Antithrombin reduces leukocyte adhesion during chronic endotoxemia by modulation of the cyclooxygenase pathway

J. N. Hoffmann1, B. Vollmar2, D. Inthorn1, F. W. Schildberg1, and M. D. Menger2

1 Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians University, D-81377 Munich; and 2 Institute for Clinical and Experimental Surgery, University of Saarland, D-66421 Homburg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antithrombin (AT) is known as the most important natural inhibitor of thrombin activity and has been shown to improve distinct clinical parameters during the course of septic (endotoxin)-induced multiple organ dysfunction. We hypothesized that AT acts by inhibiting leukocyte activation and microvascular injury via the promotion of endothelial release of PGI2, and therefore, we studied the effects of AT on leukocyte/endothelial cell interaction and microvascular perfusion during endotoxemia. In a skinfold preparation of Syrian hamsters, severe endotoxemia was induced by repeated administration of endotoxin intravenously [lipopolysaccharide (LPS), Escherichia coli, 2 mg/kg] at 0 and 48 h. AT (250 IU/kg) was administered intravenously at 0, 24, and 48 h (n = 6, AT group). In control animals (n = 5, control), LPS was given without AT supplementation. By intravital fluorescence microscopy, leukocyte-endothelial cell interaction and functional capillary density (FCD; measure of capillary perfusion) were analyzed during a 72-h period after the first LPS injection. AT significantly attenuated LPS-induced arteriolar and venular leukocyte adherence after both the first and the second LPS injection [P < 0.01, measures analysis of variance (MANOVA)]. In parallel, AT was effective in preventing LPS-induced depression of FCD after the first and the second LPS administration (P < 0.05, MANOVA). By pretreatment with the cyclooxygenase inhibitor indomethacin (n = 6), effects of AT on leukocyte adherence and FCD were found completely abolished. Thus our study indicates that AT exerts its beneficial effects in endotoxemia by reducing leukocyte-endothelial cell interaction and microvascular perfusion failure probably via liberation of prostacyclin from endothelial cells.

proteinase inhibition; endotoxemia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOST IMPORTANT INHIBITOR of thrombin and of other proteinases of the coagulation pathway is antithrombin (AT) (3). In patients with sepsis, coagulatory activation and excessive thrombin formation cause a fall in AT activity and thereby lead to a mismatch between proinflammatory mediators and natural inflammation inhibitors (natural inhibitory potential of the organism) (16). Thus, in patients with sepsis, low AT plasma activities at the admission to the intensive care unit correlate well with poor outcome (32, 39, 40). Because a secondary increase in AT activity during the clinical course of sepsis corresponds with improved survival rate (20), recent studies have established the concept of AT supplementation in the treatment of septic patients (18, 19, 36). In a prospective controlled clinical trial, we have shown that long-term AT supplementation (during a 14-day period), which aimed at supranormal AT activities (120%), ameliorates lung dysfunction and prevents the development of septic liver and kidney failure in surgical patients with severe sepsis (12). These clinical improvements were associated with a significant modulation of interleukin-6 plasma levels and downregulation of plasma activities of intercellular adhesion molecule-1 (ICAM-1), as well as E-selectin (13).

The mechanisms by which AT exerts its beneficial effects, however, are still unclear. Because AT supplementation has been demonstrated to significantly increase the survival rate in a rabbit endotoxin model without improvement of coagulopathy (34), whereas the anticoagulant heparin improves coagulopathy but without reducing mortality (33), anti-inflammatory AT properties, independent from their well-known anticoagulatory activities, have been proposed. Recently, it has been shown that AT causes an amelioration of endotoxin-induced pulmonary vascular injury (35) and a reduction of ischemia-reperfusion-induced damage of intestinal and hepatic tissue (10, 27). These effects, however, seem not to be caused solely by inhibition of thrombin because selective inhibition of thrombin generation did not prove similar effectivity (10, 35). AT actions, however, may involve the cyclooxygenase pathway, because AT is also known to promote the production of prostacyclin from endothelial cells (41). With the view that endotoxemia exerts its deleterious effects by leukocyte activation, endothelial adherence, and microvascular injury (9) and that prostacyclin has the potential to attenuate leukocytic response and to improve microvascular perfusion (4, 9), we hypothesized that AT in endotoxemia acts beneficially by reducing leukocyte-endothelial cell interaction and ameliorating microvascular perfusion failure via the anti-inflammatory and vasoactive properties of prostacyclin.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Animals. All experiments adhered to the international standards for good animal care and laboratory practice and were performed in line with the National Institutes of Health guidelines on the use of experimental animals. Syrian golden hamsters, 6-8 wk old with body weight of 60-80 g, were used for the study. The animals were housed one per cage and had free access to tap water and standard pellet food (Altromin, Lage, Germany) throughout the experiment. Fluid resuscitation during the experiment was additionally achieved by the administration of isotonic NaCl solution (2 ml ip at 24 and 56 h after induction of endotoxemia).

