Clostridium perfringens Epsilon-Toxin Increases Permeability of Single Perfused Microvessels of Rat Mesentery
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感染与免疫杂志 2005年第8期
Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, One Shields Avenue, Davis, California 95616
California Animal Health and Food Safety Laboratory, University of California, Davis, 105 W. Central Avenue, San Bernardino, California 92408
Department of Microbiology, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
ABSTRACT
Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (Lp) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 μg ml–1 the Lp increased irreversibly to more than 15 times the control value by 10 min. At 0.3 μg ml–1 no increase in Lp was observed for up to 90 min. The toxin-induced increase in Lp was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.
INTRODUCTION
One of the most potent of the clostridial toxins, epsilon-toxin of Clostridium perfringens, has been named a class B select agent of the Centers for Disease Control and Prevention and a category B priority agent of the National Institute of Allergy and Infectious Diseases. While newly regarded as a potential bioterrorism tool, epsilon-toxin has long been recognized as an important cause of mortality in livestock, particularly sheep and goats, where it is the primary virulence factor of C. perfringens type D and leads to an often-fatal enterotoxemia. C. perfringens type D can be a normal inhabitant of the intestine of sheep and several other animal species, but when large amounts of easily fermentable carbohydrates pass into the intestine, generally due to sudden changes in diet, this microorganism proliferates and produces large quantities of epsilon-toxin. Although little evidence is available in this regard, it is generally accepted that epsilon-toxin compromises the intestinal barrier and is then taken up by the vasculature of the gut, from which it is spread systemically. The effects of injecting epsilon-toxin intravenously have been studied in sheep (7), mice (10), goats (38), rats (11), and cattle (39). The toxin thus administered produces increased vascular permeability in many tissues, the most significant effects of which are acute pulmonary and cerebral edema, which is associated with vascular endothelial injury demonstrable with transmission electron microscopy (13). Thus, the toxemia has been assumed to directly cause widespread damage to the vascular endothelium, although no definitive proof has been produced about the direct action of epsilon-toxin on endothelial cells.
Of the many cultured cells that have been tested for sensitivity to epsilon-toxin, only Madin-Darby canine kidney (MDCK) cells and Caucasian renal leiomyoblastoma (G-402) cells are sensitive enough for useful investigation of the toxin (4, 32, 40). The MDCK cells are about 100-fold more sensitive than the G-402 cells and have been used in most cultured cell studies of epsilon-toxin. In MDCK cells, epsilon-toxin was found in high-molecular-weight complexes and induced increased membrane permeability to ions and small hydrophilic solutes (33). It forms an aqueous anion-selective pore permeable to solutes up to 1 kDa (35). Analysis of labeled constructs derived from epsilon-toxin clearly demonstrated that it forms heptamers when incubated with rat brain synaptosomal membranes and that cleavage of a C-terminal peptide is essential for oligomerization (26). Also in MDCK cells, epsilon-toxin associates with apical membrane in preference to basolateral membrane and forms a heptameric pore associated with detergent-resistant domains (27, 34). While it has relatively low amino acid sequence identity (ca. 14%) to the heptameric pore-forming toxin aerolysin from Aeromonas hydrophila, it does have three-dimensional structural similarity to aerolysin, one of a family of toxins known as -pore-forming toxins (8). Epsilon-toxin also has some amino acid sequence identity to a family of mosquitocidal toxins typified by Mtx2 and Mtx3 of Bacillus sphaericus (21% and 24% identity, respectively) (9, 22, 37). Because only some cultured cells are highly sensitive to the toxin it has been suggested that a specific host receptor is essential for binding and pore formation, but no specific receptor molecule has been found. A recent study demonstrated that experimental development of tolerance to epsilon-toxin by MDCK cells was associated with loss of expression of a group of membrane proteins, lending further support to the hypothesis that host proteins mediate epsilon-toxin binding and toxicity (4). Because of the assumed association of epsilon-toxin with vascular endothelium, development of an endothelial model is highly desirable. However, cultured aortic endothelial cells (goat, sheep, and cattle) failed to show any morphological response to epsilon-toxin with up to 48 h of exposure, even at concentrations much higher than were toxic for MDCK cells (40). The latter observation brought into question whether the in vivo toxicity of epsilon-toxin is due to direct action of the toxin with vascular endothelium.
Because the cultured endothelial cells were not morphologically affected by epsilon-toxin, in this study we have used single perfused microvessels in rat mesentery, which enable direct observation of permeability properties of the vascular wall during exposure to toxin under controlled conditions. While the effects of epsilon-toxin in tissue have been investigated using morphological and histochemical techniques to demonstrate accumulations of fluid and labeled proteins, there are no reports in the literature of biophysical permeability coefficients determined from directly measured fluxes and controlled driving gradients. Moreover, by presenting the toxin directly to the endothelium within an artificial perfusate, the technique enables investigation of the endothelial response in the absence of potential systemic mechanisms or interactions with blood plasma. Variations of the single perfused microvessel model have been used to investigate vascular permeability coefficients regarding convective water flow and permeability of both small and large solutes (19, 24, 25), regulation of inflammatory mechanisms (3, 16), ultrastructure of the endothelial cleft (2), and regulation of the adherens junction complex (1, 42). Here we determine the hydraulic conductivity coefficient (Lp) as a measure of the response to epsilon-toxin. We demonstrate that microvessels of the rat mesentery are highly sensitive to epsilon-toxin, and we describe an experimental system in which to investigate the direct action of bacterial toxins with vascular endothelium.
MATERIALS AND METHODS
Animal preparation. Experiments were carried out on rats (male, Sprague-Dawley, 350 to 450 g; Hilltop Laboratory Animals, Inc.) anesthetized with pentobarbital (65 mg kg–1 body weight given subcutaneously). Anesthesia, assessed by toe-pinch reflex, was maintained by giving additional pentobarbital (subcutaneous, 3 mg per dose) as needed. At the end of experiments, animals were euthanized with pentobarbital overdose. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Each rat was placed on a heating pad to maintain normal body temperature. A midline surgical incision was made in the abdominal wall, and the gut gently was taken from the abdominal cavity to spread the mesentery over a quartz pillar for transillumination and observation on the fixed stage of an inverted microscope. The upper surface of the mesentery was continuously superfused with Ringer's solution maintained at 35°C to 37°C during preparation and experimentation. Because our present techniques are not suited for use of vessels smaller than about 15 μm in diameter, we have not investigated the properties of true capillaries (diameter of ca. 6 μm). Therefore, experiments were performed on straight nonbranched segments of venular microvessels typically 25 to 40 μm in diameter and usually between 700 and 900 μm in length. Prior to cannulation, all vessels selected for experiments had brisk blood flow and were free of leukocytes sticking or rolling on the vessel wall.
