Simvastatin Inhibits Inflammatory Properties of Staphylococcus aureus α-Toxin
Background— Simvastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, has been shown to lower serum cholesterol levels in clinical use. Moreover, statins exert beneficial effects in vascular diseases by inhibition of leukocyte rolling, adherence, and transmigration. The aim of this study was to determine if pretreatment with simvastatin attenuates Staphylococcus aureus α-toxin–induced increase in leukocyte-endothelial interactions during exotoxemia.
Methods and Results— The effects of simvastatin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation. Simvastatin (50 or 100 μg/kg) was administered 18 hours before the study. Activation of microcirculation was induced by bolus administration of 40 μg/kg S aureus α-toxin. Exotoxemia resulted in a significant and time-dependent increase in leukocyte rolling, adherence, and transmigration of leukocytes as well as P-selectin expression on the intestinal vascular endothelium. Pretreatment with simvastatin significantly inhibited exotoxin-induced leukocyte rolling from 71±10 to 14±4.7 cells/min (P<0.01) and adherence from 14±3.5 to 0.4±0.2 cells (P<0.01). In addition, simvastatin pretreatment significantly inhibited transmigration of leukocytes from 10.5±1.2 to 4.2±0.9 (P<0.05) cells. Immunohistochemical detection of endothelial cell adhesion molecule P-selectin showed a 50% decrease in endothelial cell surface expression after simvastatin treatment. Furthermore, simvastatin treatment resulted in enhanced expression of endothelial cell NO synthase III in the intestinal microcirculation.
Conclusions— These results demonstrate that simvastatin interferes with exotoxin-induced leukocyte-endothelial cell interactions, which may be relevant in various infectious diseases. Statin treatment may offer a new therapeutic strategy for these clinical conditions.
Received May 30, 2002; revision received July 18, 2002; accepted July 18, 2002.
Polymorphonuclear leukocytes (PMNs) are believed to play a critical role in inflammatory states like bacterial-induced sepsis and local or systemic infectious diseases like endocarditis. Activated PMNs can release oxygen-derived free radicals, including superoxide anions, hydrogen peroxide, and proteolytic enzymes.1 The interaction between PMNs and the vascular endothelium is assumed to be a key event in the inflammatory process. After activation, leukocytes start to roll along the vascular endothelium mediated by P-selectin on the endothelial surface and constitutively expressed L-selectin on neutrophils.2 The counter-receptors for the selectins are considered to be carbohydrate-containing molecules such as sialyl Lewisx, sialyl Lewisa, or PSGL-1. Cooperation of P-selectin and platelet activating factor (PAF) expressed on the endothelium results in activation of the rolling the leukocytes with shape change, shedding of L-selectin, and upregulation of CD11/CD18 as prominent events.3 This leads to tight adhesion of leukocytes and endothelial cells. β2-integrins (CD11/CD18) on the leukocytes and intracellular adhesion molecule-1 on the vascular endothelium are responsible for the firm attachment, subsequent transendothelial migration,4 and recruitment of leukocytes into injured tissue,3 the end result of which contributes to additional tissue injury in inflammatory states.5
See p 2041
In this context, P-selectin and intracellular adhesion molecule-1 are upregulated on endothelial cells after activation with proteinaceous exotoxin (ie, Staphylococcus aureus α-toxin and Escherichia coli hemolysin).6 α-Toxin, prototype of a pore-forming exotoxin, is the major cytotoxin of Staphylococcus aureus.7–9⇓⇓ Purified α-toxin is known to provoke cardiovascular collapse in intact animals or isolated hearts, causing strong activation and mediator release of various cell types.10–14⇓⇓⇓⇓
The discovery of the 3-hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in the 1980s and their clinical use as therapeutic agents15 has resulted in widespread treatment of hyperlipidemia and coronary artery disease. The major mechanism of the HMG-CoA reductase inhibitors (ie, simvastatin, lovastatin, and pravastatin), commonly referred to as statins, is the inhibition of cholesterol synthesis in the liver by blocking the conversion of HMG-CoA to mevalonate, the rate-limiting step in cholesterol biosynthesis. Recently, statins have been reported to protect endothelial function in the absence of hypercholesterolemia in isolated endothelial cells16 and in intact organisms.17,18⇓
Because of the important role of leukocyte-endothelium interaction during inflammatory states,19 our study was designed to investigate the effects and mechanisms of the prototype statin simvastatin on leukocyte-endothelial cell interactions in vivo, using exotoxemia for mimicking septic conditions.
