American Journal of Pathology, Vol. 139, No. 2, August 1991 Copyright ©D American Association of Patbologist
Expression of Endothelial Leukocyte Adhesion Molecule-1 in Septic But Not Traumatic/Hypovolemic Shock in the Baboon Heinz Redl,* Hans P. Dinges,t Wim A. Buurman, Ces J. van der Linden, Jordan S. Pober,11 Ramzi S. Cotran,11 and Gunther Schlag* From the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology,* Vienna, Austria; the Institute of Pathology,t University of Graz, Graz, Austria; the Department of General Surgery,* University of Limburg, Maastricht, The Netherlands; and the Departments of Pathology,11 Brigham and Women's Hospital and the Harvard Medical School, Boston, Massachusetts
Baboons were subjected to septic or traumatic! hypovolemic shock and their tissues were examined for the de novo expression of endothelial leukocyte adhesion molecule I (ELAM-1), using immunohistochemical techniques. In animals with septic shock induced with live Escherichia coli there was widespread expression of EL4M-1, recognized by monoclonal antibodiesH4/18 orENA-1 in most tissues examined with strong staining in the lung, liver, and kidneys. Endothelial leukocyte adhesion molecule 1 expression was evident in capillaries, venules, small veins, arterioles, and arteries. In contrast, baboons with traumatic/hypovolemic shock had minimal levels offocal ELAM expression in all organs studied Similarly evidence of neutrophil activation measured by granulocyte elastase levels in the plasma was much more pronounced in animals with septic shock The study documents that lipopolysaccharide (LPS)- and cytokine-induced endothelial activation occurs in vivo in septic shock Much higher levels of ELAM- I expression and plasma granulocyte-elastase titer in septic shock, as contrasted with traumatic! hypovolemic shock, are consistent with the higher levels of circulating tumor necrosis factor, other cytokines, and LPS in sepsis. (Am J Pathol 1991,
139:461-466)
Septic shock is a syndrome characterized by coagulation disturbances, hemodynamic collapse, and multior-
gan damage developing in the setting of systemic infection, usually with gram-negative bacteria. Lipopolysaccharide (LPS) released from the bacterial cell wall is believed to be central to the manifestations of shock. Lipopolysaccharide acts directly and through the induction of a variety of mediators, including cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).1 Infusions of TNF into experimental animals mimic many of the hemodynamic and vascular changes of septic shock,2 including hypotension, coagulopathy, leukocyte aggregation, and vascular leakage. Anti-TNF antibodies prevent septic shock in experimental lethal Escherichia co/i septicemia.3 In vitro studies recently have shown that one of the major targets of LPS and cytokine actions is vascular endothelium, which undergoes a variety of functional and structural alterations, collectively called endothelial activation.4 Principal among these changes is increased adhesivity of endothelium to leukocytes and the induction of procoagulant activities on the surface of endothelial cells, rendering them thrombogenic. Currently, however, there is little evidence that these endothelial alterations either occur in vivo in septic shock or account for the pathologic manifestations of shock, because both LPS and cytokines have a variety of effects on monocytes, cardiac and vascular muscle cells, and other cell types. We sought to obtain evidence for endothelial cell activation in septic shock by examining for the expression of endothelial leukocyte adhesion molecule 1 (ELAM-1 ).5 This is an endothelial-specific molecule, induced by LPS, TNF, and IL-1, which mediates endothelial adhesion to neutrophils,6 eosinophils,7 and memory T cells.89 Previous studies in humans have shown that ELAM-1 is an extremely useful marker for endothelial activation,10'11
Supported by grants from 'Lorenz Bohler Fond' and the National Institutes of Health (PO1 HL 36028). Accepted for publication April 15, 1991. Address reprnt requests to Heinz Redl, PhD, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, A-1200, Vienna, Austna.
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and experiments in baboon skin show that postcapillary venular endothelial cells express ELAM-1 in response to interdermal injections of TNF12 or LPS.13 In a preliminary study, we have also described that ELAM-1 is expressed in the endothelium of baboons perfused with live E.
coil.415 The present study was designed to compare the responses of endothelial cells in septic shock, thought to be mediated by LPS and cytokines, with those in a model of traumatic/hypovolemic shock, a syndrome with similar hemodynamic alterations, but without significant cytokine release.16 We report that endothelial activation, detected by ELAM-1 expression, is strongly evident in septic shock, but not in traumatic/hypovolemic shock. These data support the interpretation that endothelial cell activation may contribute to organ damage in septic shock.