Experimental protocol. To test the efficacy of AT during double-hit endotoxemia, one group of animals [n = 5, control group (control)] was assigned to receive two intravenous endotoxin [lipopolysaccharide (LPS)] injections (2 mg/kg body wt each, Escherichia coli 0128:B12, Sigma, Deisenhofen, Germany) directly after monitoring of baseline parameters (0 h) and after 48 h. Analysis of the microcirculation by intravital microscopy was performed at baseline (0 h) and at 30 min, as well as at 3, 8, 24, 48, 56, and 72 h after the first LPS injection. In a second group of animals [n = 6, AT group (AT)], LPS was also injected twice as described above. Additionally, AT (250 IU/kg, Kybernin, Centeon, Marburg, Germany) was applicated 5 min before the first LPS injection intravenously after measurement of baseline parameters (0 h). A second AT injection (250 IU/kg) was administered after intravital microscopy at 24 h, and a third AT injection (250 IU/kg) was given 5 min before the second LPS challenge at 48 h. Analysis of the microcirculation by intravital microscopy was also performed at baseline (0 h) and at 30 min, as well as at 3, 8, 24, 48, 56, and 72 h after the first LPS injection (Fig. 1).


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Fig. 1.   Study design. One group of animals (n = 5, control group) was assigned to receive 2 intravenous endotoxin [lipopolysaccharide (LPS)] injections directly after monitoring of baseline parameters (0 min) and after 48 h. Analysis of the microcirculation by intravital microscopy (IVM) was performed at baseline and at 30 min, 3 h, 8 h, 24 h, 48 h, 56 h, and 72 h after the first LPS injection. In a second group of animals [n = 6, antithrombin (AT) group], LPS was also injected twice as described above. Additionally, AT was applicated 5 min before the first LPS injection intravenously. A second AT injection was administered after IVM at 24 h, and AT was again injected before the second LPS challenge at 48 h. Analysis of the microcirculation by IVM was performed during 72 h as described above.

To study the mechanism of AT action, we chose in additional animals a study period of 24 h with a single-hit endotoxemia (2 mg/kg body wt each, E. coli 0128:B12, Sigma). In AT animals the cyclooxygenase inhibitor indomethacin (Indo; 5 mg/kg sc) was given 30 min before LPS injection (AT+Indo, n = 6). Indomethacin pretreatment in this dose has been shown to completely abolish the beneficial effects of AT on pulmonary vascular injury during rat endotoxemia (35). Intravital microscopic parameters of the indomethacin-pretreated animals (AT+Indo) were compared with the results of the LPS-challenged animals receiving AT without indomethacin pretreatment (AT) and animals receiving solely LPS (control) during 24 h.

To test the effect of AT+Indo on laboratory parameters after 24 h of endotoxemia, AT+Indo animals were compared with an additional five animals that had received LPS in combination with AT [killed after 24 h (AT 24 h)] and five animals that had received solely LPS without AT treatment (LPS 24 h).

Microcirculation model. For intravital fluorescence microscopy, we used the dorsal skinfold chamber preparation that contains one layer of striated muscle and skin and allows the in vivo observation of the microcirculation in the awake animal over a prolonged period of time (22). The model permits the in vivo determination of leukocyte-endothelial cell interaction (rolling and adherent leukocytes) and the quantification of microhemodynamic parameters, including functional capillary density (FCD), red blood cell velocity, and diameters of the individual microvascular segments. Microcirculatory analysis was completed by determining the extravasation of macromolecules, indicating disintegration of the microvascular endothelial lining (24).