Measurement of Lp of the microvessel wall. All measurements were based on the modified Landis microperfusion technique, which enables measurement of the volume flux of water across the wall of a microvessel due to a known pressure difference between the lumen and the interstitium. The technique and its assumptions and limitations have been evaluated in detail (2, 24). Briefly, each vessel was cannulated by the tip of a glass micropipette filled with a physiological perfusate additionally containing flow marker erythrocytes (ca. 4%, vol/vol). The micropipette was positioned in the vessel to achieve free downstream perfusion with no leak to the upstream direction or to the tissue. The rear of the micropipette was in communication with a water manometer used to set the hydrostatic pressure and drive flow from the micropipette through the cannulated vessel. To measure the initial transcapillary water flow per unit area of the capillary wall (Jv/A), a glass microoccluder was lowered onto the microvessel up to 900 μm from the cannulation site, with the hydraulic pressure set at predetermined values of 30 to 60 cm H2O. When the vessels were occluded, the intravascular hydraulic pressure was equal to that of the manometer, and the motion of flow marker erythrocytes was recorded on videotape for later analysis. Microvessel Lp was calculated as the slope of the relation between Jv/A and applied hydraulic pressure. For most experiments, Jv/A was estimated from each of several occlusions using one hydraulic pressure, with the assumption that the net effective pressure determining fluid flow was equal to the applied hydraulic pressure minus 3.6 cm H2O, the approximate osmotic pressure contributed by bovine serum albumin (BSA) at 10 mg ml–1. Experimental reagents (e.g., epsilon-toxin) were added to the perfusate in the respective experiments and delivered to the vascular lumen via the micropipette continuously during Lp measurement. Changes in perfusate were accomplished by withdrawing the initial micropipette and replacing it with a second micropipette filled with new perfusate solution of the appropriate composition.
Preliminary experiments were designed to determine the effective osmotic pressure (eff) of albumin (30 mg ml–1) in the perfusate. Before each measurement the hydraulic pressure in the vessel was held at 60 cm H2O for 30 s by partial downstream occlusion to establish a high-filtration steady state in which the BSA concentration on the downstream side of the selectivity barrier was washed down to a minimal level, thus maximizing the effective oncotic pressure difference (24, 25). Jv/A was measured by monitoring the motion of flow marker erythrocytes during brief (5 to 8 s) full occlusions. With 60 cm H2O hydraulic pressure, erythrocytes moved toward the occlusion site as fluid moved out of the vessel. Measurements of Jv/A at microvessel pressure of 10 cm H2O were made after setting up steady-state filtration at 60 cm H2O, occluding, and rapidly (<0.5 s) switching the applied hydraulic pressure to 10 cm H2O. Reabsorptive water movement was seen as marker erythrocytes moved away from the occlusion site toward the cannulation site as fluid moved into the vessel. Three to five measurements were made at each pressure, yielding a control relationship between Jv/A and applied hydraulic pressure. The slope of the relationship is the hydraulic conductivity (Lp), and the intercept on the pressure axis is eff of the serum albumin. Our test solutions of BSA (10 or 30 mg ml–1) had protein osmotic pressures of 3.6 and 14 cm H2O at 37°C (calculated from published equations [23]).
Experimental protocols. Each vessel was initially perfused with a control solution containing BSA at 10 mg ml–1 in Ringer's solution (except preliminary experiments as indicated). Usually between 5 and 10 occlusions at 50 cm H2O over 15 to 20 min were used to establish a control Lp. Then, the first pipette was removed and a second pipette containing the test solution was introduced at the same cannulation site. Multiple occlusions over the subsequent 15 to 120 min enabled estimation of Lp during exposure to the test solution. Occlusions were made every 20 to 30 s during the first 5 min of test perfusion to check for rapid initial change in Jv/A and then made less frequently (three to five occlusions every 5 to 10 min) for the remainder of the experiment.
Statistics. We tested for differences from basal Lp due to the perfusion with epsilon-toxin by comparing the averaged Lp in each experimental group with the corresponding time-matched value found in a control (no-toxin) group of vessels. Because basal Lp distribution is non-Gaussian, we used the nonparametric Mann-Whitney statistic and assumed significance for a P value of <0.05. Throughout, descriptive statistics are reported as the mean ± the standard error of the mean (SEM).
Ultrastructure. To investigate the structural nature of the response to toxin, four vessels exposed to epsilon-toxin (two using 3 μg ml–1 and two using 10 μg ml–1) were fixed after a substantial rise in Lp (25 to 40 min of toxin exposure). Two other vessels were perfused with vehicle solution (control vessels) for a comparable time and also inspected using the following methods. After the final determination of Jv/A, fixation for electron microscopy was started by dripping ice-cold glutaraldehyde (3% in 0.1 mM cacodylate buffer) on the mesentery. This was done while vessels were still perfused and occluded to ensure that the final perfusion conditions and geometry were maintained during fixation. After 1 to 2 min the perfusion pipette was removed while immersion fixation continued. A diagram of the area enabled later identification of the perfused vessel segment. After 10 min in situ fixation, tissue was cut away from the animal and glutaraldehyde fixation continued on ice for a total of 1 h before changing to OsO4 (1% in veronal acetate, pH 7.2) for 1 h. Tissue was treated with tannic acid (0.5% in maleate buffer, pH 6) for 30 min to enhance membrane staining and placed in uranyl acetate (2% in maleate buffer, pH 6) overnight. After dehydration in a graded series of acetone, the tissues were flat embedded in epoxy resin. Sections (50 nm thick) from transversely sectioned vessels were collected on Formvar-coated slots and were stained with uranyl acetate and lead citrate. They were examined using transmission electron microscopy (TEM) with a Philips CM120 at 80 kV.
Solutions and reagents. Mammalian Ringer's solution was composed (in mM) of 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES and Na-HEPES. The ratio of acid-HEPES to Na-HEPES was adjusted to achieve pH 7.40 to 7.45 for Ringer's solutions. All perfusates were mammalian Ringer's solution additionally containing BSA (A4378; Sigma-Aldrich) at 10 or 30 mg ml–1 as indicated.
Epsilon-prototoxin was prepared from an overnight culture of C. perfringens type D (strain NCTC 8346) in Trypticase-yeast-glucose medium, under anaerobic conditions at 37°C. The overnight cultures were centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant containing epsilon-toxin was saved for toxin purification. The toxin was then precipitated by ammonium sulfate. Two columns were prepared with DEAE and CM Sepharose (Pharmacia, Sweden), respectively, equilibrated in 10 mM Tris, pH 7.5. The toxin was applied to the DEAE column, and the effluent was monitored at 220 nm. The initially eluted peak was saved and applied to the CM column. Again the effluent was monitored at 220 nm, and the first peak was collected, dialyzed against distilled water, and freeze-dried. This product was shown to contain pure epsilon-prototoxin when tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prior to its use in these experiments, the prototoxin was reconstituted in Ringer's and activated by incubation at 37°C during 30 min with 0.1% trypsin (Sigma).
To further establish specificity of the Lp response to toxin, we tested the effectiveness of an epsilon-toxin neutralizing mouse monoclonal antibody (immunoglobulin G; 5B7) provided by Paul Hauer (Center of Veterinary Biologics, Ames, Iowa). The antibody (final concentration, 0.13 mg ml–1) was added to perfusion solution containing epsilon-toxin (3 μg ml–1) and incubated for 30 min at room temperature before use. The effect on microvascular Lp was measured and compared to the time-matched set of measurements made with epsilon-toxin in the absence of antibody.
RESULTS
Epsilon-toxin increases microvascular Lp. We perfused single microvessels of rat mesentery with solutions containing epsilon-toxin to test for increased Lp as an indication of endothelial cell intoxication. In a few preliminary experiments the perfusate contained 30 mg ml–1 BSA, enabling determination of the effective protein osmotic pressure difference across the vessel wall. The data from a representative experiment illustrate that during control measurement in the absence of toxin the Lp (slope of the relation between Jv/A and applied pressure) was low and the effective protein osmotic pressure (intercept on the pressure axis) was between 10 and 20 cm H2O (Fig. 1). After removing the initial pipette and perfusing with a second pipette containing toxin (10 μg ml–1), the Lp increased over time and the effective protein osmotic pressure fell to near zero. Both changes are consistent with loss of endothelial barrier function. This initial technique required multiple measurements, typically taking several minutes at various pressures. When permeability changed rapidly, it was difficult to make estimates of the intercept on the pressure axis. Therefore, in remaining experiments the BSA concentration was 10 mg ml–1 and estimates of Lp were made using one applied pressure (usually 50 or 60 cm H2O) as described in Materials and Methods. The latter technique better enabled our following of rapid Lp changes.