Materials and Methods
Animals and Experimentation Male baboons (Papio ursinus) 20 to 25 kg in body weight were sedated with ketamine hydrochloride and placed in the supine position. Anesthesia was maintained with pentobarbital (2 to 5 mg/kg/hr). The depth of anesthesia was monitored by fronto occipital electroencephalogram (EEG) and by determination of the arterial C02 (35 to 40 mmHg) and end expiratory C02, and adjusted by a servo-assisted mechanism after Fourier spectrum analysis of EEG. Basal blood temperature was 370C ± 0.50C. and inspired oxygen concentration was set at 30%. Pulmonary artery pressure and mean arterial pressure were monitored, and cardiac output was measured using thermodilution techniques. Tissues were examined for ELAM-1 expression from six baboons with septic shock and 13 with traumatic/hypovolemic shock.
Traumatic/Hypovolemic Shock Traumatic hypovolemic shock was induced in anesthetized baboons as described previously.16,17 Trauma was followed by withdrawing sufficient blood to attain a mean arterial pressure adjusted between 35 and 40 mmHg, so that the cardiac output (CO) was reduced by 50% to 70%. After 3 hours, retransfusion of the withdrawn heparinized blood, together with Ringer's solution, was started. During the retransfusion phase (3 hours), CO was maintained between 15% and 20% above the baseline by the addition of Ringer's solution. Animals were killed 6 hours after trauma with an overdose of pentobarbital. Sham animals were subjected to all procedures except the infliction of trauma and hypovolemia.
Septic Shock Protocol To induce septic shock, 2 x 1010 live E. coli/kg (Hinshaw's strain B7 086a61 ATCC#33985) were infused intravenously over an 8-hour period. Intravenous fluid was given to maintain constant pulmonary arterial wedge pressure and cardiac output. Animals were then killed by pentobarbital anesthesia at either 6 or 8 hours. The studies on traumatic and septic shock models in baboons were approved by the ethics committee of the Roodeplaat Research Laboratories, according to guidelines advocated by the International Laboratory Animal Science Committee (Dr. S. J. von Rensburg, Chairman).
Immunohistochemical Studies Endothelial leukocyte adhesion molecule 1 was localized with the use of two monoclonal antibodies, H4/1818 and ENA-1,19 both of which react with ELAM-1. PBS or an unreactive mouse immunoglobulin was used as a negative control. Tissues were removed at autopsy and immediately quick frozen and kept at - 70°C until cryostat sectioning. Four-micron-thick frozen sections were fixed for 10 minutes in cold acetone (- 20°C) and air dried. Sections then were incubated with the primary antiserum diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, in a humidified chamber at room temperature. Dilutions of the primary antiserum were optimized in several pilot experiments for H4/18 1/500 and ENA-1 1/1000. Sections were incubated for 1 hour with the primary antiserum, washed, and then incubated for 30 minutes with rabbit anti-mouse immunoglobulin (Dakopatts; Glostrum, Denmark) at a dilution of 1:30 in PBS. Sections were then washed and exposed to the APAAP-complex (Dakopatts) for 30 minutes. The color reaction then was developed with substrate solution containing 10 mg naphthol-AB-MX-phosphate (Serva; Heidelberg, Federal Republic of Germany), dissolved in 0.5 ml N,Ndimethyformamide (Serva) and 50 ml barbitone buffer pH9.2, 50 mg fast red TR (Serva), and 10 mg levamisole (Sigma; St. Louis, MO). Before use, the solution was adjusted to pH 9.2 to 9.8 and filtered. The sections were incubated for 15 to 30 minutes at room temperature in this solution and then mounted and examined. Sections were examined and quantitated by two of the authors (RC and HD) without knowledge of the identity of the animal from which the tissue was derived. The intensity of endothelial staining was assessed as follows: negative; ± (equivocal or minimal staining); + 1 (mild but definite staining); + 2 (moderate staining); + 3 (strongest
staining).
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Plasma Analysis Granulocyte elastase was measured using the IMAC assay (Merck, Federal Republic of Germany) on a Hitachi 704, using human elastase standards.20
Results
Immunohistochemical Studies The results with both antibodies were qualitatively similar. The semiquantitative evaluation was done only with sections stained with the ENA-1 antibody (Table 1). All six animals with septic shock showed significant endothelial staining for ELAM-1 in the kidneys, lung, liver, intestines, and skin. In the kidneys, there was strong staining restricted to vascular endothelium, in glomeruli and peritubular capillaries, and there was strong and distinctive staining of the endothelium in arteries (Figures 1 to 3). Intraglomerular fibrin thrombi were frequent, consistent with the increased surface procoagulant activity characteristic of activated endothelium (Figure 4). In the lung, the staining was strong but patchy and involved capillaries in the alveolar walls, as well as venules and occasional arterioles in the interlobular fibrous septae (Figure 5). In the liver, staining was diffuse, involving the lining of the majority of sinusoids, as well as venules and small veins in the portal tracts (Figure 6). Endothelial leukocyte adhesion molecule 1 expression was weakest and most spotty in skeletal muscle. In contrast, animals subjected to traumatic/ hypovolemic shock had only minimal expression of ELAM-1 after 6 hours of shock (Table 1). There was virtually no staining in the lung and liver, and the intensity of staining in the kidney was significantly less than that in kidneys of animals with septic shock, being restricted to mild (+ 1) glomerular staining, except in one animal, in which there was also + 1 staining of arteries. To measure granulocyte activation, neutrophilspecific elastase levels were measured in plasma using an assay system developed for the human enzyme. Although baseline levels (1 to 20 ,ugA) are lower than nor-
Figure 1. Low-power illustration of renal cortex in septic shock showing ELAM-1 staining of endothelium of glomerular and peritubular capillaries and small artery (louer left).
