humans, about 50% of E. coli strains causing pyelonephritis and septicemia are toxin producers, while E. coli strains of the normal fecal flora usually are not (8, 36). In various animal models, Hly-producing E. coli strains are more virulent than nonproducers (13, 34). Hly is secreted as a 110,000-Da protein (11). Studies of erythrocytes and planar lipid membranes showed that Hly damages cell membranes by insertion into the lipid bilayer and formation of discrete hydrophilic transmembrane pores with effective internal diameters of 2 nm (2-4, 21). Hly was recently identified as a potent leucocidin (2) and reported to induce chemoluminescence responses as well as enzyme and leukotriene release from human neutrophils in vitro (15). Septicemia (following, for example, E. coli-induced pyelonephritis) and associated alterations of pulmonary endothelial function by bacterial toxins appear to be important in the pathogenesis of the acute respiratory distress syndrome (22). Extensive studies of the effects of endotoxin on the pulmonary microvasculature exist (7). Alterations of endothelial cell functions by important bacterial exotoxins, however, have received little attention. In the present study, the effects of Hly on two functions of cultured pulmonary endothelial cells were examined. We found that very low concentrations of Hly increased the permeability of pulmonary artery endothelial cell monolayers. In addition, Hly stimulated the arachidonate metabolism in these cells. (This paper includes part of the M.D. thesis of B. Floer.)
*
MATERIALS AND METHODS Materials. Tissue culture plasticware was obtained from Becton-Dickinson, Heidelberg, Federal Republic of Germany. Medium 199, Hanks balanced salt solution (HBSS), trypsin-EDTA solution, N-2-hydroxyethylpiperazine-N'ethansulfonic acid (HEPES), and antibiotics were from GIBCO, Karlsruhe, Federal Republiv of Germany. Fetal calf serum was purchased from Biochrom, Berlin, Federal Republic of Germany. Collagenase (CLS type II) was from Worthington Biochemical Corp., Freehold, N.J. 6-Keto-3Hprostaglandin Fla (6-keto-PGFj,,), 45CaC12 (36 Ci/g), 3Hinulin (270 mCi/g), and 3H-sucrose (10.8 Ci/mmol) were obtained from New England Nuclear, Dreieich, Federal Republic of Germany. 3H-dextran (500 mCi/g), carboxyl14C-dextran (1.2 mCi/g), 3H20 (1 mCi/g), and methyl-'4Calbumin (0.026 Ci/g) were from Amersham Buchler, Braunschweig, Federal Republic of Germany. Anti-6-keto-PGF,a was purchased from Paesel, Frankfurt, Federal Republic of Germany. All other reagents were obtained from Sigma, Munich, Federal Republic of Germany. Polycarbonate micropore filter membranes (25-mm diameter, 5-pum pore size) were purchased from Nucleopore GmbH, Tubingen, Federal Republic of Germany). Preparation of E. coli Hly. The toxin was prepared by polyethylene glycol precipitation of the culture supernatant of E. coli LE 2001 (19, 20) as described elsewhere (2, 4, 10). The precipitated protein was dissolved in water, immediately shock frozen in liquid nitrogen, and stored at -70°C. Each thawed vial was titrated immediately before experiments were conducted, and all experiments were performed within 1 h thereafter. The toxin was held on ice throughout the duration of experiments. Adherence to this protocol was essential since Hly lost activity within a few hours, even when kept at 0°C. The applied doses of Hly will be referred to in hemolytic units (HU) per milliliter. By definition, 1
Corresponding author. 3796
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HU/ml is the toxin concentration evoking 60% lysis of a suspension containing 5 x 108 erythrocytes per ml of Veronal-buffered saline (2). The Hly protein concentration was determined by a sandwich enzyme-linked immunosorbent assay that uses a monoclonal anti-Hly antibody to capture the antigen and a second, polyclonal rabbit antibody for development (10). The assay was calibrated with a toxin standard obtained by incorporation of Hly into phosphatidylcholine liposomes and subsequent isolation of the liposomes by flotation in a sucrose density gradient. The protein content of this preparation was determined by quantitative amino acid analysis. A single polypeptide band of Mr 110,000 was found upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this enzyme-linked immunosorbent assay, it was found that an Hly titer of 1 HU/ml corresponded to a toxin protein concentration of about 0.05 to 0.1 ,ug/ml. The endotoxin in the Hly preparation was quantitated with a chromogenic substrate with a commercially available test (12) and amounted to 3 ng/HU. The highest Hly concentration used in this study was 5 HU/ml for 10 min. Usually endothelial cells were exposed to 1 HU/ml for 1 h. From previous studies (26), we know that the endotoxin concentrations used within the time frame indicated are without effect on endothelial cell function (see Results). In addition, in two experiments the Hly used was allowed to age for several hours at 37°C, a procedure which leads to a rapid loss of cytotoxicity (4). Endothelial cell culture. Endothelial cells were obtained from pulmonary arteries of freshly slaughtered pigs after exposure to 0.1% collagenase as previously described (26, 29-31, 33). Two T25 flasks were inoculated with material from one artery. Cells were grown in medium 199-10% fetal calf serum. After reaching confluence, cells were dispersed and subcultured as described previously (26, 31). Studies were performed on confluent and contact-inhibited monolayers in their second to fourth passages. Endothelial cells were identified as previously described (25, 26, 28-31, 33). Radioimmunoassay of 6-keto-PGF,.. Prostacyclin (PGI2) was assayed serologically as its stable hydrolysis product 6-keto-PGF1,. Assay mixtures contained 0.1 ml of suitably diluted medium or HBSS or 0.03 to 0.5 ng of an authentic standard, 0.1 ml of antiserum, tracer 6-keto-PGFia, and phosphate-gelatin buffer in a total volume of 0.5 ml. Assay mixtures were incubated at 4°C for 16 h. Antigen-antibody complexes were separated from free- antigen by adsorption of free antigen to charcoal. This assay has been previously characterized with regard to cross-reactivity and detection limits (26, 29-31, 33). Endothelial cell monolayers on polycarbonate filter membranes. Polycarbonate filter membranes were coated with gelatin, exposed to glutaraldehyde, and sterilized as previously described (25, 28). The coated filter membranes were placed in the bottom of a petri dish, and about 2 x 106 cells were seeded on the filter in medium 199-10% fetal calf serum and allowed to adhere for 3 h. An additional 10 ml of medium was then added to the petri dish. Thereafter, the monolayers were fed every other day and used about 6 days after plating. Determination of hydraulic conductivity. A confluent filter membrane was mounted in a modified chemotaxis chamber. The upper and lower compartments were filled with HBSS. One port to the upper compartment was for substitution of filtered volume and for application of pressure, and one port was for the addition of reagents. The lower compartment of the system was part of a semiclosed perfusion system (total volume of 10 ml). A roller pump provided a flow of 10 mllmin. The circulating fluid dripped into a 1-ml capillary;
3797
the height of the fluid level in this capillary directly corresponded to the volume filtered through the cell monolayer. A filter volume of 10 RI could reliably be measured. The entire system was kept at 37°C by a heated water bath (28). A hydrostatic pressure of 10 cm H20 was continuously applied to the upper side of the cell monolayer. The filtration rate across the endothelial monolayer was continuously determined (25, 28). Reflection cofficient for dextran. In this study, dextran instead of albumin (25, 28) was used to determine the selectivity of the endothelial monolayer because the use of 14C-albumin required the presence of 0.25% albumin in the system. Albumin shifts the dose-response curve of Hlyrelated effects to the right (2) and therefore would not allow a direct comparison in terms of hemolytic units per milliliter for Hly-induced PG12 synthesis and endothelial permeabil-
ity.