A representative experiment shows the increase in Lp with time associated with toxin exposure (Fig. 2A). Perfusion with vehicle control solution for 30 min established the control Lp of 0.3 x 10–7 cm s–1 cm H2O–1. The initial micropipette was removed, and the vessel was cannulated a second time and perfused with toxin (3 μg ml–1) for the next 40 min (Fig. 2A). During the first 5 min of perfusion with toxin, there was no apparent change in Lp. However, within 10 min of exposure to epsilon-toxin the Lp increased to about 2 x 10–7 cm s–1 cm H2O–1. It continued to increase and was above 15 x 10–7 cm s–1 cm H2O–1 by 40 min of toxin exposure. Representative data from a vessel perfused for 90 min without toxin illustrate that the perfusion technique alone does not increase Lp (Fig. 2B). Averaged data from groups of experiments are described next.
To test the concentration dependence of the sensitivity to epsilon-toxin in the microvasculature, the Lp response to toxin over time was tested in four different groups of vessels (Fig. 3A). A separate group of vessels perfused without toxin for 90 min demonstrated that Lp did not change in that group (n = 15) (Fig. 3B). The Lp values from that group were used as time-matched control values for the toxin-perfused vessels. At the lowest toxin concentration (0.3 μg ml–1) no increase in Lp was observed for up to 90 min (n = 7). At 10 μg ml–1 the Lp increased rapidly, on average to more than 15 times the control value within 10 min, and was significantly greater than the time-matched control group at all times (P < 0.05, Mann-Whitney). At intermediate concentrations the response to epsilon-toxin exposure took longer to develop and the mean values of Lp did not reach as high. The threshold concentration for a measurable Lp response within 60 min was 1 μg ml–1 (significantly greater than time-matched control group at 50 min and longer). In two experiments, after the Lp had significantly increased during epsilon-toxin exposure, each vessel was recannulated with the control perfusate and Lp measurements were made for 20 min to test for recovery from the epsilon-toxin. The Lp in these vessels did not return toward control; the effect of epsilon-toxin to increase Lp was irreversible.
Antibody neutralization of the toxin effect on Lp. A separate group of vessels were perfused with solution containing a neutralizing monoclonal antibody (0.13 mg ml–1) to epsilon-toxin in addition to epsilon-toxin (3 μg ml–1). In perfusions lasting up to 40 min, the Lp in these vessels did not increase. Lp measured over time is shown in comparison with the mean response to the same concentration of toxin alone in Fig. 4. Over this time the antibody completely prevented the increase in Lp induced by toxin in the absence of antibody.
Ultrastructure of microvessels exposed to toxin. Two vessels of the group perfused with 3 μg ml–1 epsilon-toxin and two of the group perfused with 10 μg ml–1 epsilon-toxin were examined with TEM. The two vessels exposed to 3 μg ml–1 toxin (25 and 40 min, respectively) reached an Lp about 5 times the control value just before fixation; in the two vessels exposed to 10 μg ml–1 epsilon-toxin (25 and 35 min, respectively), Lp had increased to 13 and 24 times the control Lp when fixed. Features consistent with direct endothelial damage were observed in all epsilon-toxin-treated vessels (Fig. 5). Most endothelial cells were very attenuated, showing loss of cytoplasm, although the cytoplasmic membrane appeared intact in most cases (Fig. 5A, B, C, and G). Different degrees of detachment from the basement membrane were also observed. A few cells were also fragmented, with some of the fragments remaining attached to the basement membrane. Thus, in places the endothelium was discontinuous, exhibiting gaps either between or through the endothelial cells (Fig. 5A, B, D, and E). Near one such gap the subendothelial space between the basement membrane and pericyte was expanded and filled with flocculent material, possibly protein from disrupted cells (Fig. 5A and B). Sections of the basement membrane were exposed to the vascular lumen, although the basement membrane in these areas and elsewhere appeared intact. Some of the pericytes were also densely stained and highly attenuated (Fig. 5A and B). Most endothelial cells had angular shapes, and the cytoplasmic membrane presented an irregular outline, with numerous indentations and a few cytoplasmic blebs consisting of flocculent relatively electron-lucent material devoid of organelles and surrounded by cytoplasmic membrane. Most endothelial cells showed a diffusely electron-dense cytoplasm in which it was impossible to distinguish any cytoplasmic detail. A few endothelial cells, however, showed a clear cytoplasm in which dilated cisterns of endoplasmic reticulum were observed. Very few ribosomes were seen attached to the endoplasmic reticulum membranes. A few membrane-bound vacuoles with myelinic figures in front of a clear background were also seen in the cytoplasm of some cells. The nuclei of some endothelial cells were shrunken and showed large clumps of electron-dense chromatin. A few cells showed diffuse dense chromatin occupying the entire nucleus. Nuclear membranes were unaffected in most cases. Endothelial cells of control vessels perfused for comparable times with vehicle solutions in the absence of toxin did not show ultrastructural abnormalities, such as gaps in the endothelium or necrotic characteristics (Fig. 6). Specifically, the endothelium was in contact with basement membrane and formed a continuous intimal layer with intact junctional complexes. The peripheral cytoplasm was of moderate electron density and was relatively uniform in thickness (0.1 to 0.2 μm).
DISCUSSION
We set out to test the specific hypothesis that epsilon-toxin would increase the hydraulic permeability of microvessels in the rat mesentery and to answer the more general question of whether the single perfused vessel model could be used to investigate epsilon-toxin. The data demonstrated a time- and dose-dependent irreversible increase in convective water flux across the vessel walls in response to exposure to activated epsilon-toxin. Many endothelial cells from toxin-treated vessels appeared necrotic, highly attenuated, and with dense cytoplasm, indicative of extreme fluid loss associated with necrosis. Patent pathways through the endothelial layer were seen associated with fluid accumulation in the subendothelial spaces. Such pathways are never seen in control endothelium and represent a very high conductance path for both plasma water and protein. The results are consistent with the hypothesis that epsilon-toxin interacts with endothelial cells of rat venular microvessels and increases the vessel wall permeability by direct damage of the endothelium. The answer to the broad question of whether the rat mesentery model might be useful to study endothelial interaction with blood-borne toxins such as epsilon-toxin is clear. The single perfused microvessel model does enable investigation of relatively small quantities of toxin with mammalian microvessel endothelium.
While this study is the first to measure biophysical permeability coefficients in response to epsilon-toxin, there are numerous previous reports indicating fluid and protein extravasation resulting from epsilon-toxin toxemia. Edema and excess protein extravasation in various tissues have been noted at necropsy in experimentally and naturally intoxicated animals (12, 14, 30, 38, 39, 43). Morphological and histochemical methods provide endpoint observations but do not enable finer temporal characterization of the progress of intoxication. Moreover, because the toxin has been shown to stimulate vascular smooth muscle contraction and elevate arterial pressure, it is very difficult to separate changes in vessel wall permeability, tissue perfusion, and intravascular pressure in whole-animal models (28, 36). Under the conditions of our experiments, the intravascular hydraulic pressure and protein osmotic pressure are directly controlled, while extravascular hydraulic pressure and protein osmotic pressure are negligible (2, 20). The postcapillary microvessels used in this study are not invested with smooth muscle cells and maintain a constant diameter under the conditions of these experiments. Individual measurements in the single perfused vessel, taking between 5 and 8 seconds, enable characterization of permeability changes with temporal resolution comparable to cell culture methods used to measure transepithelial resistance, small solute flux, or hydraulic conductivity (18, 34, 41).