mal human levels, there is cross-reactivity with the baboon enzyme, and the shock-related proportional increases are comparable to those in the clinical situation. As seen in Table 2, sepsis caused the expected rapid elevated elastase levels; markedly less activation was noted in the animals subjected to traumatic/hypovolemic shock.
Discussion The present study demonstrates that strong widespread multiorgan expression of ELAM-1 occurs in septic shock.
Table 1. Immunohistochemical Localization of ELAM-1 in Septic and Traumatic Shock Traumatic Shock Septic Shock No. Average No. Average score examined score examined 0.03+ 2.2+ 13 6 Lung 12 0.16+ 3.0+ 5 Liver 0.8+ 2.7+ 9 3 Kidney 2 0.5+ 2 3.0+ Skin The average score denotes average level of ELAM-1 expres-
sion, on a scale of 0, ± (0.5), 1, 2 and 3+ in the group examined.
Figure 2. High magnification of glomerulus ELAM-1 staining is restricted to endothelium.
showing that the
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Figure 3. Small renal artery showing distinct endothelial staining for ELAM-1.
These observations are consistent with the current hypotheses that septic shock is mediated by systemic LPS and by induced cytokines, especially TNF and IL-1, and that LPS and these cytokines act on vascular endothelial cells to cause their activation. We chose ELAM-1 as a measure of endothelial activation for the following reasons: In vitro studies indicate that ELAM-1 is a marker of acute activation, and its time-sequence of induction is consistent with that of the induction of septic shock in this model.5'18 Secondly ELAM-1 is not constitutively present in normal endothelium, unlike other endothelial activation markers (such as ICAM-1 and INCAM-i 1 ONCAM-1),21 and is a reliable evidence of LPS- and cytokine-induced activation when it is present.22 Thirdly antibodies to hu-
Figure 4. H&E-stained glomerulus from dxhok showing fbrin thrombi.
an animal
edh septic
Figure 5. Lung from septic shock, showing ELAM-1 staining of endothelium of a venule (arrow) and spotty staining of capillaries.
I
Note adherent leukocytes (arrow beads) in venular lumen.
man ELAM-1 cross-react with baboon endothelium12; and finally we have previously used ELAM-1 expression to detect evidence of endothelial activation in human diseases in vivo10'11 and after local injections of endotoxin and cytokines in baboons.12'13 Endothelial activation was widespread, involving most
Figure 6. Section of liver with diffuse staining of sinusoidal lining
endothelium.
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Table 2. Granulocyte Elastase Levels in Plasma Measured by Immunoassay in Baboons Subjected to Hypovolemic/Traumatic (Sham) or Hyperdynamic Septic Shock (lxg/l) Hypovolemic/ traumatic Groups shock (hrs/animal) Sepsis 15 ± 2 0 6±1.3 2 30±6 270±45.0 4 62±8 262±26.0* 6 388 31.0* 77 ± 15 ND 8 304 ± 27.0 Sepsis statistically different from hypovolemic traumatic shock (P < 0.05) using Wilcoxon statistics.