For calculation of the reflection coefficient, 100 nCi of 3H20 and 100 nCi of ['4C]dextran were added to the upper compartment. The amount of 3H20 and ['4C]dextran in the lower compartment was continuously measured in a Ramona LS-5 radioactivity monitor (Raytest, Heidelberg, Federal Republic of Germany) consisting of a flowthrough cell, a splitter-mixer, and an IBM personal computer for data calculation (for details, see reference 28). The dextran reflection coefficient (RC) of the endothelial cell monolayer
system was calculated every 5 min on the basis of 3H20 and ['4C]dextran in the upper and lower compartments of the filter system. Calculations were done as follows (see also reference 28): RC = 1 - [(AIB) x (CID)]. The term AIB yields the dextran clearance, and the term CID yields the volume filtered. A and C are the differences (in counts per minute per milliliter) of ['4C]dextran and 3H20, respectively, in the lower compartment of the system between time points x and x + 5 min, multiplied by the volume of the lower compartment. B and D are the sums (in counts per minute per milliliter) of ['4C]dextran and 3H20, respectively, in the upper compartment at time points x and x + 5 min, divided
by two.
Release of lactate dehydrogenase. The release of lactate dehydrogenase was used as a marker for overt cytolysis. At the end of the experiments, the medium was removed and centrifuged at 8,000 x g for 2 min. Lactate dehydrogenase activity in the supernatant was determined by the colorimet-
ric measurement of the reduction of sodium pyruvate in the presence of NADP was measured as described elsewhere (25). Enzyme release was expressed as the percentage of total enzyme activity released from endothelial cells in the presence of 100 ,ug of mellitin per ml (31). Marker flux studies. For marker flux experiments, endothelial cells w'ere grown in T75 flasks until confluence. Cells were transferred to 3.5-ml tubes (2.5 x 10' cells per tube), washed three times with HBSS, and incubated in 0.5 ml of HBSS (containing 1.26 mM Ca2+) in the presence of 2.5 HU of Hly per ml for 5 min at 37°C. Then cells were chilled on ice and incubated with 1 ,uCi of 45Ca2+ or 2.5 ,uCi of tritiated sucrose, inulin, or dextran for 60 min at 0°C. At the end of the incubation, the total assay volume was layered on top of 700 ,ul of Versilube F-50 silicone oil in a centrifugation tube, and cells were quickly separated from the medium by centrifugation at 8,000 x g for 1 min as described previously (31). Cell pellets were solubilized by the addition of 100 jig of mellitin per ml (31, 32) and processed for determination of
radioactivity. Morphology. Because actin is concentrated along the margins of endothelial cells, rhodamine-labeled phalloidin
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INFECT. IMMUN.
Up to 100 ng of endotoxin (lipopolysaccharide from Salmonella abortus-equi) per ml did not induce significant PGI2 synthesis within 60 min (4.1 ± 0.09 ng of 6-keto-PGF10, per ml at 60 min (mean ± SE, n = 7). These data do not rule out the possibility of a synergistic interaction between Hly and endotoxin which is responsible for the effects observed. Hly and permeability of endothelial cell monolayers. Undisturbed endothelial cell monolayers exposed to a hydrostatic pressure of 10 cm H20 (25, 28) displayed a hydraulic conductivity of 4 x 10-6 cm s-1 cm H20-1. Addition of 0.5 to 5 HU of Hly per ml resulted in a dose- and timedependent increase in the hydraulic conductivity (Fig. 2A). With 5 HU of Hly per ml, this parameter increased about 30-fold within 10 min. The reflection coefficient for dextran, a measure of endothelial monolayer selectivity, amounted to 0.71 in resting monolayers. This parameter decreased in a dose- and time-dependent manner in the presence of Hly, indicating that the cell monolayer lost its selective perme-
-
40
30
20-
10-
O0J 10 20 Min: 30 40 50 60 FIG. 1. Time- and dose-dependent PGI2 synthesis by Hly. Pulmonary artery endothelial cells were incubated with 0, 0.05, 0.1, 0.5, and 1 HU of Hly per ml for 1 to 60 min, and then the medium was taken for determination of PGI2 levels (measured radioimmunologically as 6-keto-PGF10,,). Data presented are means SE from seven separate experiments. ±
used to visualize interendothelial gap formation. Monolayers were fixed at room temperature for 5 min at a hydrostatic pressure of 10 cm H20 in HBSS (pH 7.4) containing 2% formaldehyde. After three washes with HBSS, monolayers were permeabilized by exposure to acetone at -20°C for 1 min. Cells were then washed with HBSS and incubated for 30 min with rhodamine-labeled phalloidin (1.4 ,ug/ml). After a brief wash, the preparations were mounted in 60% glycerol (containing 1.5% n-propyl gallate) and examined by fluorescence microscopy as previously described (25, 28). Statistics. Data were analyzed for unbalanced data by a one-way and two-way analysis of variance (9). was
RESULTS Hly and endothelial cell PGI2 synthesis. Hly stimulated PGI2-synthesis in cultured pulmonary artery endothelial cells in a dose- and time-dependent manner (Fig. 1). The lowest toxin concentration which induced significant PGI2 formation within 30 min was 0.05 HU of Hly per ml. The effects of Hly occurred in the absence of overt cell lysis, as indicated by the lack of lactate dehydrogenase release (data not shown). No increased PGI2 synthesis was noted in experiments using, per ml, 1 HU of hemolysin which was allowed to age for 8 h at 37°C (4.2 + 1.4 ng of 6-keto-PGF1c per ml at 30 min and 5.1 + 1.6 ng/ml at 60 min, mean standard error [SE], n = 2; no significant difference from control cells). ±