The chamber and its implantation procedure have been described previously by Endrich et al. (8) in detail. The preparation used in this study was similar, except for minor modifications, and has been described also in earlier reports (23). Briefly, under pentobarbital sodium anesthesia (50 mg/kg body wt ip; Nembutal, Abbott, Wiesbaden, Germany), the animals were fitted with two symmetrical titanium frames, positioned on the dorsal skinfold, sandwiching the extended double layer of skin. One layer of skin was completely removed in a circular area of 15 mm in diameter, and the remaining layers (consisting of striated skin muscle and subcutaneous tissue) were covered with a removable coverslip incorporated into one of the titanium frames. Fine polyethylene catheters (PE-10, 0.28-mm internal diameter) were inserted into the jugular vein, passed subcutaneously to the dorsal site of the neck, and fixed to the titanium frames. The chambers and catheters were implanted at least 72 h before the experiments. This allowed us to eliminate the effect of surgical trauma and anesthesia on the chamber tissue.

Intravital fluorescence microscopy. For the in vivo microscopic analysis, the awake hamsters were immobilized in Plexiglas tubes, and the skinfold preparation was attached to the microscope stage. The stage was then placed on a computer-controlled microscope desk that allowed repeated scanning of identical segments of microvessels during the experiment. After intravenous injection of 0.2 ml 5% FITC-labeled dextran (mol mass 150 kDa; Pharmacia, Uppsala, Sweden), which stains blood plasma, and in vivo staining of leukocytes by rhodamine 6G (0.15 mg/kg body wt; Sigma), intravital microscopy was performed using a modified Leitz Orthoplan microscope with a 100-W HBO mercury lamp attached to a Ploemo-Pak illuminator with an I2 blue and an N2 green filter block (Leitz, Wetzlar, Germany) for epi-illumination. The observations were recorded by means of a charge-coupled device video camera (FK 6990; Cohu, Prospective Measurements, San Diego, CA) and transferred to a video system for offline evaluation. The microscopic images were recorded on videotape and analyzed during playback by using a special computer software (CAPIMAGE version 6.0; Dr. Zeintl, Heidelberg, Germany). A 25-fold water-immersion objective (Leitz), resulting in a total magnification of ×690 at a 14-inch monitor screen, was used to select at least four regions of interest per chamber, whereby each site contained at least six postcapillary or collecting venules, and at least three terminal arterioles and capillary fields. These regions were followed during 72 h.

Microcirculatory analysis. Vascular diameters (micrometers) were analyzed by length measurements, orientated perpendicularly to the vessel path (CAPIMAGE). Microvascular permeability was determined by analyzing the extravasation of FITC-dextran 10 min after intravenous injection. Extravasation was quantified densitometrically and is given as the ratio of fluorescence intensity outside the vessel segment (directly adjacent to the vessel wall) vs. that inside the vessel segment (CAPIMAGE) (23). FCD, which served as the measure of quality of microvascular perfusion, was defined as the length of all red blood cell-perfused nutritive capillaries per observation area (CAPIMAGE) and is given in cm-1 (25).

Leukocytes were classified as nonadherent, rolling, or adherent cells, according to their interaction with the microvascular endothelium. Nonadherent leukocytes were defined as cells passing the observed vessel segment without contact to the endothelial lining and are expressed as the number of cells traversing the observed vessel segment per 20 s. Rolling leukocytes were defined as cells moving along the endothelial lining at a velocity slower than two-fifths of the surrounding red blood cell column and are given as percentage of all nonadherent leukocytes traversing the observed vessel segment within 20 s (23). Adherent leukocytes were defined as cells that did not detach from the endothelial lining within a 20-s observation period and are given as number of cells/mm2 of endothelial surface, calculated from the respective vessel diameter and length (100 µm) of the vessel segment studied, assuming cylindrical geometry.