organs examined, but was particularly strong in the kidney, liver, and lung. Of great interest is that ELAM-1 was expressed not only in venules, as previously noted in the setting of local immune inflammation in humans in vivo,5,11 or after the administration of endotoxin and cytokines in baboon skin in vivo,12'13 but also was observed on capillaries, arterioles, arteries, and small veins. This apparently aberrant expression could be due to organ differences (for example, lung capillaries like venules allow leukocyte traffic) and to the extraordinarily high levels of cytokines found in systemic sepsis. Endothelial leukocyte adhesion molecule 1 expression was much greater in septic shock than in traumatic/ hypovolemic shock. This is consistent with previous studies that have shown massive cytokine release in the septic but not traumatic baboon models.16 Furthermore, circulating LPS levels in the plasma are several logs higher in sepsis than in trauma (S. Bahrami, unpublished results). Nevertheless, small amounts of LPS are seen after trauma due to bacterial translocation,23 perhaps accounting for the low level of ELAM-1 expression observed in the traumatic/hypovolemic shock animals. Endothelial leukocyte adhesion molecule 1 expression was used in this model as a marker for an activated endothelial state, and should not be construed as a necessary or sufficient mediator of leukocyte adhesion. In vitro studies with transfected ELAM-1 cDNA as well as antibody-blocking studies have established that ELAM-1 contrbutes to neutrophil adhesion in acutely activated endothelium.5 However ELAM-1 expression is not necessary for neutrophil adhesion, as shown by the inflammatory response to neutrophil-directed mediators, such as leukotriene B4,13 nor is it likely to be sufficient. Previous studies with ELAM-1 localization in vivo have shown that although ELAM-1 expression is associated with neutrophil adhesion under some conditions-such as after local injection of cytokines and LPS,12.13 or in acute appendicitis-ELAM-1 in vivo frequently occurs in the absence of leukocyte adhesion.511 In the current experiments, adherent leukocytes were frequently found in lung capillaries in association with ELAM-1 staining, but many
vessels, including arteries, sinusoids, and glomerular capillaries, exhibited minimal leukocyte adhesion in the presence of ELAM-1 staining. In addition to endothelial activation, septic shock or infusions of TNF induce vascular leakage, which accounts in part for the hemodynamic collapse characteristic of these states. This vascular leakiness can be due either to the direct effects of TNF on the endothelium, or to indirect effects mediated by activated neutrophils. Tumor necrosis factor in vitro causes endothelial cell retraction and increased permeability by a direct mechanism that, it has been suggested, involves a pertussis toxinsensitive G protein.24 Human recombinant TNF produces levels of pulmonary edema in neutropenic sheep equivalent to that seen in normal animals.25 Tumor necrosis factor, however, also causes aggregation and activation of neutrophils, and these, when adherent to the endothelium, may be responsible for the majority of the vascular leakiness that is associated with shock. It has been suggested that cytokine-treated endothelial cells are more susceptible both to direct lysis and to lysis by activated neutrophils.2627 The high plasma elastase levels documented in this study in animals with septic shock suggest a high degree of neutrophil activation, not seen in animals with traumatic shock. Finally although these studies are consistent with the possibility that ELAM-1 itself may contnbute to leukocyte adhesion and eventual organ damage in septic shock, the extent of this contribution will require the use of inhibitory agents such as blocking antibodies or ELAM-1 soluble forms in vivo. The results of such studies will be of great interest.
Note Added in Proof Furthermore, we observed that in cynomolgous monkeys treated with a lethal dose of endotoxin, ELAM-1 expression was also not restricted to post-capillary venules, but was widely expressed on the vasculature of the main organs including heart and brain (Engelberts 1, Samyo SK, Leeuwenberg JFM, van der Linden CJ, Buurman WA: A role for ELAM-1 in the pathogenesis of MOF during septic shock. J Surg Res (In press).
References 1. Beutler B, Cerami A: The biology of cachectirVTNF-a primary mediator of the host response. Ann Rev Immunol
1989, 7:625-655 2. Tracey K, Beutler B, Lowry S, Merryweather J, Wolpe S, Milsark 1, Hariri R, Fahey T, Zentella A, Albert J, Shires G,