The single vessel technique was used here to measure an increase in water flux crossing the microvessel wall resulting from epsilon-toxin exposure and was also used to infer loss of the barrier to serum albumin (decrease of effective protein osmotic pressure shown in Fig. 1). Such changes in each of these parameters are directly important in the development of edema. However, one cannot specify what level of Lp measured in a single vessel would be correlated with organ failure or death. We can say that the ultrastructural examination of all vessels perfused with epsilon-toxin revealed endothelial cells that were severely damaged and regions of basement membrane exposed directly to the perfusate. These endothelial gaps were not found in control vessels. The latter control data agree with results of a much larger examination of normal endothelium in a recent publication from our group in which nearly 700 micrographs were analyzed for tight junction structure using rat mesentery microvessels that were perfused for 15 to 30 min with vehicle solutions similar to that in the present study (2). We also noted that once the Lp began to increase, removing perfusate containing toxin and reverting to control perfusate failed to return the Lp toward control. Indeed, in many instances when the perfusing pipette was removed at the end of an experiment and blood was allowed to return to the vessel, we observed hemostasis in vessels that had been perfused with toxin; vessels not perfused with toxin almost always resumed normal blood flow.
We also note that the effects of epsilon-toxin were not similar to an acute inflammatory response, such as that stimulated by platelet activating factor or other acute inflammatory mediators. The typical response of mesenteric postcapillary venules to platelet activating factor is an increase to a peak value within 10 to 15 min followed by a recovery phase lasting another 10 to 60 min (3). Similar patterns have been described for the inflammatory mediators histamine (21) and bradykinin (15) in these vessels. Because the epsilon-toxin effect exhibited a continuous Lp increase and did not return toward control, it did not have the characteristic pattern associated with an acute inflammatory response.
Previous studies of epsilon-toxin using MDCK cells have examined parameters different from the water flux and loss of protein osmotic pressure measured in the present study. Loss of intracellular K+ (29, 33, 35), decreased cell viability (27, 29, 33-35), increased entry of propidium iodide (34, 35), and a fall in trans-epithelial resistance (34) all have been used to infer epsilon-toxin toxicity toward MDCK cell monolayers. None of these measures are directly comparable to increased Lp in microvessels. However, all these measures are indicative of damaged cell membrane and were correlated with compromised epithelial monolayer barrier function. The MDCK cell model is particularly sensitive to epsilon-toxin, and the concentrations of epsilon-toxin that elicited effects in the cited MDCK studies ranged from near 10–10 M to 10–8 M. In the in vivo mesentery microvessel, we found a threshold effect at 1 μg ml–1 (approximately 3 x 10–8 M) when measuring increases in Lp. Endothelial gaps seen with TEM and correlated with increased Lp could have formed as endothelial tight and adherens junctions released or could have resulted from cell disruption. Some clostridial toxins inactivate small GTPases, leading to the breakdown of adherens and tight junctions in both epithelial and endothelial cells (1, 5, 42). In a recent study using MDCK monolayers, at a time and dose of epsilon-toxin that elicited a complete loss of trans-epithelial resistance, the monolayer permeability to a solute (dextran 38500) restricted to the paracellular pathway was not increased and the immunofluorescent distribution of peripheral band actin and components of the tight junction complex was not different from control (34). These data indicated that direct cell disruption by pore formation on the apical membrane preceded any possible direct effect on the junctional complex. Thus, it is likely that the broad areas of basement membrane exposed during epsilon-toxin treatment of the mesentery microvessels result from the disruption of the endothelial cells rather than from disassembly of cell-cell junctions.
The present results using in vivo endothelium and epsilon-toxin concentrations up to 10 μg ml–1 contrast with those of our previous study, in which the toxin at up to 120 μg ml–1 did not induce morphological changes in cultured aortic endothelial cells (tested from cattle, goats, and sheep) (40). One possibility for this difference was that activity of the toxin toward endothelium was dependent on a factor in blood not present in the culture medium. In our previous study we addressed that possibility by testing for toxicity against the endothelial cells in the presence as well as the absence of serum proteins, but we found that in either case there was no morphological change induced by the toxin. While most of the present experiments were carried out in physiological saline (Ringer's) solution supplemented with bovine serum albumin, in several experiments in which toxin was diluted into Ringer's solution without BSA, epsilon-toxin also induced large increases in Lp (not shown). Therefore, our results indicate that no other component of circulating blood is required for toxicity against the mesenteric venular microvessel endothelium.
A specific receptor for epsilon-toxin has not been identified. However, the presence of a receptor, particularly in brain and kidney, has been suggested by uptake of labeled toxin (6, 30). Also, in vitro studies showed that treatment of rat brain homogenates with neuraminidase or lipase reduced binding of epsilon-toxin, indicating that a receptor could be a sialoglycoprotein within a specific lipid environment (31). Following these earlier studies, recent work demonstrated that heptamers of epsilon-toxin were found exclusively associated with the detergent-resistant membrane fraction from MDCK cells, further demonstrating that epsilon-toxin binding is dependent on specific cholesterol-containing microdomains, possibly lipid rafts or caveolae (27). Therefore, it is possible that the aortic endothelial cells which showed no morphological response to epsilon-toxin did not express the appropriate receptor or associated microdomain. We cannot say at present whether those cells express such a domain in vivo or could do so under different culture conditions. The demonstration in the present experiments of the sensitivity of postcapillary microvessel endothelium to epsilon-toxin suggests that cultured endothelial monolayers from other sources or under different culture conditions should be tested and that an appropriate endothelial culture model could be found.
In our experiments we also noted damage to pericytes of the toxin-perfused microvessels. While some studies have suggested that pericytes promote vascular permeability through contractility, recent studies have focused on the role of pericytes in the maturation of microvessels (17). Loss of pericytes, in addition to endothelial cells, due to epsilon-toxin could potentially exacerbate local permeability increases and delay return to normal vascular function in cases of sublethal intoxication.
The present study uses the mesenteric microvessel as a model for the direct vascular effects of epsilon-toxin. However, the principal lethal pathology in sheep is associated with brain and lungs. The traditionally accepted model to explain brain lesions due to epsilon-toxin (6) suggests that such lesions are due to the action of this toxin on endothelial cells, which generates perivascular edema followed by hypoxia and necrosis of the perivascular tissues. However, a recent study showed that epsilon-toxin can also leak into the brain and act directly on rat neurons, producing degeneration and necrosis of these cells in cases of subacute or chronic epsilon-toxin intoxication (11). The results of the present experiments confirm that direct damage does occur in endothelial cells exposed to epsilon-toxin. However, it is possible that epsilon-toxin acts both on endothelial cells and neurons alike. Moreover, it is possible and as yet unknown whether differences exist between various vascular beds in regard to toxin sensitivity and pathogenesis.
Summary. We have shown that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromises the barrier function of venular microvessel endothelium in the rat mesentery. The toxin appeared to directly damage vascular endothelium, inducing formation of gaps in the endothelial cell layer correlated with loss of effective protein osmotic pressure and large increases in measured Lp. These changes are consistent with those expected to lead to rapid edema formation and would also promote access of the toxin to cells in the surrounding tissue. The single perfused vessel model will enable further investigation of epsilon-toxin interaction with the vascular endothelium in situ.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants from the National Heart, Lung, and Blood Institute (HL28607 and HL44485) and National Institute of Allergy and Infectious Diseases (AI56177).