ability (Fig. 2B). Morphological studies. Morphological studies indicated that resting cells were tightly packed, without any evidence of interendothelial gap formation. Figure 3a shows the typical marginal microfilament network which is closely associated with endothelial cell junctions. Addition of 1 HU of Hly per ml for 25 min to endothelial cell monolayers resulted in the formation of small interendothelial gaps (Fig. 3b). These morphological alterations were even more pronounced after exposure to 5 HU of Hly per ml for 10 min (Fig. 3c). These morphological alterations suggested that Hly increased endothelial monolayer permeability by enhancing the paracellular fluid flux. Characterization of Hly-induced effects. Hly has been shown to create small hydrophilic pores of 2-nm effective diameter in erythrocyte membranes (4) and in planar lipid bilayers (21). This pore-forming property of Hly has not been shown for endothelial cells. We determined the influx pattern of four differently sized marker molecules (45Ca2 , tritiated sucrose, inulin, and dextran) in Hly-exposed endothelial cells. There was a significant passive influx of the small molecules 45Ca2+ and 3H-sucrose but not of the large tritiated inulin or dextran (Fig. 4). These observations are compatible with the presence of Hly-induced pores with an effective internal diameter of 2 nm in the cell membranes. In previous studies (27, 29-32), we proposed that toxincreated transmembrane pores act as nonphysiological Ca2` bypass gates which allow an influx of extracellular Ca2+ into the cell, thereby activating Ca2'-dependent cellular processes. In this study, we found that Hly-induced endothelial PGI2-synthesis is dependent on the extracellular concentration of Ca2+ but not of Mg2+ (Fig. 5). Toxin-treated endothelial cells were exposed to 0.9 mM Mg2+, and the extracellular Ca2+ concentration was varied. Hly-induced formation of PGI2 was strictly dependent on the presence of micromolar concentrations of extracellular Ca2+ (Fig. 5). Vice versa, experiments wherein the Ca2+ concentration was set at 2.5 mM and the Mg2" concentration was varied were conducted. In this case, stimulation of PGI2 synthesis occurred to a similar extent throughout. Ca2+ channel antagonists (maximally 100 ,uM diltiazem or 50 ,uM nimodipin) were without effect, while 25 ,uM trifluoperazine, a nonspecific inhibitor of calmodulin function, reduced Hly-induced PGI2 formation by 80% (data not shown). Indomethacin (1 ,uM) inhibited Hly-related PGI2 synthesis by 95% (data not
shown).
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FIG. 2. Time- and dose-dependent increase in permeability of endothelial cell monolayers caused by Hly. Confluent cell monolayers on polycarbonate filter membranes were mounted in a modified chemotaxis chamber and exposed to a continuous hydrostatic pressure of 10 cm H20 (see Materials and Methods for details). After a steady state was reached, 0, 0.5, 1, 2.5, or 5 HU of Hly per ml was added. Hly induced a time- and dose-dependent increase in the hydraulic conductivity (A) and a decrease of the reflection coefficient (RC) for dextran (B), indicating severe damage to the barrier function of the endothelial cell monolayers. Data presented are means + SE from 10 separate experiments.
DISCUSSION This study demonstrates that two important alterations of endothelial cell function are induced by low concentrations of Hly: generation of PGI2 and enhanced permeability of endothelial cell monolayers. These effects were observed at very low toxin concentrations (.0.05 HU/ml; .5 ng/ml) and proceeded in the absence of overt cell lysis. Endothelial cells may, together with granulocytes and monocytes (2), repre-
sent preferred targets for attack by Hly in the host organism. A similarly high susceptibility of pulmonary endothelium to Hly in isolated, buffer-perfused rabbit lungs has recently been noted (24). Current evidence suggests that Hly acts by forming small hydrophilic transmembrane pores (4, 9). In the present study, we noted an influx of Ca2+ and sucrose but not of inulin or dextran into Hly-treated endothelial cells. This
FIG. 3. Hly-induced morphological alterations of endothelial cell monolayers. At the end of the experiments, cells were stained with rhodamine-labeled phalloidin (highly specific for filamentous actin) to visualize interendothelial gaps. (a) Cell monolayers without Hly showed the typical marginal microfilament network (arrowheads) which is closely associated with endothelial cell junctions. (b) After exposure to 1 HU of Hly per ml for 25 min, small interendothelial gaps were noted. (c) There was a dramatic increase in interendothelial gaps (large arrows) after exposure to 5 HU of Hly per ml for 10 min. A remaining cell bridge between neighboring cells is indicated by the small arrows. Bar 10 ,um.
INFECT. IMMUN.
SUTTORP ET AL.
3800
700, %
600*
40- 6-keto PGFg f
TI
(ng/ml)
30-
50020/r
400-
I~~~~~~~~~ 0.5 HU Toxin,Mg+0.9mM 0.5 HU Toxin,Ca*+2.5mM I no Toxin,Mg++0.9mM no
300-
Toxin,Ca++2.5mM
10-
f-L ----
.1,
2000-
100-
45Ca3H-Sucro 3H-Inulln 3H-Dextran FIG. 4. Influx pattern of small (45Ca2' and 3H-sucrose [3HSucro]) and large (3H-inulin and 3H-dextran) marker molecules in Hly-exposed endothelial cells. Cells were incubated with buffer or with 2.5 HU of Hly per ml at 37°C for 5 min, and then cells were chilled on ice and incubated with 1 ,uCi of 45Ca2+ or 2.5 ,uCi of tritiated sucrose, inulin, or dextran for 60 min at 0°C. Cells were washed, and pellets were processed for determination of radioactivity. The amount of labeled marker molecules in Hly-free cell pellets was set at 100%. There was a significant influx of small (45Ca2' and 3H-sucrose) but not of large (tritiated inulin and dextran) marker molecules in toxin-treated endothelial cells. Data presented are means + SE from eight separate experiments. influx pattern is similar to that observed by Bhakdi et al. (4) with rabbit erythrocytes and is compatible with the concept of toxin-induced transmembrane pores with internal diameters of about 2 nm. Hly extends the list of pore-forming proteins such as staphylococcal alpha-toxin (1, 6), Pseudo-
aeruginosa cytotoxin (18), terminal complement complexes (5), and lymphocytolysins (14). Previous studies addressed the hypothesis that these transmembrane pores act as nonspecific Ca2+ bypass gates which allow an influx of Ca2+ into the cell (26, 27, 29-32). We demonstrated an increase in intracellular free Ca2+ in Staphylococcus aureus alpha-toxin-treated polymorphonuclear leukocytes and showed that this effect was the result of an influx of extracellular Ca2+ and not of a mobilization of intracellularly stored Ca2+ (27). This intracellular Ca2+ signal would be expected to initiate many Ca2'-dependent secondary cellular responses. In this context, we demonstrated staphylococcal alpha-toxin and terminal complement complex C5b-8 induced leukotriene B4 synthesis in polymorphonuclear leukocytes (23, 31) and PGI2 synthesis in endothelial cells (29, 31). In the present study, a strict dependency of hemolysin-induced PGI2 synthesis on extracellular Ca2+ was also found. The effect of Hly was inhibitable by monas
-2.5 -3 -5 -4 Ca++ or -6 log Ca++ or Mg++ (M) Mg++ free FIG. 5. Dependence of Hly-induced endothelial PGI2 synthesis on increasing concentrations of extracellular Ca2' but not on increasing concentrations of extracellular Mg2+. Data presented are SE from seven separate experiments (four experiments means involving variation of extracellular Mg2"). ±