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Cerami A: Shock and tissue injury induced by recombinant human cachectin. Science 1986, 243:470 3. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A: Anti-cachectin/TNF antibodies prevent septic shock during lethal bacteremia. Nature (Lond) 1987, 330:662-664 4. PoberJS, Cotran RS: Cytokines and endothelial cell biology. Physiol Rev 1990, 70:427 5. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA Jr: lnterleukin-1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocytic cell lines. J Clin Invest 1985, 76:2003-2011 6. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA Jr: Interleukin 1 (IL-1) activation of vascular endothelium: Effects on procoagulant activity and leukocyte adhesion. Am J Pathol 1985,121:393-403 7. Kyan-Aung U, Haskard DO, Poston RN, Thomhill MH, Lee TH: Endothelial leukocyte adhesion molecule-1 mediate the adhesion of eosinophils to endothelial cells in vitro and are expressed by endothelium in allergic cutaneous inflammation in vivo. J Immunol 1991, 146:521-528 8. Picker U, Kishimoto TK, Smith CW, Wamock RA, Butcher EC: ELAM-1 is an adhesion molecule for skin-homing T-cells. Nature 1991, 28:793 9. Shimizu Y, Shaw S, Graber N, Gopan TV, Horgan KJ, Van Seventer GA, Newman W: Activation-independent binding of human membory T-cells to adhesion molecule ELAM-1. Nature 1991, 349:796 10. Cotran RS, Gimbrone MA Jr, Bevilacqua MP, Mendrick DL, Pober JS: Induction and detection of a human endothelial activation antigen in vivo. J Exp Med 1986,164:661-666 11. Cotran RS, PoberJS: Endothelial activation. Its role in inflammatory and immune reactions, Endothelial Cell Biology. Edited by N Simionescu, M Simionescu. New York, Plenum, 1988, pp 335-347 12. Munro JM, Pober HS, Cotran RS: Tumor necrosis factor and interferon-y induce distinct pattems of endothelial activation and leukocyte accumulation in skin of Papio anubis. Am J Pathol 1989, 135:121-133 13. Munro JM, Pober JS, Cotran RS: Recruitment of neutrophils in the local endotoxin response: Association with de novo endothelial expression of the adhesion molecule ELAM-1. Lab Invest 1991, 64:295-299 14. Cotran RS, Pober JS: Effects of cytokines on vascular en-
dothelium: Their role in vascular and immune injury. Kidney Int 1989, 35:969 15. Cotran RS, Pober JS, Tracey KJ, Lowry SF, Cerami A: (Unpublished observations) 16. Redl H, Schlag G, Paul E, Davies J: Monocyte/macrophage activation with cytokine release after polytrauma and sepsis in baboon. Circ Shock 1989, 27:308 17. Pretorius JP, Schlag G, Redl H, Botha WS, Goosen DJ, Boisman H, van Eeden AF: The 'lung in shock' as a result of hypovolemic/traumatic shock in baboons. J Trauma 1987, 27:1344 18. Pober JS, Bevilacqua MP, Mendrick DL, Lapierre LA, Fiers W, Gimbrone MA Jr: Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J Immunol 1986,136:1680-1687 19. Leeuwenberg FM, Jeunhomme TMAA, Buurman WA: Induction of an activation antigen on human endothelial cells in vitro. Eur J Immunol 1989, 19:715 20. Lang H, Jochum PM, Fritz H, Redl H: Validity of the elastase assay in intensive care medicine, Progress in Clinical and Biological Research. Vol 308. Edited by G Schlag, H Redl. New York, Alan R. Liss, 1989, pp 701-706 21. Osborn L: Leukocyte adhesion to endothelium in inflammation. Cell 1990, 62:3-6 22. Cotran RS, Pober JS: Cytokine-endothelial interactions in inflammation, immunity and vascular injury. J Am Soc Nephrol 1990,1:225-235 23. Schlag G, Redl H, Radmore K, Davies J: Bacterial translocation in a baboon model of hypovolemic/traumatic shock. Circ Shock 1989, 27:331 24. Brett J, Gerlach H, Nawroth P, Steinberg S, Godman G, Stem D: Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J Exp Med 1989,169:1977-1991 25. Horvath CJ, Ferro TJ, Jesmok G, Malik AB: Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils. Proc Natl Acad Sci (USA) 1988, 85:9212-9223 26. Varani J, Bendelow MJ, Sealey DE, Kunkel SL, Gonnon DE, Ryan US, Ward PA. Tumor necrosis factor enhances susceptibility of vascular endothelial cells to neutrophilmediated killing. Lab Invest 1988, 59:292-295 27. Ward PA, Varani J: Mechanisms of neutrophil-mediated killing of endothelial cells. J Leuk Biol 1990, 48:97-102
Expression of Endothelial Leukocyte Adhesion Molecule-1 in Septic But Not Traumatic/Hypovolemic Shock in the Baboon Heinz Redl,* Hans P. Dinges,t Wim A. Buurman, Ces J. van der Linden, Jordan S. Pober,11 Ramzi S. Cotran,11 and Gunther Schlag* From the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology,* Vienna, Austria; the Institute of Pathology,t University of Graz, Graz, Austria; the Department of General Surgery,* University of Limburg, Maastricht, The Netherlands; and the Departments of Pathology,11 Brigham and Women's Hospital and the Harvard Medical School, Boston, Massachusetts
Baboons were subjected to septic or traumatic! hypovolemic shock and their tissues were examined for the de novo expression of endothelial leukocyte adhesion molecule I (ELAM-1), using immunohistochemical techniques. In animals with septic shock induced with live Escherichia coli there was widespread expression of EL4M-1, recognized by monoclonal antibodiesH4/18 orENA-1 in most tissues examined with strong staining in the lung, liver, and kidneys. Endothelial leukocyte adhesion molecule 1 expression was evident in capillaries, venules, small veins, arterioles, and arteries. In contrast, baboons with traumatic/hypovolemic shock had minimal levels offocal ELAM expression in all organs studied Similarly evidence of neutrophil activation measured by granulocyte elastase levels in the plasma was much more pronounced in animals with septic shock The study documents that lipopolysaccharide (LPS)- and cytokine-induced endothelial activation occurs in vivo in septic shock Much higher levels of ELAM- I expression and plasma granulocyte-elastase titer in septic shock, as contrasted with traumatic! hypovolemic shock, are consistent with the higher levels of circulating tumor necrosis factor, other cytokines, and LPS in sepsis. (Am J Pathol 1991,