We gratefully acknowledge the expert technical work of Joyce Lenz.
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California Animal Health and Food Safety Laboratory, University of California, Davis, 105 W. Central Avenue, San Bernardino, California 92408
Department of Microbiology, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
ABSTRACT
Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (Lp) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 μg ml–1 the Lp increased irreversibly to more than 15 times the control value by 10 min. At 0.3 μg ml–1 no increase in Lp was observed for up to 90 min. The toxin-induced increase in Lp was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.
INTRODUCTION
One of the most potent of the clostridial toxins, epsilon-toxin of Clostridium perfringens, has been named a class B select agent of the Centers for Disease Control and Prevention and a category B priority agent of the National Institute of Allergy and Infectious Diseases. While newly regarded as a potential bioterrorism tool, epsilon-toxin has long been recognized as an important cause of mortality in livestock, particularly sheep and goats, where it is the primary virulence factor of C. perfringens type D and leads to an often-fatal enterotoxemia. C. perfringens type D can be a normal inhabitant of the intestine of sheep and several other animal species, but when large amounts of easily fermentable carbohydrates pass into the intestine, generally due to sudden changes in diet, this microorganism proliferates and produces large quantities of epsilon-toxin. Although little evidence is available in this regard, it is generally accepted that epsilon-toxin compromises the intestinal barrier and is then taken up by the vasculature of the gut, from which it is spread systemically. The effects of injecting epsilon-toxin intravenously have been studied in sheep (7), mice (10), goats (38), rats (11), and cattle (39). The toxin thus administered produces increased vascular permeability in many tissues, the most significant effects of which are acute pulmonary and cerebral edema, which is associated with vascular endothelial injury demonstrable with transmission electron microscopy (13). Thus, the toxemia has been assumed to directly cause widespread damage to the vascular endothelium, although no definitive proof has been produced about the direct action of epsilon-toxin on endothelial cells.
Of the many cultured cells that have been tested for sensitivity to epsilon-toxin, only Madin-Darby canine kidney (MDCK) cells and Caucasian renal leiomyoblastoma (G-402) cells are sensitive enough for useful investigation of the toxin (4, 32, 40). The MDCK cells are about 100-fold more sensitive than the G-402 cells and have been used in most cultured cell studies of epsilon-toxin. In MDCK cells, epsilon-toxin was found in high-molecular-weight complexes and induced increased membrane permeability to ions and small hydrophilic solutes (33). It forms an aqueous anion-selective pore permeable to solutes up to 1 kDa (35). Analysis of labeled constructs derived from epsilon-toxin clearly demonstrated that it forms heptamers when incubated with rat brain synaptosomal membranes and that cleavage of a C-terminal peptide is essential for oligomerization (26). Also in MDCK cells, epsilon-toxin associates with apical membrane in preference to basolateral membrane and forms a heptameric pore associated with detergent-resistant domains (27, 34). While it has relatively low amino acid sequence identity (ca. 14%) to the heptameric pore-forming toxin aerolysin from Aeromonas hydrophila, it does have three-dimensional structural similarity to aerolysin, one of a family of toxins known as -pore-forming toxins (8). Epsilon-toxin also has some amino acid sequence identity to a family of mosquitocidal toxins typified by Mtx2 and Mtx3 of Bacillus sphaericus (21% and 24% identity, respectively) (9, 22, 37). Because only some cultured cells are highly sensitive to the toxin it has been suggested that a specific host receptor is essential for binding and pore formation, but no specific receptor molecule has been found. A recent study demonstrated that experimental development of tolerance to epsilon-toxin by MDCK cells was associated with loss of expression of a group of membrane proteins, lending further support to the hypothesis that host proteins mediate epsilon-toxin binding and toxicity (4). Because of the assumed association of epsilon-toxin with vascular endothelium, development of an endothelial model is highly desirable. However, cultured aortic endothelial cells (goat, sheep, and cattle) failed to show any morphological response to epsilon-toxin with up to 48 h of exposure, even at concentrations much higher than were toxic for MDCK cells (40). The latter observation brought into question whether the in vivo toxicity of epsilon-toxin is due to direct action of the toxin with vascular endothelium.
Because the cultured endothelial cells were not morphologically affected by epsilon-toxin, in this study we have used single perfused microvessels in rat mesentery, which enable direct observation of permeability properties of the vascular wall during exposure to toxin under controlled conditions. While the effects of epsilon-toxin in tissue have been investigated using morphological and histochemical techniques to demonstrate accumulations of fluid and labeled proteins, there are no reports in the literature of biophysical permeability coefficients determined from directly measured fluxes and controlled driving gradients. Moreover, by presenting the toxin directly to the endothelium within an artificial perfusate, the technique enables investigation of the endothelial response in the absence of potential systemic mechanisms or interactions with blood plasma. Variations of the single perfused microvessel model have been used to investigate vascular permeability coefficients regarding convective water flow and permeability of both small and large solutes (19, 24, 25), regulation of inflammatory mechanisms (3, 16), ultrastructure of the endothelial cleft (2), and regulation of the adherens junction complex (1, 42). Here we determine the hydraulic conductivity coefficient (Lp) as a measure of the response to epsilon-toxin. We demonstrate that microvessels of the rat mesentery are highly sensitive to epsilon-toxin, and we describe an experimental system in which to investigate the direct action of bacterial toxins with vascular endothelium.
MATERIALS AND METHODS
Animal preparation. Experiments were carried out on rats (male, Sprague-Dawley, 350 to 450 g; Hilltop Laboratory Animals, Inc.) anesthetized with pentobarbital (65 mg kg–1 body weight given subcutaneously). Anesthesia, assessed by toe-pinch reflex, was maintained by giving additional pentobarbital (subcutaneous, 3 mg per dose) as needed. At the end of experiments, animals were euthanized with pentobarbital overdose. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Each rat was placed on a heating pad to maintain normal body temperature. A midline surgical incision was made in the abdominal wall, and the gut gently was taken from the abdominal cavity to spread the mesentery over a quartz pillar for transillumination and observation on the fixed stage of an inverted microscope. The upper surface of the mesentery was continuously superfused with Ringer's solution maintained at 35°C to 37°C during preparation and experimentation. Because our present techniques are not suited for use of vessels smaller than about 15 μm in diameter, we have not investigated the properties of true capillaries (diameter of ca. 6 μm). Therefore, experiments were performed on straight nonbranched segments of venular microvessels typically 25 to 40 μm in diameter and usually between 700 and 900 μm in length. Prior to cannulation, all vessels selected for experiments had brisk blood flow and were free of leukocytes sticking or rolling on the vessel wall.
Measurement of Lp of the microvessel wall. All measurements were based on the modified Landis microperfusion technique, which enables measurement of the volume flux of water across the wall of a microvessel due to a known pressure difference between the lumen and the interstitium. The technique and its assumptions and limitations have been evaluated in detail (2, 24). Briefly, each vessel was cannulated by the tip of a glass micropipette filled with a physiological perfusate additionally containing flow marker erythrocytes (ca. 4%, vol/vol). The micropipette was positioned in the vessel to achieve free downstream perfusion with no leak to the upstream direction or to the tissue. The rear of the micropipette was in communication with a water manometer used to set the hydrostatic pressure and drive flow from the micropipette through the cannulated vessel. To measure the initial transcapillary water flow per unit area of the capillary wall (Jv/A), a glass microoccluder was lowered onto the microvessel up to 900 μm from the cannulation site, with the hydraulic pressure set at predetermined values of 30 to 60 cm H2O. When the vessels were occluded, the intravascular hydraulic pressure was equal to that of the manometer, and the motion of flow marker erythrocytes was recorded on videotape for later analysis. Microvessel Lp was calculated as the slope of the relation between Jv/A and applied hydraulic pressure. For most experiments, Jv/A was estimated from each of several occlusions using one hydraulic pressure, with the assumption that the net effective pressure determining fluid flow was equal to the applied hydraulic pressure minus 3.6 cm H2O, the approximate osmotic pressure contributed by bovine serum albumin (BSA) at 10 mg ml–1. Experimental reagents (e.g., epsilon-toxin) were added to the perfusate in the respective experiments and delivered to the vascular lumen via the micropipette continuously during Lp measurement. Changes in perfusate were accomplished by withdrawing the initial micropipette and replacing it with a second micropipette filled with new perfusate solution of the appropriate composition.