Laboratory analysis. Laboratory analysis was performed at the end of the respective observation periods (24 and 72 h) in plasma samples obtained by laparotomy and blood withdrawal from the abdominal aorta. Repeated plasma sampling was not possible due to the limited circulating blood volume of the hamster. In the animals of groups AT, control, AT 24 h, AT+Indo, and LPS 24 h, blood gases were analyzed (BGA 348, Chiron Diagnostics) in heparinized samples that were taken at the end of the study period by puncture of the abdominal aorta. Total red blood cell, leukocyte, and platelet counts, as well as hemoglobin, were determined by a Coulter Counter (AcTdiff, Coulter, Hamburg, Germany). Additionally, the coagulatory parameters thromboplastin time, partial thromboplastin time, fibrinogen, antithrombin, and protein C were measured in citrated plasma by standard activity assays. Five healthy animals in which blood was taken by puncture of the abdominal aorta under pentobarbital anesthesia served for assessment of physiological values.

Statistical analysis. All parameters are given as means ± SE. If not indicated otherwise, mean values of the observed microscopic parameters were calculated for each animal (6 venules, >3 arterioles per site of observation), and statistical analysis was performed between experimental groups by using the mean values of the respective animals. To compare intravital microscopic parameters at baseline between groups, a one-way ANOVA or Kruskal-Wallis analysis was performed where appropriate. To analyze for different responses to the first LPS injection, intravital microscopic parameters of AT and control animals during 48 h after the first LPS injection were analyzed by a two-way, repeated- measures analysis of variance (MANOVA; SPSS, version 6.0.1; Chicago, IL), which takes into account the multiplicity of measurements over time. To test different responses between AT and control animals after the second LPS injection, the time points 48 h until 72 h were also analyzed by MANOVA.

To study the mechanism of AT, a one-way ANOVA was performed at 0 and 24 h, and the Student-Newman-Keuls test was used for post hoc comparison only at baseline, 8 h, and at 24 h to avoid multiple testing. To detect potential differences in laboratory parameters between experimental groups, parameters were analyzed by ANOVA at 24 h (AT 24 h, AT+Indo, LPS 24 h, healthy control animals) and at 72 h (AT, control, healthy control animals), respectively. Adequate tests were used for post hoc analysis. A significance level of P < 0.05 was used throughout the study.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of AT supplementation on arteriolar and venular diameters. At baseline, arteriolar and venular diameters did not differ between control and AT animals (Table 1). In both groups, LPS did not influence arteriolar diameters during 48 h. Correspondingly, there was no significant difference between AT and control animals during 48 h after the first LPS injection (Table 1, P = 0.93, MANOVA for repeated measurements). Also, between 48 and 72 h, AT did not influence arteriolar diameters (Table 1, P = 0.47, MANOVA).

                              
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Table 1.   Effect of antithrombin on arteriolar and venular diameters during endotoxemia

In venules, the first LPS challenge caused vasodilation in both groups (AT and control), which was obvious after 3 and 8 h, but normalized after 24 h. Comparison between the two groups did not reveal significant differences in this LPS response during the first 48-h observation period (P = 0.80, MANOVA). The second LPS injection led to a significant and persisting increase in venular diameters in control animals (Fig. 2) that was prevented by AT supplementation (Table 1, P = 0.048, MANOVA).


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Fig. 2.   Induction of arteriolar and venular leukocyte adherence by endotoxin. Precapillary arterioles (left) and postcapillary collecting venules (right) within hamster dorsal skinfold preparation before (top) and after (bottom) 56 h of endotoxemia. Note massive venular dilation (bottom right) after the second endotoxin administration. White dots in the vascular lumen represent adherent leukocytes that massively accumulate after endotoxin stimulation in arterioles and venules. Leukocytes and endothelial cells are stained in vivo with the fluorescent marker rhodamine 6G. At baseline, only a few leukocytes interact with the vascular endothelium (top), whereas after 56 h of endotoxemia, many leukocytes adhere to the dilated vessel walls (bottom). In vivo fluorescence microscopy. Total magnification, 240-fold.

Effect of AT supplementation on leukocyte/endothelial cell interaction. At baseline, the number of nonadherent leukocytes did not differ between AT and control animals (Table 2). The first LPS administration induced an impressive reduction in the amount of nonadherent leukocytes to less than 10% of baseline that was not prevented by AT supplementation. In both groups, baseline conditions were reached again after 8 h, followed by an increase of the number of nonadherent leukocytes during 24 h until 72 h (Table 2) without major differences between the groups during the first 48 h (P = 0.31, MANOVA) and during 48 h until 72 h (P = 0.41, MANOVA).