139:461-466)
Septic shock is a syndrome characterized by coagulation disturbances, hemodynamic collapse, and multior-
gan damage developing in the setting of systemic infection, usually with gram-negative bacteria. Lipopolysaccharide (LPS) released from the bacterial cell wall is believed to be central to the manifestations of shock. Lipopolysaccharide acts directly and through the induction of a variety of mediators, including cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).1 Infusions of TNF into experimental animals mimic many of the hemodynamic and vascular changes of septic shock,2 including hypotension, coagulopathy, leukocyte aggregation, and vascular leakage. Anti-TNF antibodies prevent septic shock in experimental lethal Escherichia co/i septicemia.3 In vitro studies recently have shown that one of the major targets of LPS and cytokine actions is vascular endothelium, which undergoes a variety of functional and structural alterations, collectively called endothelial activation.4 Principal among these changes is increased adhesivity of endothelium to leukocytes and the induction of procoagulant activities on the surface of endothelial cells, rendering them thrombogenic. Currently, however, there is little evidence that these endothelial alterations either occur in vivo in septic shock or account for the pathologic manifestations of shock, because both LPS and cytokines have a variety of effects on monocytes, cardiac and vascular muscle cells, and other cell types. We sought to obtain evidence for endothelial cell activation in septic shock by examining for the expression of endothelial leukocyte adhesion molecule 1 (ELAM-1 ).5 This is an endothelial-specific molecule, induced by LPS, TNF, and IL-1, which mediates endothelial adhesion to neutrophils,6 eosinophils,7 and memory T cells.89 Previous studies in humans have shown that ELAM-1 is an extremely useful marker for endothelial activation,10'11
Supported by grants from 'Lorenz Bohler Fond' and the National Institutes of Health (PO1 HL 36028). Accepted for publication April 15, 1991. Address reprnt requests to Heinz Redl, PhD, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, A-1200, Vienna, Austna.
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and experiments in baboon skin show that postcapillary venular endothelial cells express ELAM-1 in response to interdermal injections of TNF12 or LPS.13 In a preliminary study, we have also described that ELAM-1 is expressed in the endothelium of baboons perfused with live E.
coil.415 The present study was designed to compare the responses of endothelial cells in septic shock, thought to be mediated by LPS and cytokines, with those in a model of traumatic/hypovolemic shock, a syndrome with similar hemodynamic alterations, but without significant cytokine release.16 We report that endothelial activation, detected by ELAM-1 expression, is strongly evident in septic shock, but not in traumatic/hypovolemic shock. These data support the interpretation that endothelial cell activation may contribute to organ damage in septic shock.
Materials and Methods
Animals and Experimentation Male baboons (Papio ursinus) 20 to 25 kg in body weight were sedated with ketamine hydrochloride and placed in the supine position. Anesthesia was maintained with pentobarbital (2 to 5 mg/kg/hr). The depth of anesthesia was monitored by fronto occipital electroencephalogram (EEG) and by determination of the arterial C02 (35 to 40 mmHg) and end expiratory C02, and adjusted by a servo-assisted mechanism after Fourier spectrum analysis of EEG. Basal blood temperature was 370C ± 0.50C. and inspired oxygen concentration was set at 30%. Pulmonary artery pressure and mean arterial pressure were monitored, and cardiac output was measured using thermodilution techniques. Tissues were examined for ELAM-1 expression from six baboons with septic shock and 13 with traumatic/hypovolemic shock.
Traumatic/Hypovolemic Shock Traumatic hypovolemic shock was induced in anesthetized baboons as described previously.16,17 Trauma was followed by withdrawing sufficient blood to attain a mean arterial pressure adjusted between 35 and 40 mmHg, so that the cardiac output (CO) was reduced by 50% to 70%. After 3 hours, retransfusion of the withdrawn heparinized blood, together with Ringer's solution, was started. During the retransfusion phase (3 hours), CO was maintained between 15% and 20% above the baseline by the addition of Ringer's solution. Animals were killed 6 hours after trauma with an overdose of pentobarbital. Sham animals were subjected to all procedures except the infliction of trauma and hypovolemia.