Preliminary experiments were designed to determine the effective osmotic pressure (eff) of albumin (30 mg ml–1) in the perfusate. Before each measurement the hydraulic pressure in the vessel was held at 60 cm H2O for 30 s by partial downstream occlusion to establish a high-filtration steady state in which the BSA concentration on the downstream side of the selectivity barrier was washed down to a minimal level, thus maximizing the effective oncotic pressure difference (24, 25). Jv/A was measured by monitoring the motion of flow marker erythrocytes during brief (5 to 8 s) full occlusions. With 60 cm H2O hydraulic pressure, erythrocytes moved toward the occlusion site as fluid moved out of the vessel. Measurements of Jv/A at microvessel pressure of 10 cm H2O were made after setting up steady-state filtration at 60 cm H2O, occluding, and rapidly (<0.5 s) switching the applied hydraulic pressure to 10 cm H2O. Reabsorptive water movement was seen as marker erythrocytes moved away from the occlusion site toward the cannulation site as fluid moved into the vessel. Three to five measurements were made at each pressure, yielding a control relationship between Jv/A and applied hydraulic pressure. The slope of the relationship is the hydraulic conductivity (Lp), and the intercept on the pressure axis is eff of the serum albumin. Our test solutions of BSA (10 or 30 mg ml–1) had protein osmotic pressures of 3.6 and 14 cm H2O at 37°C (calculated from published equations [23]).
Experimental protocols. Each vessel was initially perfused with a control solution containing BSA at 10 mg ml–1 in Ringer's solution (except preliminary experiments as indicated). Usually between 5 and 10 occlusions at 50 cm H2O over 15 to 20 min were used to establish a control Lp. Then, the first pipette was removed and a second pipette containing the test solution was introduced at the same cannulation site. Multiple occlusions over the subsequent 15 to 120 min enabled estimation of Lp during exposure to the test solution. Occlusions were made every 20 to 30 s during the first 5 min of test perfusion to check for rapid initial change in Jv/A and then made less frequently (three to five occlusions every 5 to 10 min) for the remainder of the experiment.
Statistics. We tested for differences from basal Lp due to the perfusion with epsilon-toxin by comparing the averaged Lp in each experimental group with the corresponding time-matched value found in a control (no-toxin) group of vessels. Because basal Lp distribution is non-Gaussian, we used the nonparametric Mann-Whitney statistic and assumed significance for a P value of <0.05. Throughout, descriptive statistics are reported as the mean ± the standard error of the mean (SEM).
Ultrastructure. To investigate the structural nature of the response to toxin, four vessels exposed to epsilon-toxin (two using 3 μg ml–1 and two using 10 μg ml–1) were fixed after a substantial rise in Lp (25 to 40 min of toxin exposure). Two other vessels were perfused with vehicle solution (control vessels) for a comparable time and also inspected using the following methods. After the final determination of Jv/A, fixation for electron microscopy was started by dripping ice-cold glutaraldehyde (3% in 0.1 mM cacodylate buffer) on the mesentery. This was done while vessels were still perfused and occluded to ensure that the final perfusion conditions and geometry were maintained during fixation. After 1 to 2 min the perfusion pipette was removed while immersion fixation continued. A diagram of the area enabled later identification of the perfused vessel segment. After 10 min in situ fixation, tissue was cut away from the animal and glutaraldehyde fixation continued on ice for a total of 1 h before changing to OsO4 (1% in veronal acetate, pH 7.2) for 1 h. Tissue was treated with tannic acid (0.5% in maleate buffer, pH 6) for 30 min to enhance membrane staining and placed in uranyl acetate (2% in maleate buffer, pH 6) overnight. After dehydration in a graded series of acetone, the tissues were flat embedded in epoxy resin. Sections (50 nm thick) from transversely sectioned vessels were collected on Formvar-coated slots and were stained with uranyl acetate and lead citrate. They were examined using transmission electron microscopy (TEM) with a Philips CM120 at 80 kV.
Solutions and reagents. Mammalian Ringer's solution was composed (in mM) of 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES and Na-HEPES. The ratio of acid-HEPES to Na-HEPES was adjusted to achieve pH 7.40 to 7.45 for Ringer's solutions. All perfusates were mammalian Ringer's solution additionally containing BSA (A4378; Sigma-Aldrich) at 10 or 30 mg ml–1 as indicated.
Epsilon-prototoxin was prepared from an overnight culture of C. perfringens type D (strain NCTC 8346) in Trypticase-yeast-glucose medium, under anaerobic conditions at 37°C. The overnight cultures were centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant containing epsilon-toxin was saved for toxin purification. The toxin was then precipitated by ammonium sulfate. Two columns were prepared with DEAE and CM Sepharose (Pharmacia, Sweden), respectively, equilibrated in 10 mM Tris, pH 7.5. The toxin was applied to the DEAE column, and the effluent was monitored at 220 nm. The initially eluted peak was saved and applied to the CM column. Again the effluent was monitored at 220 nm, and the first peak was collected, dialyzed against distilled water, and freeze-dried. This product was shown to contain pure epsilon-prototoxin when tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prior to its use in these experiments, the prototoxin was reconstituted in Ringer's and activated by incubation at 37°C during 30 min with 0.1% trypsin (Sigma).
To further establish specificity of the Lp response to toxin, we tested the effectiveness of an epsilon-toxin neutralizing mouse monoclonal antibody (immunoglobulin G; 5B7) provided by Paul Hauer (Center of Veterinary Biologics, Ames, Iowa). The antibody (final concentration, 0.13 mg ml–1) was added to perfusion solution containing epsilon-toxin (3 μg ml–1) and incubated for 30 min at room temperature before use. The effect on microvascular Lp was measured and compared to the time-matched set of measurements made with epsilon-toxin in the absence of antibody.
RESULTS
Epsilon-toxin increases microvascular Lp. We perfused single microvessels of rat mesentery with solutions containing epsilon-toxin to test for increased Lp as an indication of endothelial cell intoxication. In a few preliminary experiments the perfusate contained 30 mg ml–1 BSA, enabling determination of the effective protein osmotic pressure difference across the vessel wall. The data from a representative experiment illustrate that during control measurement in the absence of toxin the Lp (slope of the relation between Jv/A and applied pressure) was low and the effective protein osmotic pressure (intercept on the pressure axis) was between 10 and 20 cm H2O (Fig. 1). After removing the initial pipette and perfusing with a second pipette containing toxin (10 μg ml–1), the Lp increased over time and the effective protein osmotic pressure fell to near zero. Both changes are consistent with loss of endothelial barrier function. This initial technique required multiple measurements, typically taking several minutes at various pressures. When permeability changed rapidly, it was difficult to make estimates of the intercept on the pressure axis. Therefore, in remaining experiments the BSA concentration was 10 mg ml–1 and estimates of Lp were made using one applied pressure (usually 50 or 60 cm H2O) as described in Materials and Methods. The latter technique better enabled our following of rapid Lp changes.