Intravital Microscopy of the Rat Mesentery
Male Sprague-Dawley rats, weighing 250 to 275 g, were anesthetized with sodium pentobarbital (30 to 60 mg/kg) injected intraperitoneally. A tracheotomy was performed to maintain a patent airway throughout the experiment. A polyethylene catheter was inserted in the left carotid artery to monitor mean arterial blood pressure by a Hellige Servomed (Hellige).20 All experiments were approved by the state and the Johannes Gutenberg-University Animal Care Committee.
A loop of ileal mesentery was exteriorized through the midline incision and placed in a temperature-controlled fluid-filled plexiglass chamber for observation of the mesenteric microcirculation via intravital microscopy, as described previously.20 The ileum and mesentery were superfused throughout the experiment with a buffered saline solution warmed to 37°C. A microscope and a ×40 water immersion lens (Zeiss-Axioplan) were used to visualize the mesenteric microcirculation and the mesenteric tissue. The image was projected by a CCD video camera onto a high-resolution monitor, and the image was recorded with a videocassette recorder. All images were then analyzed using computerized imaging software. Red blood cell velocity was determined online using an optical Doppler velocimeter (Microcirculation Research Institute) to calculate shear rates. This method gives an average red blood cell velocity, which is digitally displayed and allows calculation of shear rates [formula: shear rate g=8(Vmean/D); (Vmean=Vrbc/1.6, red blood cell velocity (V), venular diameter (D)].21
The rats were allowed to stabilize for 20 to 30 minutes after surgery. After stabilization, a 30- to 45-μm-diameter postcapillary venule was chosen for observation. The number of rolling and adherent leukocytes was determined offline by playback analysis of videotape. Leukocytes were considered to be rolling if they were moving at a velocity significantly slower than the red blood cells and expressed as the number of cells moving past a designated point per minute. A leukocyte was judged to be adherent if it remained stationary for >30 seconds.22 Adherence is expressed as the number of leukocytes adhering to the endothelium per 100 μm of vessel length. To quantify the number of transmigrated leukocytes, the tissue area adjacent to the 100-μm length of postcapillary venule over a distance of 20 μm from the vessel wall was used. The number of extravasated leukocytes was counted and normalized with respect to this area.
Immunolocalization of P-Selectin and Endothelial Cell NO Synthase in the Microvasculature
After completion of intravital microscopy, the immunohistochemical localization of P-selectin was determined in ileal samples, as described above.20 A segment of ileum was isolated from the intestine and fixed in 4.5% paraformaldehyde in PBS (pH 7.0). After dehydration, the sections were embedded and cut to 5-μm tissue sections. Immunohistochemical localization of P-selectin was accomplished using the avidin/biotin immunoperoxidase technique (Vectasin ABC Reagent, Vector Laboratories), using a monoclonal antibody against P-selectin (Pharmingen, Hamburg, Germany) exposed on the endothelial cell surface and expressed as percentage of positively staining venules. Similarly, immunohistochemical analysis of constitutive endothelial cell NO synthase (NOS III) was performed using a monoclonal antibody against constitutive ecNOS (NOS III, Pharmingen, Hamburg, Germany) expressed in the vascular endothelium. Because ecNOS is expressed constitutively, we used an intensity score from 0 to 5, where 0 expressed no staining and 5 expressed intense staining. Intensity was calculated from sections of the vehicle- and simvastatin-treated intestinal tissue.
Rats were randomly divided into one of the following groups: (a) buffer-superfused mesenteries (control group); (b) rats injected with 50 μg/kg simvastatin in 1.0 mL 0.9% NaCl IP 18 hours before intravital microscopy; (c) rats injected with 100 μg/kg simvastatin as above; or (d) rats injected with 1.0 mL of 0.9% NaCl as above. A baseline recording was made to establish basal values for leukocyte rolling, adherence, and transmigration (time 0). Immediately thereafter, in groups b, c, and d, 40 μg/kg S aureus α-toxin (provided by Professor Bhakdi, Mainz, Germany, isolation was performed as described previously23) or its vehicle (NaCl 0.9%) were administered systemically as bolus injection. Video recordings were made at 30, 60, 90, and 120 minutes after initiation of exotoxemia for quantification of leukocyte rolling, adherence, and transmigration.