calmodulin but not by Ca2+ channel antagonists. These results are compatible with the concept of stimulus-response coupling through toxin-created transmembrane pores. Besides stimulation of arachidonate metabolism, an enhanced permeability of endothelial monolayers was observed following the addition of Hly. The toxin not only increased the fluid filtration rate but also impaired the reflecting properties of the endothelial cell monolayers for large molecules (dextran), indicating a loss of the barrier function of the endothelium. Morphologically, endothelial cells appeared to contract upon exposure to Hly, thereby opening gaps as paracellular routes for enhanced fluid exchange across the cell monolayer. Ca2+ influx also appears to be important for this process (25). Current evidence suggests that an increase in intracellular free Ca2" may cause alterations in the cytoskeleton, leading to contraction of the endothelial cells (Schnittler et al., J. Physiol., in press). It is of interest that Hly-induced enhanced endothelial permeability occurred at higher toxin concentrations than the increase in endothelial PGI2 synthesis. A comparable finding was made when Hly was perfused through isolated rabbit lungs (24). It is conceivable that a higher intracellular free Ca2+ concentration is required for endothelial cell retraction than for phospholipase activation. In conclusion, low concentrations of Hly induced endothelial PGI2 synthesis and damaged the barrier function of endothelial cell monolayers. These observations are relevant to human biology (for example, hemolytic E. coli-induced pyelonephritis with subsequent septicemia) because synthesis of mediators and increased permeability of the pulmonary microvasculature are hallmarks of the acute respiratory
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E. COLI HEMOLYSIN-ENDOTHELIAL CELL INTERACTION
distress syndrome (22). Several other medically relevant exotoxins of gram-negative organisms have recently been recognized to share structural homologies with Hly (16, 17, 35), and they may exert similar toxic effects on endothelial cell function. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 249). We thank P. Rohrig, M. Muhly, and S. Tannert-Otto for excellent technical assistance and P. Muller for skillful graphic illustrations. LITERATURE CITED 1. Bhakdi, S., R. Fussle, and J. Tranum-Jensen. 1981. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc. Natl. Acad. Sci. USA 78:5475-5479. 2. Bhakdi, S., S. Greulich, M. Muhly, B. Eberspacher, H. Becker, A. Thiele, and F. Hugo. 1989. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J. Exp. Med. 169:737-754. 3. Bhakdi, S., N. Mackman, G. Menestrina, L. Gray, F. Hugo, W. Seeger, and I. B. Holland. 1988. The hemolysin of Escherichia coli. Eur. J. Epidemiol. 4:135-143. 4. Bhakdi, S., N. Mackman, J.-M. Nicaud, and I. B. Holland. 1986. Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. Infect. Immun. 52:63-69. 5. Bhakdi, S., and J. Tranum-Jensen. 1984. Mechanism of complement cytolysis and the concept of channel-forming proteins. Philos. Trans. R. Soc. London B Biol. Sci. 306:311-324. 6. Bhakdi, S., and J. Tranum-Jensen. 1987. Damage to mammalian cells by proteins that form transmembrane pores. Rev. Physiol.
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coli. J. Bacteriol. 169:1509-1515. 17. Lo, R. Y. C., C. A. Strathdee, and P. E. Shewen. 1987. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica Al. Infect. Immun. 55:1987-1996. 18. Lutz, F., M. Maurer, and K. Failing. 1987. Cytotoxic protein from Pseudomonas aeruginosa: formation of hydrophilic pores in Ehrlich ascites tumor cells and effect on cell viability. Toxicon 25:293-305. 19. Mackman, N., and I. B. Holland. 1984. Functional characterization of a cloned hemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide. Mol. Gen. Genet. 196:123-134. 20. Mackman, N., J. M. Nicaud, L. Gray, and I. B. Holland. 1986. Secretion of hemolysin by E. coli. Curr. Top. Microbiol. Immunol. 125:159-181. 21. Menestrina, G., N. Mackman, I. B. Holland, and S. Bhakdi. 1987. E. coli hemolysin forms voltage-dependent ion channels in lipid membranes. Biochim. Biophys. Acta 905:109-117. 22. Rinaldo, J. E., and R. M. Rogers. 1986. Adult respiratory distress syndrome. N. Engl. J. Med. 315:578-579. 23. Seeger, W., N. Suttorp, A. Hellwig, and S. Bhakdi. 1986. Noncytolytical terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes. J. Immunol. 137:1286-1293. 24. Seeger, W., H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi. 1989. Thromboxane-mediated hypertension and vascular leakage evoked by low doses of Escherichia coli hemolysin in rabbit lungs. J. Clin. Invest. 84:220-227. 25. Suttorp, N., T. Fuchs, W. Seeger, A. Wilcke, and D. Drenckhahn. 1989. Role of Ca2" and Mg2" for endothelial permeability of water and albumin in vitro. Lab. Invest. 61:183-191. 26. Suttorp, N., C. Galanos, and H. Neuhof. 1987. Endotoxin alters arachidonate metabolism in pulmonary endothelial cells. Am. J. Physiol. 253:C384-C390. 27. Suttorp, N., and E. Habben. 1988. Effect of staphylococcal alpha-toxin on intracellular Ca2" in polymorphonuclear leukocytes. Infect. Immun. 56:2228-2234. 28. Suttorp, N., T. Hessz, W. Seeger, A. Wilke, R. Koob, F. Lutz, and D. Drenckhahn. 1988. Bacterial exotoxins and endothelial permeability for water and albumin in vitro. Am. J. Physiol.
255:C368-C376. 29. Suttorp, N., W. Seeger, E. Dewein, S. Bhakdi, and L. Roka. 1985. Staphylococcal alpha-toxin-induced PGI2 production in endothelial cells: role of calcium. Am. J. Physiol. 248:C127C134. 30. Suttorp, N., W. Seeger, J. Uhl, F. Lutz, and L. Roka. 1985. Pseudomonas aeruginosa cytotoxin stimulates prostacyclin production in cultured pulmonary artery endothelial cells: membrane attack and calcium influx. J. Cell. Physiol. 123:64-72. 31. Suttorp, N., W. Seeger, S. Zinsky, and S. Bhakdi. 1987. Complement complex CSb-8 induces PGI2 formation in cultured endothelial cells. Am. J. Physiol. 253:C13-C21. 32. Suttorp, N., W. Seeger, J. Zucker-Raimann, L. Roka, and S. Bhakdi. 1987. Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin. Infect. Immun. 55:104-110. 33. Suttorp, N., W. Toepfer, and L. Roka. 1986. Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase. Am. J. Physiol. 251:C671-C680. 34. van den Bosch, J., L. Emody, and I. Ketyi. 1982. Virulence of haemolytic strains of Escherichia coli in various animal models. FEMS Microbiol. Lett. 13:427-430. 35. Welch, R. A. 1987. Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect. Immun.