A representative experiment shows the increase in Lp with time associated with toxin exposure (Fig. 2A). Perfusion with vehicle control solution for 30 min established the control Lp of 0.3 x 10–7 cm s–1 cm H2O–1. The initial micropipette was removed, and the vessel was cannulated a second time and perfused with toxin (3 μg ml–1) for the next 40 min (Fig. 2A). During the first 5 min of perfusion with toxin, there was no apparent change in Lp. However, within 10 min of exposure to epsilon-toxin the Lp increased to about 2 x 10–7 cm s–1 cm H2O–1. It continued to increase and was above 15 x 10–7 cm s–1 cm H2O–1 by 40 min of toxin exposure. Representative data from a vessel perfused for 90 min without toxin illustrate that the perfusion technique alone does not increase Lp (Fig. 2B). Averaged data from groups of experiments are described next.
To test the concentration dependence of the sensitivity to epsilon-toxin in the microvasculature, the Lp response to toxin over time was tested in four different groups of vessels (Fig. 3A). A separate group of vessels perfused without toxin for 90 min demonstrated that Lp did not change in that group (n = 15) (Fig. 3B). The Lp values from that group were used as time-matched control values for the toxin-perfused vessels. At the lowest toxin concentration (0.3 μg ml–1) no increase in Lp was observed for up to 90 min (n = 7). At 10 μg ml–1 the Lp increased rapidly, on average to more than 15 times the control value within 10 min, and was significantly greater than the time-matched control group at all times (P < 0.05, Mann-Whitney). At intermediate concentrations the response to epsilon-toxin exposure took longer to develop and the mean values of Lp did not reach as high. The threshold concentration for a measurable Lp response within 60 min was 1 μg ml–1 (significantly greater than time-matched control group at 50 min and longer). In two experiments, after the Lp had significantly increased during epsilon-toxin exposure, each vessel was recannulated with the control perfusate and Lp measurements were made for 20 min to test for recovery from the epsilon-toxin. The Lp in these vessels did not return toward control; the effect of epsilon-toxin to increase Lp was irreversible.
Antibody neutralization of the toxin effect on Lp. A separate group of vessels were perfused with solution containing a neutralizing monoclonal antibody (0.13 mg ml–1) to epsilon-toxin in addition to epsilon-toxin (3 μg ml–1). In perfusions lasting up to 40 min, the Lp in these vessels did not increase. Lp measured over time is shown in comparison with the mean response to the same concentration of toxin alone in Fig. 4. Over this time the antibody completely prevented the increase in Lp induced by toxin in the absence of antibody.
Ultrastructure of microvessels exposed to toxin. Two vessels of the group perfused with 3 μg ml–1 epsilon-toxin and two of the group perfused with 10 μg ml–1 epsilon-toxin were examined with TEM. The two vessels exposed to 3 μg ml–1 toxin (25 and 40 min, respectively) reached an Lp about 5 times the control value just before fixation; in the two vessels exposed to 10 μg ml–1 epsilon-toxin (25 and 35 min, respectively), Lp had increased to 13 and 24 times the control Lp when fixed. Features consistent with direct endothelial damage were observed in all epsilon-toxin-treated vessels (Fig. 5). Most endothelial cells were very attenuated, showing loss of cytoplasm, although the cytoplasmic membrane appeared intact in most cases (Fig. 5A, B, C, and G). Different degrees of detachment from the basement membrane were also observed. A few cells were also fragmented, with some of the fragments remaining attached to the basement membrane. Thus, in places the endothelium was discontinuous, exhibiting gaps either between or through the endothelial cells (Fig. 5A, B, D, and E). Near one such gap the subendothelial space between the basement membrane and pericyte was expanded and filled with flocculent material, possibly protein from disrupted cells (Fig. 5A and B). Sections of the basement membrane were exposed to the vascular lumen, although the basement membrane in these areas and elsewhere appeared intact. Some of the pericytes were also densely stained and highly attenuated (Fig. 5A and B). Most endothelial cells had angular shapes, and the cytoplasmic membrane presented an irregular outline, with numerous indentations and a few cytoplasmic blebs consisting of flocculent relatively electron-lucent material devoid of organelles and surrounded by cytoplasmic membrane. Most endothelial cells showed a diffusely electron-dense cytoplasm in which it was impossible to distinguish any cytoplasmic detail. A few endothelial cells, however, showed a clear cytoplasm in which dilated cisterns of endoplasmic reticulum were observed. Very few ribosomes were seen attached to the endoplasmic reticulum membranes. A few membrane-bound vacuoles with myelinic figures in front of a clear background were also seen in the cytoplasm of some cells. The nuclei of some endothelial cells were shrunken and showed large clumps of electron-dense chromatin. A few cells showed diffuse dense chromatin occupying the entire nucleus. Nuclear membranes were unaffected in most cases. Endothelial cells of control vessels perfused for comparable times with vehicle solutions in the absence of toxin did not show ultrastructural abnormalities, such as gaps in the endothelium or necrotic characteristics (Fig. 6). Specifically, the endothelium was in contact with basement membrane and formed a continuous intimal layer with intact junctional complexes. The peripheral cytoplasm was of moderate electron density and was relatively uniform in thickness (0.1 to 0.2 μm).
DISCUSSION
We set out to test the specific hypothesis that epsilon-toxin would increase the hydraulic permeability of microvessels in the rat mesentery and to answer the more general question of whether the single perfused vessel model could be used to investigate epsilon-toxin. The data demonstrated a time- and dose-dependent irreversible increase in convective water flux across the vessel walls in response to exposure to activated epsilon-toxin. Many endothelial cells from toxin-treated vessels appeared necrotic, highly attenuated, and with dense cytoplasm, indicative of extreme fluid loss associated with necrosis. Patent pathways through the endothelial layer were seen associated with fluid accumulation in the subendothelial spaces. Such pathways are never seen in control endothelium and represent a very high conductance path for both plasma water and protein. The results are consistent with the hypothesis that epsilon-toxin interacts with endothelial cells of rat venular microvessels and increases the vessel wall permeability by direct damage of the endothelium. The answer to the broad question of whether the rat mesentery model might be useful to study endothelial interaction with blood-borne toxins such as epsilon-toxin is clear. The single perfused microvessel model does enable investigation of relatively small quantities of toxin with mammalian microvessel endothelium.
While this study is the first to measure biophysical permeability coefficients in response to epsilon-toxin, there are numerous previous reports indicating fluid and protein extravasation resulting from epsilon-toxin toxemia. Edema and excess protein extravasation in various tissues have been noted at necropsy in experimentally and naturally intoxicated animals (12, 14, 30, 38, 39, 43). Morphological and histochemical methods provide endpoint observations but do not enable finer temporal characterization of the progress of intoxication. Moreover, because the toxin has been shown to stimulate vascular smooth muscle contraction and elevate arterial pressure, it is very difficult to separate changes in vessel wall permeability, tissue perfusion, and intravascular pressure in whole-animal models (28, 36). Under the conditions of our experiments, the intravascular hydraulic pressure and protein osmotic pressure are directly controlled, while extravascular hydraulic pressure and protein osmotic pressure are negligible (2, 20). The postcapillary microvessels used in this study are not invested with smooth muscle cells and maintain a constant diameter under the conditions of these experiments. Individual measurements in the single perfused vessel, taking between 5 and 8 seconds, enable characterization of permeability changes with temporal resolution comparable to cell culture methods used to measure transepithelial resistance, small solute flux, or hydraulic conductivity (18, 34, 41).