                              
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Table 2.   Effect of AT administration on leukocyte-endothelial cell interaction during double-hit endotoxemia

In arterioles, 1-2% of leukocytes showed loose interaction with the vascular endothelium, representing the fraction of spontaneously rolling leukocytes (Table 2) at baseline. In both groups, the first LPS administration led to an increase in arteriolar rolling over time that was most impressive (40-70% rolling leukocytes) after 8 h of endotoxemia (Table 2). There was no significant difference between AT and control animals during the first 48 h (P = 0.46, MANOVA). The second LPS injection led to a two- to threefold increase in the rolling leukocyte fraction at 56 h but decreased again until 72 h, independent from AT administration (Table 2, P = 0.41, MANOVA). In venules, 20% of the leukocytes studied at baseline showed loose interaction with the vascular endothelium, representing the fraction of rolling leukocytes (Table 2). In both groups, the first LPS administration led to a fall in the fraction of rolling leukocytes as early as after 30 min. Whereas baseline conditions were reached again after 3 h, a secondary increase in venular rolling leukocyte fraction was noted at 8 h and persisted until 72 h in both groups (0 h until 48 h: P = 0.56, MANOVA; 48 h until 72 h: P = 0.53, MANOVA). During 24 h until 72 h, 60-90% of leukocytes showed loose interaction with the venular endothelium.

The number of adherent leukocytes in arterioles and venules did not differ at baseline between AT and control animals (Table 3). In control animals, the first LPS injection led to a profound increase of arteriolar and venular leukocyte adherence that peaked at 8 h and persisted during 48 h (until 48 h). This increase in leukocyte-endothelial cell interaction was significantly attenuated during the first 48 h by AT supplementation both in arterioles (P = 0.001, MANOVA) and venules (P = 0.004, MANOVA). In control animals, the second LPS challenge resulted in a persisting enhancement of leukocyte adherence with a maximum response at 56 h (Fig. 2), whereas in AT animals the number of adherent leukocytes was found decreased between 48 and 72 h (Table 3). Correspondingly, comparison of the two groups showed a significant difference both in arterioles (P = 0.001, MANOVA) and in venules (P = 0.003, MANOVA).

                              
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Table 3.   Effect of AT administration on leukocyte-endothelial cell interaction during double-hit endotoxemia

Effect of AT supplementation on macromolecular extravasation and FCD. At baseline, arteriolar and venular extravasation of the macromolecule FITC-dextran did not differ between AT and control animals (Table 4). Extravasation increased stepwise during 48 h without significant differences between the two groups (arterioles: P = 0.63, MANOVA; venules: P = 0.47, MANOVA). During 48 h until 72 h (after the second LPS administration), however, arteriolar extravasation in AT animals was found significantly lower compared with that analyzed from controls (P = 0.019, MANOVA), whereas in venules, extravasation was only slightly but not significantly reduced by the AT supplementation (Table 4, P = 0.36, MANOVA).

                              
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Table 4.   Effect of AT administration on plasma extravasation and capillary perfusion during double-hit endotoxemia

Analysis of FCD revealed that baseline capillary perfusion did not differ between AT and control animals (Table 4, Fig. 3). However, in control animals, the first LPS administration reduced FCD significantly by ~50% during the first 48-h period of endotoxemia, whereas FCD in AT animals was not affected (P = 0.03, MANOVA). The second LPS administration led to a further deterioration of FCD to ~10% of baseline values in controls, whereas FCD remained almost constant in AT animals (Table 4, Fig. 3). Accordingly, comparison between the two groups showed a significant difference also after the second LPS administration (P = 0.005, MANOVA).


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Fig. 3.   Prevention of endotoxin-induced capillary perfusion failure by AT. Striated muscle capillaries within hamster dorsal skinfold preparation in representative animals before endotoxin (baseline, A and B) and after 72 h of endotoxemia (C and D). Note endotoxin-induced decrease in the amount of erythrocyte-perfused capillaries (that horizontally transverse the panel) after 72 h without AT supplementation (C). Seventy-two hours after the beginning of endotoxemia, AT completely prevented the reduction of erythrocyte-perfused capillaries (D). In vivo fluorescence microscopy, contrast enhancement by FITC-dextran. Total magnification, 160-fold.