55:2183-2190. 36. Welch, R. A., E. P. Dellinger, B. Minshew, and S. Falkow. 1981. Haemolysin contributes to virulence of extra-intestinal E. coli infections. Nature (London) 294:665-667.
Vol. 58, No. 11
0019-9567/90/113796-06$02.00/0
Effects of Escherichia coli Hemolysin
on
Endothelial Cell Function
NORBERT SUTTORP,l* BARBARA FLOER,1 HANS SCHNITTLER,2 WERNER SEEGER,' AND SUCHARIT BHAKDI3 Department of Internal MedicinelDivision of Clinical Pathophysiology and Experimental Medicine' and Department of Medical Microbiology,3 Justus Liebig University, D-6300 Giessen, and Department of Anatomy and Cell Biology, Philipps University, D-3550 Marburg,2 Federal Republic of Germany Received 2 March 1990/Accepted 30 August 1990
Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (-0.05 hemolytic units/ml; .5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2' but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initihting different Ca2+dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin. Several lines of evidence suggest that Escherichia coli hemolysin (Hly) is an important virulence factor in extraintestinal E. coli infections. In humans, about 50% of E. coli strains causing pyelonephritis and septicemia are toxin producers, while E. coli strains of the normal fecal flora usually are not (8, 36). In various animal models, Hly-producing E. coli strains are more virulent than nonproducers (13, 34). Hly is secreted as a 110,000-Da protein (11). Studies of erythrocytes and planar lipid membranes showed that Hly damages cell membranes by insertion into the lipid bilayer and formation of discrete hydrophilic transmembrane pores with effective internal diameters of 2 nm (2-4, 21). Hly was recently identified as a potent leucocidin (2) and reported to induce chemoluminescence responses as well as enzyme and leukotriene release from human neutrophils in vitro (15). Septicemia (following, for example, E. coli-induced pyelonephritis) and associated alterations of pulmonary endothelial function by bacterial toxins appear to be important in the pathogenesis of the acute respiratory distress syndrome (22). Extensive studies of the effects of endotoxin on the pulmonary microvasculature exist (7). Alterations of endothelial cell functions by important bacterial exotoxins, however, have received little attention. In the present study, the effects of Hly on two functions of cultured pulmonary endothelial cells were examined. We found that very low concentrations of Hly increased the permeability of pulmonary artery endothelial cell monolayers. In addition, Hly stimulated the arachidonate metabolism in these cells. (This paper includes part of the M.D. thesis of B. Floer.)
*
MATERIALS AND METHODS Materials. Tissue culture plasticware was obtained from Becton-Dickinson, Heidelberg, Federal Republic of Germany. Medium 199, Hanks balanced salt solution (HBSS), trypsin-EDTA solution, N-2-hydroxyethylpiperazine-N'ethansulfonic acid (HEPES), and antibiotics were from GIBCO, Karlsruhe, Federal Republiv of Germany. Fetal calf serum was purchased from Biochrom, Berlin, Federal Republic of Germany. Collagenase (CLS type II) was from Worthington Biochemical Corp., Freehold, N.J. 6-Keto-3Hprostaglandin Fla (6-keto-PGFj,,), 45CaC12 (36 Ci/g), 3Hinulin (270 mCi/g), and 3H-sucrose (10.8 Ci/mmol) were obtained from New England Nuclear, Dreieich, Federal Republic of Germany. 3H-dextran (500 mCi/g), carboxyl14C-dextran (1.2 mCi/g), 3H20 (1 mCi/g), and methyl-'4Calbumin (0.026 Ci/g) were from Amersham Buchler, Braunschweig, Federal Republic of Germany. Anti-6-keto-PGF,a was purchased from Paesel, Frankfurt, Federal Republic of Germany. All other reagents were obtained from Sigma, Munich, Federal Republic of Germany. Polycarbonate micropore filter membranes (25-mm diameter, 5-pum pore size) were purchased from Nucleopore GmbH, Tubingen, Federal Republic of Germany). Preparation of E. coli Hly. The toxin was prepared by polyethylene glycol precipitation of the culture supernatant of E. coli LE 2001 (19, 20) as described elsewhere (2, 4, 10). The precipitated protein was dissolved in water, immediately shock frozen in liquid nitrogen, and stored at -70°C. Each thawed vial was titrated immediately before experiments were conducted, and all experiments were performed within 1 h thereafter. The toxin was held on ice throughout the duration of experiments. Adherence to this protocol was essential since Hly lost activity within a few hours, even when kept at 0°C. The applied doses of Hly will be referred to in hemolytic units (HU) per milliliter. By definition, 1
Corresponding author. 3796
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E. COLI HEMOLYSIN-ENDOTHELIAL CELL INTERACTION
HU/ml is the toxin concentration evoking 60% lysis of a suspension containing 5 x 108 erythrocytes per ml of Veronal-buffered saline (2). The Hly protein concentration was determined by a sandwich enzyme-linked immunosorbent assay that uses a monoclonal anti-Hly antibody to capture the antigen and a second, polyclonal rabbit antibody for development (10). The assay was calibrated with a toxin standard obtained by incorporation of Hly into phosphatidylcholine liposomes and subsequent isolation of the liposomes by flotation in a sucrose density gradient. The protein content of this preparation was determined by quantitative amino acid analysis. A single polypeptide band of Mr 110,000 was found upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this enzyme-linked immunosorbent assay, it was found that an Hly titer of 1 HU/ml corresponded to a toxin protein concentration of about 0.05 to 0.1 ,ug/ml. The endotoxin in the Hly preparation was quantitated with a chromogenic substrate with a commercially available test (12) and amounted to 3 ng/HU. The highest Hly concentration used in this study was 5 HU/ml for 10 min. Usually endothelial cells were exposed to 1 HU/ml for 1 h. From previous studies (26), we know that the endotoxin concentrations used within the time frame indicated are without effect on endothelial cell function (see Results). In addition, in two experiments