Septic Shock Protocol To induce septic shock, 2 x 1010 live E. coli/kg (Hinshaw's strain B7 086a61 ATCC#33985) were infused intravenously over an 8-hour period. Intravenous fluid was given to maintain constant pulmonary arterial wedge pressure and cardiac output. Animals were then killed by pentobarbital anesthesia at either 6 or 8 hours. The studies on traumatic and septic shock models in baboons were approved by the ethics committee of the Roodeplaat Research Laboratories, according to guidelines advocated by the International Laboratory Animal Science Committee (Dr. S. J. von Rensburg, Chairman).
Immunohistochemical Studies Endothelial leukocyte adhesion molecule 1 was localized with the use of two monoclonal antibodies, H4/1818 and ENA-1,19 both of which react with ELAM-1. PBS or an unreactive mouse immunoglobulin was used as a negative control. Tissues were removed at autopsy and immediately quick frozen and kept at - 70°C until cryostat sectioning. Four-micron-thick frozen sections were fixed for 10 minutes in cold acetone (- 20°C) and air dried. Sections then were incubated with the primary antiserum diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, in a humidified chamber at room temperature. Dilutions of the primary antiserum were optimized in several pilot experiments for H4/18 1/500 and ENA-1 1/1000. Sections were incubated for 1 hour with the primary antiserum, washed, and then incubated for 30 minutes with rabbit anti-mouse immunoglobulin (Dakopatts; Glostrum, Denmark) at a dilution of 1:30 in PBS. Sections were then washed and exposed to the APAAP-complex (Dakopatts) for 30 minutes. The color reaction then was developed with substrate solution containing 10 mg naphthol-AB-MX-phosphate (Serva; Heidelberg, Federal Republic of Germany), dissolved in 0.5 ml N,Ndimethyformamide (Serva) and 50 ml barbitone buffer pH9.2, 50 mg fast red TR (Serva), and 10 mg levamisole (Sigma; St. Louis, MO). Before use, the solution was adjusted to pH 9.2 to 9.8 and filtered. The sections were incubated for 15 to 30 minutes at room temperature in this solution and then mounted and examined. Sections were examined and quantitated by two of the authors (RC and HD) without knowledge of the identity of the animal from which the tissue was derived. The intensity of endothelial staining was assessed as follows: negative; ± (equivocal or minimal staining); + 1 (mild but definite staining); + 2 (moderate staining); + 3 (strongest
staining).
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Plasma Analysis Granulocyte elastase was measured using the IMAC assay (Merck, Federal Republic of Germany) on a Hitachi 704, using human elastase standards.20
Results
Immunohistochemical Studies The results with both antibodies were qualitatively similar. The semiquantitative evaluation was done only with sections stained with the ENA-1 antibody (Table 1). All six animals with septic shock showed significant endothelial staining for ELAM-1 in the kidneys, lung, liver, intestines, and skin. In the kidneys, there was strong staining restricted to vascular endothelium, in glomeruli and peritubular capillaries, and there was strong and distinctive staining of the endothelium in arteries (Figures 1 to 3). Intraglomerular fibrin thrombi were frequent, consistent with the increased surface procoagulant activity characteristic of activated endothelium (Figure 4). In the lung, the staining was strong but patchy and involved capillaries in the alveolar walls, as well as venules and occasional arterioles in the interlobular fibrous septae (Figure 5). In the liver, staining was diffuse, involving the lining of the majority of sinusoids, as well as venules and small veins in the portal tracts (Figure 6). Endothelial leukocyte adhesion molecule 1 expression was weakest and most spotty in skeletal muscle. In contrast, animals subjected to traumatic/ hypovolemic shock had only minimal expression of ELAM-1 after 6 hours of shock (Table 1). There was virtually no staining in the lung and liver, and the intensity of staining in the kidney was significantly less than that in kidneys of animals with septic shock, being restricted to mild (+ 1) glomerular staining, except in one animal, in which there was also + 1 staining of arteries. To measure granulocyte activation, neutrophilspecific elastase levels were measured in plasma using an assay system developed for the human enzyme. Although baseline levels (1 to 20 ,ugA) are lower than nor-
Figure 1. Low-power illustration of renal cortex in septic shock showing ELAM-1 staining of endothelium of glomerular and peritubular capillaries and small artery (louer left).
mal human levels, there is cross-reactivity with the baboon enzyme, and the shock-related proportional increases are comparable to those in the clinical situation. As seen in Table 2, sepsis caused the expected rapid elevated elastase levels; markedly less activation was noted in the animals subjected to traumatic/hypovolemic shock.
Discussion The present study demonstrates that strong widespread multiorgan expression of ELAM-1 occurs in septic shock.