In a second set of experiments, rats were anesthetized with pentobarbital, and a polyethylene catheter was inserted in the left carotid artery. Mean arterial blood pressure and heart rate were recorded continuously over 4 hours. Rats were randomly divided into one of two groups: vehicle rats treated with 40 μg/kg S aureus α-toxin or rats pretreated with 100 μg/kg simvastatin in 1.0 mL 0.9% NaCl IP 18 hours before intravenous injection of 40 μg/kg S aureus α-toxin. Hemodynamic monitoring and survival rate were determined. Administration of 100 μg/kg simvastatin intraperitoneally did not change serum cholesterol or subtype (LDL or HDL) levels.
All data are presented as mean±SEM. Data were compared by ANOVA using post hoc analysis with Fisher’s corrected t test. P≤0.05 was considered statistically significant.
We found no significant differences in the initial mean arterial blood pressures (MABPs) among the experimental groups of rats after all surgical procedures (range, 101 to 123 mm Hg in all groups). S aureus α-toxin injection only slightly diminished MABP and increased heart rate, but this effect was statistically not significant (Table). Similar, no significant differences were observed for the shear rates in the different groups (range, 630±50 to 720±60 s−1). Thus, the adhesive interactions observed between leukocytes and endothelial cells were not attributable to any hemodynamic factors.
A low baseline number of rolling leukocytes was observed in the mesenteric microvasculature for all experimental groups. After infusion of vehicle, PMN rolling and adherence did not change significantly. However, infusion of S aureus α-toxin (40 μg/kg) resulted in a rapid and significant increase in leukocyte rolling compared with buffer-control animals (Figure 1). The increase in leukocyte rolling was statistically significant 30 minutes after injection of S aureus α-toxin and reached values from 7- to 8-fold above baseline values at 120 minutes (P<0.01). The increase in leukocyte adherence was statistically significant 30 minutes after toxin administration (P<0.05 versus control rats), reaching values from 6- to 7-fold above baseline values at 120 minutes (P<0.01) (Figure 2).
Pretreatment with simvastatin significantly inhibited both leukocyte rolling and adherence by virtually almost complete inhibition after α-toxin administration. The effects of simvastatin on the time course of S aureus α-toxin–induced rolling and adherence are shown in Figures 1 and 2⇑. Administration of 50 and 100 μg/kg simvastatin significantly inhibited the number of rolling and adherent leukocytes in a dose-dependent manner. Furthermore, the range of rolling velocity (10 to 100 μm/s) among the different groups did not change. Interestingly, when pretreatment with simvastatin was performed just 1 hour before exotoxin challenge, no protective effects could be observed. Similarly, intravenous administration of the P-selectin–neutralizing monoclonal antibody humanized PB1.3 (1 mg/kg) significantly decreased leukocyte rolling and adherence after S aureus α-toxin challenge similar to the degree of simvastatin-treated animals (68.4±9.3 versus 19.4±4.2 rolling PMN, 16.3±2.6 versus 2.9±1.2 adhering PMN, P<0.05). Furthermore, we could confirm the reduction of cell-cell interaction when animals were pretreated with simvastatin and mesenterium was superfused with thrombin.
In rats receiving vehicle injection, very few transmigrated leukocytes were observed at 120 minutes in the mesenteric extravascular space (Figure 4). However, in rats injected with S aureus α-toxin (40 μg/kg), the number of migrated leukocytes in the surrounding tissue was markedly increased 4- to 5-fold. Pretreatment with 100 μg/kg simvastatin 18 hours before study significantly attenuated the number of extravasated leukocytes after toxin injection (10.5±1.2 to 4.2±0.9, P<0.05). In histological analysis, we predominantly observed extravasation of neutrophils at the site of venules and only few lymphocytes and eosinophils.
When we observed the hemodynamic effects in the second set of experiments over a longer time period (ie, 4 hours), we were able to see a decrease in blood pressure that started to occur after 120 minutes and was profound in some animals. Three of the 7 vehicle animals (42%) treated with S aureus α-toxin survived the 4-hour observation period (mean survival time, 170±15 minutes), whereas all of the rats pretreated with simvastatin survived the whole 4-hour observation period (7 of 7, 100%, P<0.05).
Immunolocalization of P-Selectin in the Rat Mesenteric Microvasculature
Localization of P-selectin was accomplished using a modified avidin-biotin immunoperoxidase technique. The percentages of venules staining positive for P-selectin in ileal sections from control rats were consistently low, in the range of 10% (Figure 5). S aureus α-toxin infusion resulted in a significant increase of P-selectin expression up to 50% positive vessels. The increase in expression of P-selectin on ileal venules was significantly suppressed by pretreatment with simvastatin (P<0.05).