the Hly used was allowed to age for several hours at 37°C, a procedure which leads to a rapid loss of cytotoxicity (4). Endothelial cell culture. Endothelial cells were obtained from pulmonary arteries of freshly slaughtered pigs after exposure to 0.1% collagenase as previously described (26, 29-31, 33). Two T25 flasks were inoculated with material from one artery. Cells were grown in medium 199-10% fetal calf serum. After reaching confluence, cells were dispersed and subcultured as described previously (26, 31). Studies were performed on confluent and contact-inhibited monolayers in their second to fourth passages. Endothelial cells were identified as previously described (25, 26, 28-31, 33). Radioimmunoassay of 6-keto-PGF,.. Prostacyclin (PGI2) was assayed serologically as its stable hydrolysis product 6-keto-PGF1,. Assay mixtures contained 0.1 ml of suitably diluted medium or HBSS or 0.03 to 0.5 ng of an authentic standard, 0.1 ml of antiserum, tracer 6-keto-PGFia, and phosphate-gelatin buffer in a total volume of 0.5 ml. Assay mixtures were incubated at 4°C for 16 h. Antigen-antibody complexes were separated from free- antigen by adsorption of free antigen to charcoal. This assay has been previously characterized with regard to cross-reactivity and detection limits (26, 29-31, 33). Endothelial cell monolayers on polycarbonate filter membranes. Polycarbonate filter membranes were coated with gelatin, exposed to glutaraldehyde, and sterilized as previously described (25, 28). The coated filter membranes were placed in the bottom of a petri dish, and about 2 x 106 cells were seeded on the filter in medium 199-10% fetal calf serum and allowed to adhere for 3 h. An additional 10 ml of medium was then added to the petri dish. Thereafter, the monolayers were fed every other day and used about 6 days after plating. Determination of hydraulic conductivity. A confluent filter membrane was mounted in a modified chemotaxis chamber. The upper and lower compartments were filled with HBSS. One port to the upper compartment was for substitution of filtered volume and for application of pressure, and one port was for the addition of reagents. The lower compartment of the system was part of a semiclosed perfusion system (total volume of 10 ml). A roller pump provided a flow of 10 mllmin. The circulating fluid dripped into a 1-ml capillary;
3797
the height of the fluid level in this capillary directly corresponded to the volume filtered through the cell monolayer. A filter volume of 10 RI could reliably be measured. The entire system was kept at 37°C by a heated water bath (28). A hydrostatic pressure of 10 cm H20 was continuously applied to the upper side of the cell monolayer. The filtration rate across the endothelial monolayer was continuously determined (25, 28). Reflection cofficient for dextran. In this study, dextran instead of albumin (25, 28) was used to determine the selectivity of the endothelial monolayer because the use of 14C-albumin required the presence of 0.25% albumin in the system. Albumin shifts the dose-response curve of Hlyrelated effects to the right (2) and therefore would not allow a direct comparison in terms of hemolytic units per milliliter for Hly-induced PG12 synthesis and endothelial permeabil-
ity.
For calculation of the reflection coefficient, 100 nCi of 3H20 and 100 nCi of ['4C]dextran were added to the upper compartment. The amount of 3H20 and ['4C]dextran in the lower compartment was continuously measured in a Ramona LS-5 radioactivity monitor (Raytest, Heidelberg, Federal Republic of Germany) consisting of a flowthrough cell, a splitter-mixer, and an IBM personal computer for data calculation (for details, see reference 28). The dextran reflection coefficient (RC) of the endothelial cell monolayer
system was calculated every 5 min on the basis of 3H20 and ['4C]dextran in the upper and lower compartments of the filter system. Calculations were done as follows (see also reference 28): RC = 1 - [(AIB) x (CID)]. The term AIB yields the dextran clearance, and the term CID yields the volume filtered. A and C are the differences (in counts per minute per milliliter) of ['4C]dextran and 3H20, respectively, in the lower compartment of the system between time points x and x + 5 min, multiplied by the volume of the lower compartment. B and D are the sums (in counts per minute per milliliter) of ['4C]dextran and 3H20, respectively, in the upper compartment at time points x and x + 5 min, divided
by two.
Release of lactate dehydrogenase. The release of lactate dehydrogenase was used as a marker for overt cytolysis. At the end of the experiments, the medium was removed and centrifuged at 8,000 x g for 2 min. Lactate dehydrogenase activity in the supernatant was determined by the colorimet-
ric measurement of the reduction of sodium pyruvate in the presence of NADP was measured as described elsewhere (25). Enzyme release was expressed as the percentage of total enzyme activity released from endothelial cells in the presence of 100 ,ug of mellitin per ml (31). Marker flux studies. For marker flux experiments, endothelial cells w'ere grown in T75 flasks until confluence. Cells were transferred to 3.5-ml tubes (2.5 x 10' cells per tube), washed three times with HBSS, and incubated in 0.5 ml of HBSS (containing 1.26 mM Ca2+) in the presence of 2.5 HU of Hly per ml for 5 min at 37°C. Then cells were chilled on ice and incubated with 1 ,uCi of 45Ca2+ or 2.5 ,uCi of tritiated sucrose, inulin, or dextran for 60 min at 0°C. At the end of the incubation, the total assay volume was layered on top of 700 ,ul of Versilube F-50 silicone oil in a centrifugation tube, and cells were quickly separated from the medium by centrifugation at 8,000 x g for 1 min as described previously (31). Cell pellets were solubilized by the addition of 100 jig of mellitin per ml (31, 32) and processed for determination of
radioactivity. Morphology. Because actin is concentrated along the margins of endothelial cells, rhodamine-labeled phalloidin
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SUTTORP ET AL.
50
INFECT. IMMUN.