Effect of AT supplementation on laboratory parameters. AT and control animals did not show major disturbances of blood gases, electrolytes, and hemoglobin values compared with healthy controls (data not shown). AT effectively prevented the decrease in thromboplastin time and plasma AT activity when compared with control animals (Table 5). However, no effects of AT supplementation on fibrinogen, partial thromboplastin time, protein C activity, and platelet count were observed after the 72-h observation period.

                              
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Table 5.   Effect of AT administration on laboratory parameters after double-hit endotoxemia at 72 h

Effect of indomethacin on AT modulation of microvascular and inflammatory response. At baseline, there was no difference in arteriolar and venular diameters, rolling leukocyte fractions, and the nonadherent leukocytes between AT animals, control animals, and animals that received AT in combination with indomethacin pretreatment (AT+Indo; data not shown). Also, arteriolar and venular leukocyte adherence and capillary perfusion (FCD) did not differ between these groups at baseline (Figs. 4 and 5).


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Fig. 4.   Effect of indomethacin pretreatment on AT modulation of arteriolar (A) and venular (B) endotoxin-induced leukocyte-endothelial cell interaction. Endotoxin (control, black-triangle) induced a significant increase in arteriolar and venular leukocyte adherence [adherent leukocytes: leukocytes that did not detach from the endothelial cell lining within a 20-s observation period (given as number of cells/mm2)] with a peak after 8 h. AT (open circle ) significantly attenuated endotoxin-induced leukocyte adherence over time both in arterioles and venules. Indomethacin (AT+Indo, ; 5 mg/kg sc) pretreatment completely abolished the modulating effects of AT on arteriolar and venular leukocyte adherence. Data are the mean ± SE. # P < 0.05 vs. control; * P < 0.05 vs. control and AT+Indo.



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Fig. 5.   Effect of indomethacin pretreatment on AT modulation of endotoxin-induced microvascular perfusion injury. Endotoxin (control, black-triangle) induced a significant decrease in functional capillary density (measured as the length of all erythrocyte-perfused nutritive capillaries per observation area and given in cm-1). AT (open circle ) significantly attenuated endotoxin-induced microvascular perfusion injury over time. Indomethacin (AT+Indo, ; 5 mg/kg sc) pretreatment completely abolished the modulating effects of AT and even decreased capillary perfusion compared with controls. Data are the mean ± SE. # P < 0.05 vs. AT+Indo; * P < 0.05 vs. control and AT+Indo.

Indomethacin pretreatment, however, abolished the modulating effects of AT on LPS-induced leukocyte adherence both in arterioles and venules. In arterioles, indomethacin pretreatment inhibited the AT action, leading to a significantly higher number of adherent leukocytes in AT+Indo animals at 24 h, compared with solely AT-treated animals (Fig. 4A). In venules, AT+Indo animals showed a significant increase in leukocyte adherence at 8 and 24 h, which was comparable to that in control animals, whereas AT alone effectively attenuated the increase in venular leukocyte adherence (Fig. 4B).

The inhibition of AT effects on leukocyte adherence in arterioles and venules by indomethacin was associated with a significant deterioration of capillary perfusion in AT+Indo animals. The LPS-induced fall in capillary perfusion (measured by FCD) was not prevented by the combination of AT with indomethacin treatment when compared with AT alone (Fig. 5).

Effect of indomethacin on laboratory parameters. AT, AT+Indo, and LPS 24 h animals did not show major disturbances of blood gases and electrolytes after 24 h of LPS challenge when compared with healthy controls (data not shown). However, the beneficial effects of AT on partial thromboplastin time, plasma AT activity, and protein C activity were almost abrogated by the additional pretreatment with indomethacin (Table 6).