Immunolocalization of ecNOS in the Rat Mesenteric Microvasculature
Positive ecNOS staining was observed on the vascular endothelium in the rat intestine of vehicle rats. Immunolocalization of ecNOS was seen for almost the whole circumference of the vessels consistently with low intensity (1.7±0.4 intensity score). Pretreatment with simvastatin resulted in significant intensity increase of ecNOS within the vasculature (3.5±0.7, intensity score, P<0.05) (Figure 6). Interestingly, treatment with S aureus α-toxin did not change immunohistochemical-analyzed ecNOS expression.
The aim of the study was to characterize the acute effects of S aureus α-toxin challenge on leukocyte-endothelium interaction, to relate these changes to the expression of cell-adhesion molecules, and to determine the effects of simvastatin, a HMG-CoA reductase inhibitor (statin), on the exotoxin-induced abnormalities. To our knowledge, our results are the first to demonstrate a protective effect of a statin under conditions of acute inflammation induced by an exotoxin within the microcirculation. In particular, we provide strong evidence that simvastatin is able to attenuate enhanced leukocyte-endothelium interaction after S aureus α-toxin administration. Pore-forming S aureus α-toxin9 is known to provoke cardiovascular collapse as well as inflammatory acctivation.1,10,12–14⇓⇓⇓⇓
HMG-CoA reductase inhibitors are used clinically for lowering hypercholesterolemia because of their inhibitory effect on hepatic biosynthesis of cholesterol at the mevalonate step. Several animal and human studies24,25⇓ indicate improved endothelial function associated with a lowering of serum cholesterol. Recently, the statins have been reported to preserve endothelial function in the absence of hypercholesterolemia in isolated endothelial cells16 and in intact organisms.18,20,26⇓⇓ Because of the important role of endothelial function during inflammatory states, our study was designed to investigate the effects and mechanisms of a prototype statin on leukocyte-endothelial cell interaction in vivo after bacterial toxin challenge (ie, S aureus α-toxin).
The present study clearly demonstrates that simvastatin, given 18 hours before toxin administration in pharmacologic concentrations (0.1 mg/kg translated to ≈10 mg in humans), is able to attenuate exotoxin-induced leukocyte-endothelial cell interactions, most probably via a P-selectin–dependent mechanism. Interestingly, 1-hour pretreatment with simvastatin did not demonstrate this protective effect. These anti-adhesive effects may not be explained by the cholesterol-lowering effects of simvastatin, because in our model, cholesterol levels did not change. This might point toward other mechanisms of HMG-CoA reductase inhibitors. Moreover, these effects occurred without significant changes of venular shear rates and the range of rolling velocity among the different groups. Furthermore, the anti-inflammatory effect of simvastatin on exotoxin challenge could be translated to significant improvement of survival.
Recent studies on leukocyte and endothelial adhesion molecules involved in the acute inflammatory response indicate a complex pattern of leukocyte-endothelium interaction that precedes emigration of leukocytes from the vasculature into the surrounding tissue. Leukocyte endothelium interaction is known to be a multistep process involving sequential activation of specific cell adhesion molecules. An important early event during these conditions is endothelial dysfunction.14 This is characterized by reduced synthesis and release of NO, an important endothelium-derived substance involved in the inhibition of platelet aggregation, attenuation of neutrophil adherence, and maintenance of microvascular integrity.27 Moreover, Davenpeck et al28 demonstrated that reduced endogenous NO synthesis results in the upregulation of P-selectin on the endothelial cell surface of mesenteric microvessels. In support of this view, P-selectin upregulation can be decreased by administration of a NO donor.29 In this regard, statins have been shown to increase expression of endothelial NOS, leading to enhanced release of NO, which could be responsible for non–lipid-lowering actions of the statins.16 In this line, simvastatin treatment of mice increased the catalytic activity of calcium-dependent NOS by 2- to 3-fold.30 Moreover, statins were found to significantly increase NO generation by 2- to 3-fold (measured by direct NO electrode).31 Similarly, we were able to observe enhanced ecNOS expression within the intestinal vasculature after simvastatin treatment.