Up to 100 ng of endotoxin (lipopolysaccharide from Salmonella abortus-equi) per ml did not induce significant PGI2 synthesis within 60 min (4.1 ± 0.09 ng of 6-keto-PGF10, per ml at 60 min (mean ± SE, n = 7). These data do not rule out the possibility of a synergistic interaction between Hly and endotoxin which is responsible for the effects observed. Hly and permeability of endothelial cell monolayers. Undisturbed endothelial cell monolayers exposed to a hydrostatic pressure of 10 cm H20 (25, 28) displayed a hydraulic conductivity of 4 x 10-6 cm s-1 cm H20-1. Addition of 0.5 to 5 HU of Hly per ml resulted in a dose- and timedependent increase in the hydraulic conductivity (Fig. 2A). With 5 HU of Hly per ml, this parameter increased about 30-fold within 10 min. The reflection coefficient for dextran, a measure of endothelial monolayer selectivity, amounted to 0.71 in resting monolayers. This parameter decreased in a dose- and time-dependent manner in the presence of Hly, indicating that the cell monolayer lost its selective perme-
-
40
30
20-
10-
O0J 10 20 Min: 30 40 50 60 FIG. 1. Time- and dose-dependent PGI2 synthesis by Hly. Pulmonary artery endothelial cells were incubated with 0, 0.05, 0.1, 0.5, and 1 HU of Hly per ml for 1 to 60 min, and then the medium was taken for determination of PGI2 levels (measured radioimmunologically as 6-keto-PGF10,,). Data presented are means SE from seven separate experiments. ±
used to visualize interendothelial gap formation. Monolayers were fixed at room temperature for 5 min at a hydrostatic pressure of 10 cm H20 in HBSS (pH 7.4) containing 2% formaldehyde. After three washes with HBSS, monolayers were permeabilized by exposure to acetone at -20°C for 1 min. Cells were then washed with HBSS and incubated for 30 min with rhodamine-labeled phalloidin (1.4 ,ug/ml). After a brief wash, the preparations were mounted in 60% glycerol (containing 1.5% n-propyl gallate) and examined by fluorescence microscopy as previously described (25, 28). Statistics. Data were analyzed for unbalanced data by a one-way and two-way analysis of variance (9). was
RESULTS Hly and endothelial cell PGI2 synthesis. Hly stimulated PGI2-synthesis in cultured pulmonary artery endothelial cells in a dose- and time-dependent manner (Fig. 1). The lowest toxin concentration which induced significant PGI2 formation within 30 min was 0.05 HU of Hly per ml. The effects of Hly occurred in the absence of overt cell lysis, as indicated by the lack of lactate dehydrogenase release (data not shown). No increased PGI2 synthesis was noted in experiments using, per ml, 1 HU of hemolysin which was allowed to age for 8 h at 37°C (4.2 + 1.4 ng of 6-keto-PGF1c per ml at 30 min and 5.1 + 1.6 ng/ml at 60 min, mean standard error [SE], n = 2; no significant difference from control cells). ±
ability (Fig. 2B). Morphological studies. Morphological studies indicated that resting cells were tightly packed, without any evidence of interendothelial gap formation. Figure 3a shows the typical marginal microfilament network which is closely associated with endothelial cell junctions. Addition of 1 HU of Hly per ml for 25 min to endothelial cell monolayers resulted in the formation of small interendothelial gaps (Fig. 3b). These morphological alterations were even more pronounced after exposure to 5 HU of Hly per ml for 10 min (Fig. 3c). These morphological alterations suggested that Hly increased endothelial monolayer permeability by enhancing the paracellular fluid flux. Characterization of Hly-induced effects. Hly has been shown to create small hydrophilic pores of 2-nm effective diameter in erythrocyte membranes (4) and in planar lipid bilayers (21). This pore-forming property of Hly has not been shown for endothelial cells. We determined the influx pattern of four differently sized marker molecules (45Ca2 , tritiated sucrose, inulin, and dextran) in Hly-exposed endothelial cells. There was a significant passive influx of the small molecules 45Ca2+ and 3H-sucrose but not of the large tritiated inulin or dextran (Fig. 4). These observations are compatible with the presence of Hly-induced pores with an effective internal diameter of 2 nm in the cell membranes. In previous studies (27, 29-32), we proposed that toxincreated transmembrane pores act as nonphysiological Ca2` bypass gates which allow an influx of extracellular Ca2+ into the cell, thereby activating Ca2'-dependent cellular processes. In this study, we found that Hly-induced endothelial PGI2-synthesis is dependent on the extracellular concentration of Ca2+ but not of Mg2+ (Fig. 5). Toxin-treated endothelial cells were exposed to 0.9 mM Mg2+, and the extracellular Ca2+ concentration was varied. Hly-induced formation of PGI2 was strictly dependent on the presence of micromolar concentrations of extracellular Ca2+ (Fig. 5). Vice versa, experiments wherein the Ca2+ concentration was set at 2.5 mM and the Mg2" concentration was varied were conducted. In this case, stimulation of PGI2 synthesis occurred to a similar extent throughout. Ca2+ channel antagonists (maximally 100 ,uM diltiazem or 50 ,uM nimodipin) were without effect, while 25 ,uM trifluoperazine, a nonspecific inhibitor of calmodulin function, reduced Hly-induced PGI2 formation by 80% (data not shown). Indomethacin (1 ,uM) inhibited Hly-related PGI2 synthesis by 95% (data not
shown).
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E. COLI HEMOLYSIN-ENDOTHELIAL CELL INTERACTION
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FIG. 2. Time- and dose-dependent increase in permeability of endothelial cell monolayers caused by Hly. Confluent cell monolayers on polycarbonate filter membranes were mounted in a modified chemotaxis chamber and exposed to a continuous hydrostatic pressure of 10 cm H20 (see Materials and Methods for details). After a steady state was reached, 0, 0.5, 1, 2.5, or 5 HU of Hly per ml was added. Hly induced a time- and dose-dependent increase in the hydraulic conductivity (A) and a decrease of the reflection coefficient (RC) for dextran (B), indicating severe damage to the barrier function of the endothelial cell monolayers. Data presented are means + SE from 10 separate experiments.
DISCUSSION This study demonstrates that two important alterations of endothelial cell function are induced by low concentrations of Hly: generation of PGI2 and enhanced permeability of endothelial cell monolayers. These effects were observed at very low toxin concentrations (.0.05 HU/ml; .5 ng/ml) and proceeded in the absence of overt cell lysis. Endothelial cells may, together with granulocytes and monocytes (2), repre-
sent preferred targets for attack by Hly in the host organism. A similarly high susceptibility of pulmonary endothelium to Hly in isolated, buffer-perfused rabbit lungs has recently been noted (24). Current evidence suggests that Hly acts by forming small hydrophilic transmembrane pores (4, 9). In the present study, we noted an influx of Ca2+ and sucrose but not of inulin or dextran into Hly-treated endothelial cells. This
FIG. 3. Hly-induced morphological alterations of endothelial cell monolayers. At the end of the experiments, cells were stained with rhodamine-labeled phalloidin (highly specific for filamentous actin) to visualize interendothelial gaps. (a) Cell monolayers without Hly showed the typical marginal microfilament network (arrowheads) which is closely associated with endothelial cell junctions. (b) After exposure to 1 HU of Hly per ml for 25 min, small interendothelial gaps were noted. (c) There was a dramatic increase in interendothelial gaps (large arrows) after exposure to 5 HU of Hly per ml for 10 min. A remaining cell bridge between neighboring cells is indicated by the small arrows. Bar 10 ,um.
INFECT. IMMUN.
SUTTORP ET AL.