                              
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Table 6.   Effect of AT administration and AT administration in combination with indomethacin pretreatment on laboratory parameters after 24 h of endotoxemia


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gram-negative sepsis is a major cause of death in postoperative and posttraumatic intensive care unit patients (2). Endotoxin is known to activate different cascade systems [complement system, coagulation system, bradykinin-kinin system (5)] and to induce adhesion molecule synthesis and shedding by the activation of inflammatory cytokines (31). The mediators and end products of these systems are known to exhibit their deleterious effects on the microcirculatory level (microvascular permeability, capillary perfusion) leading to endothelial damage and microvascular thrombosis (1, 28). In this context, the adhesion of circulating leukocytes to the vascular endothelium is of major interest (21). Activated leukocytes decrease their flow velocities and adhere to the vascular endothelium to emigrate to the septic focus. They subsequently release an array of inflammatory mediators, cytotoxic enzymes, and oxygen radicals (17). These substances may result in reduced capillary perfusion that finally leads to the development of organ dysfunction (15, 21, 28).

To test the AT effects on leukocyte/endothelial cell interaction, we used a novel chronic model of a double-hit endotoxemia in awake animals (11). This model allows the repeated intravital microscopic analysis of leukocyte-endothelial cell interaction and capillary perfusion under conditions of persistent endotoxemia-induced inflammatory response. A double-hit model of endotoxemia may more effectively mimic the clinical situation where endotoxin is released in more than one episode during a long-term period. In our hands, the skinfold chamber preparation is well characterized by the chronic analysis of leukocyte-endothelial cell interaction and capillary perfusion in a variety of experimental settings, such as ischemia-reperfusion (23, 24). Although endotoxemia/sepsis-induced microvascular alterations (leukocyte recruitment, capillary perfusion failure) are known to be similar in nature in different vascular beds, including the mesentery/intestine, liver, and striated muscle (15, 29, 30, 37, 38), we are aware that our study of striated muscle microcirculation may not necessarily be representative for the magnitude of microcirculatory changes in splanchnic organs, because the different organ systems may show a variable endotoxin response within the microvasculature (21). Although it would have been of major interest to study chronic inflammatory response to endotoxin stimuli also in lung, liver, and splanchnic microcirculation, this was impossible because these organs cannot be prepared for chronic/repeated analysis (3 days) by intravital microscopy.

AT was administered repeatedly (every 24 h) in a dose (250 IU/kg) that previously was shown to improve outcome in several short-term sepsis models (6). High AT activities were attained because various animal studies showed that AT activities must be maintained substantially higher than normal to be efficacious during endotoxemia (7).

In control animals, the first endotoxin challenge ("first-hit") after initial leukopenia resulted in a significant increase in both arteriolar and venular leukocyte adherence, associated with an increase in leukocyte rolling that occurred 8 h after the first endotoxin administration and persisted during 48 h. The second endotoxin challenge ("second-hit") provoked a secondary increase in both arteriolar and venular leukocyte rolling and adherence. In AT animals, however, we noted a significant attenuation of endotoxin-induced leukocyte adherence in arterioles and venules. The observation that AT inhibits endotoxin-induced leukocyte adherence is in line with a recent report from Ostrovsky et al. (26), who showed by intravital microscopy in a cat model that AT effectively reduced leukocyte adherence during postischemic reperfusion. However, in contrast to the findings from Ostrovsky et al., who showed attenuation of leukocyte rolling besides the decrease in leukocyte adherence, we could not observe a fall in the fraction of rolling leukocytes after AT and endotoxemia. This may indicate that during endotoxemia, additional mechanisms are active that are capable to induce leukocyte rolling and that cannot be blocked by AT supplementation.

Because AT supplementation showed almost no effect on leukocyte rolling despite a clear suppression of leukocyte adherence, some speculation on the mechanisms of AT action is possible. Leukocyte rolling and adherence during endotoxemia are mediated by differential regulation of cellular adhesion molecules. The multistep process of leukocyte recruitment is initiated by adhesion molecules from the selectin family (E-selectin, P-selectin) that mediate transient leukocyte-endothelial cell interaction that manifests as leukocyte rolling (14). In contrast, the later firm leukocyte adhesion involves endothelial adhesion molecules from the IgG superfamily (ICAM-1, vascular cell adhesion molecule-1, platelet/endothelial cell adhesion molecule-1) and leukocytic beta 2-integrins (CD11/CD18) (9). Because in the present experiments with endotoxemia AT reduced leukocyte adherence without significant effects on leukocyte rolling, its anti-inflammatory effects may be attributed to inhibition of beta 2-integrins or IgG superfamily adhesion molecules.