P-selectin, a member of the selectin family of adhesion molecules, is believed to play a significant role in the initial phase of leukocyte capture, which is rolling of leukocytes along the vascular endothelial surface.2,28⇓ PMN rolling serves to tether the neutrophil to the activated endothelium, thus bringing the neutrophil in closer contact with the endothelial cells, allowing firm adherence to occur.3,32⇓ Once firmly adhered through the interaction of the β2-integrins with their endothelial counter-receptor, intercellular adhesion molecule-1, leukocytes can then undergo additional activation, migrate across the endothelium, and release free radicals and proteolytic enzymes with subsequent tissue injury.5,33⇓ It has been previously shown that lovastatin decreases CD11b expression and CD11b-dependent adhesion of monocytes to endothelium in humans, independent of cholesterol-lowering effects.34 Additionally, Lefer et al31 demonstrated that simvastatin downregulates CD18 on stimulated polymorphonuclear cells in normocholesterolemic rats. However, S aureus α-toxin is known to be a weak activator of leukocytes. Nevertheless, this also could contribute to the observed endothelium-protective effects in the present study occurring independently of the well-known lipid-lowering effects of the HMG-CoA reductase inhibitors.
Previously, Buerke et al14 demonstrated endothelium dysfunction and enhanced PMN adherence to the vascular endothelium after stimulation with S aureus α-toxin. An interesting aspect of this study was the inhibitory role of a PAF receptor antagonist in PMN-induced vasocontraction and endothelial dysfunction. PAF is coexpressed with P-selectin on the endothelial surface after stimulation with thrombin, oxygen-derived radicals, or Staphylococcus aureus α-toxin.12 Lorant et al3 demonstrated that PAF coexpressed with P-selectin on the endothelial surface facilitates the subsequent activation of PMNs after P-selectin–mediated leukocyte rolling. Therefore, it is possible that simvastatin interferes with PAF-mediated activation as an additional beneficial effect.
Furthermore, simvastatin may also inhibit the release of potent PMN chemoattractants and cytokines. Activated neutrophils produce proinflammatory modulators, including tumor necrosis factor-α and interleukin-6, which serve to activate additional neutrophils and propagate inflammation. In this regard, it has been demonstrated that simvastatin exerts marked anti-inflammatory effects via reduction of tumor necrosis factor-α and interleukin-6 levels.35 This property of simvastatin may be an additional mechanism by which statins afforded anti-inflammatory effects in vivo.
In conclusion, this is the first study to demonstrate in vivo that administration of simvastatin, a HMG-CoA reductase inhibitor, inhibits leukocyte rolling, adherence, and transmigration in acute inflammatory states induced by the exotoxin S aureus α-toxin. This effect was found to be mediated via decreased upregulation of P-selectin on endothelial cells as well as enhanced NOS III expression within the vasculature. These observations may open a new field of pharmaceutical use of statins, such as prevention of tissue damage mediated in states of infectious diseases.
This work was supported in part by grant Bu 819/5-1 and Si 560/4-1 of Deutsche Forschungsgemeinschaft and Robert Mueller-Foundation, Germany.
- ↵Lorant DE, Patel KD, McIntyre TM, et al. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils. J Cell Biol. 1991; 115: 223–234.
- ↵Krull M, Dold C, Hippenstiel S, et al. Escherichia coli hemolysin and Staphylococcus aureus α-toxin potently induce neutrophil adhesion to cultured human endothelial cells. J Immunol. 1996; 157: 4133–4140.
- ↵Bhakdi S, Tranum-Jensen J. α-Toxin of Staphylococcus aureus. Microbiol Rev. 1991; 55: 733–751.
- ↵Grimminger F, Rose F, Sibelius U, et al. Human endothelial cell activation and mediator release in response to the bacterial exotoxins Escherichia coli hemolysin and staphylococcal α-toxin. J Immunol. 1997; 159: 1909–1916.
- ↵Sibelius U, Grandel U, Buerke M, et al. Staphylococcal α-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts: role of thromboxane generation. Circulation. 2000; 101: 78–85.
- ↵Laufs U, La Fata V, Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998; 97: 1129–1135.
- ↵O’Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation. 1997; 95: 1126–1131.
- ↵Pruefer D, Scalia R, Lefer AM. Simvastatin inhibits leukocyte-endothelial cell interactions and protects against inflammatory processes in normocholesterolemic rats. Arterioscler Thromb Vasc Biol. 1999; 19: 2894–2900.
- ↵Palmer M, Jursch R, Weller U, et al. Staphylococcus aureus α-toxin: production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. J Biol Chem. 1993; 268: 11959–11962.
- ↵Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991; 88: 4651–4655.
- ↵Endres M, Laufs U, Huang Z, et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 8880–8885.
- ↵Lefer AM, Campbell B, Shin YK, et al. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation. 1999; 100: 178–184.
- ↵Weber C, Erl W, Weber KS, et al. HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia. J Am Coll Cardiol. 1997; 30: 1212–1217.