3800
700, %
600*
40- 6-keto PGFg f
TI
(ng/ml)
30-
50020/r
400-
I~~~~~~~~~ 0.5 HU Toxin,Mg+0.9mM 0.5 HU Toxin,Ca*+2.5mM I no Toxin,Mg++0.9mM no
300-
Toxin,Ca++2.5mM
10-
f-L ----
.1,
2000-
100-
45Ca3H-Sucro 3H-Inulln 3H-Dextran FIG. 4. Influx pattern of small (45Ca2' and 3H-sucrose [3HSucro]) and large (3H-inulin and 3H-dextran) marker molecules in Hly-exposed endothelial cells. Cells were incubated with buffer or with 2.5 HU of Hly per ml at 37°C for 5 min, and then cells were chilled on ice and incubated with 1 ,uCi of 45Ca2+ or 2.5 ,uCi of tritiated sucrose, inulin, or dextran for 60 min at 0°C. Cells were washed, and pellets were processed for determination of radioactivity. The amount of labeled marker molecules in Hly-free cell pellets was set at 100%. There was a significant influx of small (45Ca2' and 3H-sucrose) but not of large (tritiated inulin and dextran) marker molecules in toxin-treated endothelial cells. Data presented are means + SE from eight separate experiments. influx pattern is similar to that observed by Bhakdi et al. (4) with rabbit erythrocytes and is compatible with the concept of toxin-induced transmembrane pores with internal diameters of about 2 nm. Hly extends the list of pore-forming proteins such as staphylococcal alpha-toxin (1, 6), Pseudo-
aeruginosa cytotoxin (18), terminal complement complexes (5), and lymphocytolysins (14). Previous studies addressed the hypothesis that these transmembrane pores act as nonspecific Ca2+ bypass gates which allow an influx of Ca2+ into the cell (26, 27, 29-32). We demonstrated an increase in intracellular free Ca2+ in Staphylococcus aureus alpha-toxin-treated polymorphonuclear leukocytes and showed that this effect was the result of an influx of extracellular Ca2+ and not of a mobilization of intracellularly stored Ca2+ (27). This intracellular Ca2+ signal would be expected to initiate many Ca2'-dependent secondary cellular responses. In this context, we demonstrated staphylococcal alpha-toxin and terminal complement complex C5b-8 induced leukotriene B4 synthesis in polymorphonuclear leukocytes (23, 31) and PGI2 synthesis in endothelial cells (29, 31). In the present study, a strict dependency of hemolysin-induced PGI2 synthesis on extracellular Ca2+ was also found. The effect of Hly was inhibitable by monas
-2.5 -3 -5 -4 Ca++ or -6 log Ca++ or Mg++ (M) Mg++ free FIG. 5. Dependence of Hly-induced endothelial PGI2 synthesis on increasing concentrations of extracellular Ca2' but not on increasing concentrations of extracellular Mg2+. Data presented are SE from seven separate experiments (four experiments means involving variation of extracellular Mg2"). ±
calmodulin but not by Ca2+ channel antagonists. These results are compatible with the concept of stimulus-response coupling through toxin-created transmembrane pores. Besides stimulation of arachidonate metabolism, an enhanced permeability of endothelial monolayers was observed following the addition of Hly. The toxin not only increased the fluid filtration rate but also impaired the reflecting properties of the endothelial cell monolayers for large molecules (dextran), indicating a loss of the barrier function of the endothelium. Morphologically, endothelial cells appeared to contract upon exposure to Hly, thereby opening gaps as paracellular routes for enhanced fluid exchange across the cell monolayer. Ca2+ influx also appears to be important for this process (25). Current evidence suggests that an increase in intracellular free Ca2" may cause alterations in the cytoskeleton, leading to contraction of the endothelial cells (Schnittler et al., J. Physiol., in press). It is of interest that Hly-induced enhanced endothelial permeability occurred at higher toxin concentrations than the increase in endothelial PGI2 synthesis. A comparable finding was made when Hly was perfused through isolated rabbit lungs (24). It is conceivable that a higher intracellular free Ca2+ concentration is required for endothelial cell retraction than for phospholipase activation. In conclusion, low concentrations of Hly induced endothelial PGI2 synthesis and damaged the barrier function of endothelial cell monolayers. These observations are relevant to human biology (for example, hemolytic E. coli-induced pyelonephritis with subsequent septicemia) because synthesis of mediators and increased permeability of the pulmonary microvasculature are hallmarks of the acute respiratory
VOL. 58, 1990
E. COLI HEMOLYSIN-ENDOTHELIAL CELL INTERACTION
distress syndrome (22). Several other medically relevant exotoxins of gram-negative organisms have recently been recognized to share structural homologies with Hly (16, 17, 35), and they may exert similar toxic effects on endothelial cell function. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 249). We thank P. Rohrig, M. Muhly, and S. Tannert-Otto for excellent technical assistance and P. Muller for skillful graphic illustrations. LITERATURE CITED 1. Bhakdi, S., R. Fussle, and J. Tranum-Jensen. 1981. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc. Natl. Acad. Sci. USA 78:5475-5479. 2. Bhakdi, S., S. Greulich, M. Muhly, B. Eberspacher, H. Becker, A. Thiele, and F. Hugo. 1989. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J. Exp. Med. 169:737-754. 3. Bhakdi, S., N. Mackman, G. Menestrina, L. Gray, F. Hugo, W. Seeger, and I. B. Holland. 1988. The hemolysin of Escherichia coli. Eur. J. Epidemiol. 4:135-143. 4. Bhakdi, S., N. Mackman, J.-M. Nicaud, and I. B. Holland. 1986. Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. Infect. Immun. 52:63-69. 5. Bhakdi, S., and J. Tranum-Jensen. 1984. Mechanism of complement cytolysis and the concept of channel-forming proteins. Philos. Trans. R. Soc. London B Biol. Sci. 306:311-324. 6. Bhakdi, S., and J. Tranum-Jensen. 1987. Damage to mammalian cells by proteins that form transmembrane pores. Rev. Physiol.
Biochem. Pharmacol. 107:148-223. 7. Brigham, K. L., and B. Meyrick. 1986. Endotoxin and lung injury. Am. Rev. Respir. Dis. 133:913-927. 8. Cavalieri, S. J., G. A. Bohach, and I. S. Snyder. 1984. Escherichia coli a-hemolysin: characteristics and probable role in
pathogenicity. Microbiol. Rev. 48:326-343. 9. Dunn, 0. J., and V. A. Clark. 1974. Applied statistics: analysis of variance and regression. John Wiley & Sons, Inc., New York. 10. Eberspacher, B., F. Hugo, and S. Bhakdi. 1989. Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin. Infect. Immun. 57:983-988.
11. Felmlee, T., S. Pellet, and R. Welch. 1985. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol.
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12. Friberger, P. 1982. A quantitative endotoxin assay utilizing LAL and a chromogenic substrate. Prog. Clin. Biol. Res. 93:195-206. 13. Hacker, J., C. Hughes, H. Hof, and W. Goebel. 1983. Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect.
Immun. 42:57-63. 14. Henkart, P. 1985. Mechanism of lymphocyte-mediated cytotox-
icity. Annu. Rev. Immunol. 3:31-58.
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