Anti-inflammatory properties of AT were observed not only during the first 24 h after LPS exposure but also after the second LPS challenge, because no significant increase in leukocyte adherence was observed in AT animals, whereas control animals responded impressively at 56 h. This effect of repeated, high-dose AT supplementation is in line with the clinical observation that continuous high-dose AT supplementation results in amelioration of septic organ dysfunction and reduction of inflammatory mediator release (12, 13).

To analyze the mechanism of the anti-inflammatory properties of AT in endotoxemia, we simultaneously administered AT and indomethacin in a dose that is known to prevent prostacyclin formation during endotoxemia by inhibition of the cyclooxygenase pathway (35). We found that the combination of AT with indomethacin completely abolished the effects of AT on the microcirculation. Our results correspond to experimental findings where 1) AT has been shown to promote prostacyclin release from endothelial cells (41), and 2) prostacyclin is effective in inhibiting leukocyte adherence in conditions of endotoxemia (4, 9).

Interestingly, indomethacin pretreatment simultaneously induced a significant fall in AT activity and protein C activity compared with AT animals, although identical amounts of AT were administered at baseline. The simultaneous decrease in AT and protein C activity may reflect a higher extent of coagulation factor and coagulation inhibitor consumption during endotoxemia upon indomethacin pretreatment. The most likely explanation for this phenomenon is that the inhibition of AT-related microcirculatory effects simultaneously led to a massive increase in coagulation factor consumption with consecutive coagulation inhibitor consumption.

Because neutrophil adherence to activated endothelium is known to result in microvascular and tissue injury, we also evaluated the effects of AT on capillary perfusion. Microvascular perfusion injury was quantified by determining FCD that has been shown to be a reliable measure of nutritive capillary perfusion in various intravital microscopic preparations (25). In non-AT-treated control animals, the increase in leukocyte-endothelial cell interaction was associated with a deterioration of capillary blood perfusion over time (50% of baseline value at 48 h). After the second endotoxin challenge, microvascular perfusion even decreased to 10% of baseline values. AT significantly attenuated the endotoxin-induced depression of microvascular perfusion and resulted in a fivefold higher FCD at the end of the study period. The fact that amelioration of microvascular perfusion was also abolished by indomethacin indicates that AT-induced prostacyclin release not only prevents endotoxin-mediated inflammatory response, but also manifestation of microvascular perfusion failure, probably by prostacyclin-inherent vasoactive properties.

The present model of double-hit endotoxemia mimics the clinical situation in patients with sepsis, where endotoxin is periodically released from the septic focus (e.g., during treatment of peritonitis). Our experiments clearly show that 1) AT is effective in preventing leukocyte adherence during endotoxemia, 2) this is associated with attenuation of capillary perfusion failure, and 3) that prevention of leukocytic response and attenuation of microvascular injury are mediated by products of the cyclooxygenase pathway. These mechanisms may explain the downregulation of inflammatory mediators by AT and the simultaneously observed protective AT action on sepsis-induced multiple organ failure in clinical studies (12, 13).


    ACKNOWLEDGEMENTS

Coagulatory parameters were measured by Dr. P. Goehring (Institut of Clinical Chemistry, Klinikum Grosshadern; Director: Prof. Dr. Seidel). The skillful technical assistance of Beate Wolf is gratefully acknowledged.


    FOOTNOTES

This study was supported by European Union Grant BMH-4-CT 95-0875 and by Wilhelm Sander Stiftung Grant 93.019.02. B. Vollmar is a recipient of a Heisenberg-Stipendium of the Deutsche Forschungsgemeinschaft (Vo 450/6-1).

Address for reprint requests and other correspondence: J. N. Hoffmann, Chirurgische Klinik, Klinikum Grosshadern, Ludwig-Maximilians Universität, Marchioninistr. 15, D-81377 München, Germany.

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

Received 22 September 1999; accepted in final form 26 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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