The single vessel technique was used here to measure an increase in water flux crossing the microvessel wall resulting from epsilon-toxin exposure and was also used to infer loss of the barrier to serum albumin (decrease of effective protein osmotic pressure shown in Fig. 1). Such changes in each of these parameters are directly important in the development of edema. However, one cannot specify what level of Lp measured in a single vessel would be correlated with organ failure or death. We can say that the ultrastructural examination of all vessels perfused with epsilon-toxin revealed endothelial cells that were severely damaged and regions of basement membrane exposed directly to the perfusate. These endothelial gaps were not found in control vessels. The latter control data agree with results of a much larger examination of normal endothelium in a recent publication from our group in which nearly 700 micrographs were analyzed for tight junction structure using rat mesentery microvessels that were perfused for 15 to 30 min with vehicle solutions similar to that in the present study (2). We also noted that once the Lp began to increase, removing perfusate containing toxin and reverting to control perfusate failed to return the Lp toward control. Indeed, in many instances when the perfusing pipette was removed at the end of an experiment and blood was allowed to return to the vessel, we observed hemostasis in vessels that had been perfused with toxin; vessels not perfused with toxin almost always resumed normal blood flow.
We also note that the effects of epsilon-toxin were not similar to an acute inflammatory response, such as that stimulated by platelet activating factor or other acute inflammatory mediators. The typical response of mesenteric postcapillary venules to platelet activating factor is an increase to a peak value within 10 to 15 min followed by a recovery phase lasting another 10 to 60 min (3). Similar patterns have been described for the inflammatory mediators histamine (21) and bradykinin (15) in these vessels. Because the epsilon-toxin effect exhibited a continuous Lp increase and did not return toward control, it did not have the characteristic pattern associated with an acute inflammatory response.
Previous studies of epsilon-toxin using MDCK cells have examined parameters different from the water flux and loss of protein osmotic pressure measured in the present study. Loss of intracellular K+ (29, 33, 35), decreased cell viability (27, 29, 33-35), increased entry of propidium iodide (34, 35), and a fall in trans-epithelial resistance (34) all have been used to infer epsilon-toxin toxicity toward MDCK cell monolayers. None of these measures are directly comparable to increased Lp in microvessels. However, all these measures are indicative of damaged cell membrane and were correlated with compromised epithelial monolayer barrier function. The MDCK cell model is particularly sensitive to epsilon-toxin, and the concentrations of epsilon-toxin that elicited effects in the cited MDCK studies ranged from near 10–10 M to 10–8 M. In the in vivo mesentery microvessel, we found a threshold effect at 1 μg ml–1 (approximately 3 x 10–8 M) when measuring increases in Lp. Endothelial gaps seen with TEM and correlated with increased Lp could have formed as endothelial tight and adherens junctions released or could have resulted from cell disruption. Some clostridial toxins inactivate small GTPases, leading to the breakdown of adherens and tight junctions in both epithelial and endothelial cells (1, 5, 42). In a recent study using MDCK monolayers, at a time and dose of epsilon-toxin that elicited a complete loss of trans-epithelial resistance, the monolayer permeability to a solute (dextran 38500) restricted to the paracellular pathway was not increased and the immunofluorescent distribution of peripheral band actin and components of the tight junction complex was not different from control (34). These data indicated that direct cell disruption by pore formation on the apical membrane preceded any possible direct effect on the junctional complex. Thus, it is likely that the broad areas of basement membrane exposed during epsilon-toxin treatment of the mesentery microvessels result from the disruption of the endothelial cells rather than from disassembly of cell-cell junctions.
The present results using in vivo endothelium and epsilon-toxin concentrations up to 10 μg ml–1 contrast with those of our previous study, in which the toxin at up to 120 μg ml–1 did not induce morphological changes in cultured aortic endothelial cells (tested from cattle, goats, and sheep) (40). One possibility for this difference was that activity of the toxin toward endothelium was dependent on a factor in blood not present in the culture medium. In our previous study we addressed that possibility by testing for toxicity against the endothelial cells in the presence as well as the absence of serum proteins, but we found that in either case there was no morphological change induced by the toxin. While most of the present experiments were carried out in physiological saline (Ringer's) solution supplemented with bovine serum albumin, in several experiments in which toxin was diluted into Ringer's solution without BSA, epsilon-toxin also induced large increases in Lp (not shown). Therefore, our results indicate that no other component of circulating blood is required for toxicity against the mesenteric venular microvessel endothelium.
A specific receptor for epsilon-toxin has not been identified. However, the presence of a receptor, particularly in brain and kidney, has been suggested by uptake of labeled toxin (6, 30). Also, in vitro studies showed that treatment of rat brain homogenates with neuraminidase or lipase reduced binding of epsilon-toxin, indicating that a receptor could be a sialoglycoprotein within a specific lipid environment (31). Following these earlier studies, recent work demonstrated that heptamers of epsilon-toxin were found exclusively associated with the detergent-resistant membrane fraction from MDCK cells, further demonstrating that epsilon-toxin binding is dependent on specific cholesterol-containing microdomains, possibly lipid rafts or caveolae (27). Therefore, it is possible that the aortic endothelial cells which showed no morphological response to epsilon-toxin did not express the appropriate receptor or associated microdomain. We cannot say at present whether those cells express such a domain in vivo or could do so under different culture conditions. The demonstration in the present experiments of the sensitivity of postcapillary microvessel endothelium to epsilon-toxin suggests that cultured endothelial monolayers from other sources or under different culture conditions should be tested and that an appropriate endothelial culture model could be found.
In our experiments we also noted damage to pericytes of the toxin-perfused microvessels. While some studies have suggested that pericytes promote vascular permeability through contractility, recent studies have focused on the role of pericytes in the maturation of microvessels (17). Loss of pericytes, in addition to endothelial cells, due to epsilon-toxin could potentially exacerbate local permeability increases and delay return to normal vascular function in cases of sublethal intoxication.
The present study uses the mesenteric microvessel as a model for the direct vascular effects of epsilon-toxin. However, the principal lethal pathology in sheep is associated with brain and lungs. The traditionally accepted model to explain brain lesions due to epsilon-toxin (6) suggests that such lesions are due to the action of this toxin on endothelial cells, which generates perivascular edema followed by hypoxia and necrosis of the perivascular tissues. However, a recent study showed that epsilon-toxin can also leak into the brain and act directly on rat neurons, producing degeneration and necrosis of these cells in cases of subacute or chronic epsilon-toxin intoxication (11). The results of the present experiments confirm that direct damage does occur in endothelial cells exposed to epsilon-toxin. However, it is possible that epsilon-toxin acts both on endothelial cells and neurons alike. Moreover, it is possible and as yet unknown whether differences exist between various vascular beds in regard to toxin sensitivity and pathogenesis.
Summary. We have shown that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromises the barrier function of venular microvessel endothelium in the rat mesentery. The toxin appeared to directly damage vascular endothelium, inducing formation of gaps in the endothelial cell layer correlated with loss of effective protein osmotic pressure and large increases in measured Lp. These changes are consistent with those expected to lead to rapid edema formation and would also promote access of the toxin to cells in the surrounding tissue. The single perfused vessel model will enable further investigation of epsilon-toxin interaction with the vascular endothelium in situ.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants from the National Heart, Lung, and Blood Institute (HL28607 and HL44485) and National Institute of Allergy and Infectious Diseases (AI56177).
We gratefully acknowledge the expert technical work of Joyce Lenz.
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