Table 1. Immunohistochemical Localization of ELAM-1 in Septic and Traumatic Shock Traumatic Shock Septic Shock No. Average No. Average score examined score examined 0.03+ 2.2+ 13 6 Lung 12 0.16+ 3.0+ 5 Liver 0.8+ 2.7+ 9 3 Kidney 2 0.5+ 2 3.0+ Skin The average score denotes average level of ELAM-1 expres-
sion, on a scale of 0, ± (0.5), 1, 2 and 3+ in the group examined.
Figure 2. High magnification of glomerulus ELAM-1 staining is restricted to endothelium.
showing that the
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Figure 3. Small renal artery showing distinct endothelial staining for ELAM-1.
These observations are consistent with the current hypotheses that septic shock is mediated by systemic LPS and by induced cytokines, especially TNF and IL-1, and that LPS and these cytokines act on vascular endothelial cells to cause their activation. We chose ELAM-1 as a measure of endothelial activation for the following reasons: In vitro studies indicate that ELAM-1 is a marker of acute activation, and its time-sequence of induction is consistent with that of the induction of septic shock in this model.5'18 Secondly ELAM-1 is not constitutively present in normal endothelium, unlike other endothelial activation markers (such as ICAM-1 and INCAM-i 1 ONCAM-1),21 and is a reliable evidence of LPS- and cytokine-induced activation when it is present.22 Thirdly antibodies to hu-
Figure 4. H&E-stained glomerulus from dxhok showing fbrin thrombi.
an animal
edh septic
Figure 5. Lung from septic shock, showing ELAM-1 staining of endothelium of a venule (arrow) and spotty staining of capillaries.
I
Note adherent leukocytes (arrow beads) in venular lumen.
man ELAM-1 cross-react with baboon endothelium12; and finally we have previously used ELAM-1 expression to detect evidence of endothelial activation in human diseases in vivo10'11 and after local injections of endotoxin and cytokines in baboons.12'13 Endothelial activation was widespread, involving most
Figure 6. Section of liver with diffuse staining of sinusoidal lining
endothelium.
ELAM-1 Expression in Septic and Traumatic Shock 465 AJP August 1991, Vol. 139, No. 2
Table 2. Granulocyte Elastase Levels in Plasma Measured by Immunoassay in Baboons Subjected to Hypovolemic/Traumatic (Sham) or Hyperdynamic Septic Shock (lxg/l) Hypovolemic/ traumatic Groups shock (hrs/animal) Sepsis 15 ± 2 0 6±1.3 2 30±6 270±45.0 4 62±8 262±26.0* 6 388 31.0* 77 ± 15 ND 8 304 ± 27.0 Sepsis statistically different from hypovolemic traumatic shock (P < 0.05) using Wilcoxon statistics.
organs examined, but was particularly strong in the kidney, liver, and lung. Of great interest is that ELAM-1 was expressed not only in venules, as previously noted in the setting of local immune inflammation in humans in vivo,5,11 or after the administration of endotoxin and cytokines in baboon skin in vivo,12'13 but also was observed on capillaries, arterioles, arteries, and small veins. This apparently aberrant expression could be due to organ differences (for example, lung capillaries like venules allow leukocyte traffic) and to the extraordinarily high levels of cytokines found in systemic sepsis. Endothelial leukocyte adhesion molecule 1 expression was much greater in septic shock than in traumatic/ hypovolemic shock. This is consistent with previous studies that have shown massive cytokine release in the septic but not traumatic baboon models.16 Furthermore, circulating LPS levels in the plasma are several logs higher in sepsis than in trauma (S. Bahrami, unpublished results). Nevertheless, small amounts of LPS are seen after trauma due to bacterial translocation,23 perhaps accounting for the low level of ELAM-1 expression observed in the traumatic/hypovolemic shock animals. Endothelial leukocyte adhesion molecule 1 expression was used in this model as a marker for an activated endothelial state, and should not be construed as a necessary or sufficient mediator of leukocyte adhesion. In vitro studies with transfected ELAM-1 cDNA as well as antibody-blocking studies have established that ELAM-1 contrbutes to neutrophil adhesion in acutely activated endothelium.5 However ELAM-1 expression is not necessary for neutrophil adhesion, as shown by the inflammatory response to neutrophil-directed mediators, such as leukotriene B4,13 nor is it likely to be sufficient. Previous studies with ELAM-1 localization in vivo have shown that although ELAM-1 expression is associated with neutrophil adhesion under some conditions-such as after local injection of cytokines and LPS,12.13 or in acute appendicitis-ELAM-1 in vivo frequently occurs in the absence of leukocyte adhesion.511 In the current experiments, adherent leukocytes were frequently found in lung capillaries in association with ELAM-1 staining, but many
vessels, including arteries, sinusoids, and glomerular capillaries, exhibited minimal leukocyte adhesion in the presence of ELAM-1 staining. In addition to endothelial activation, septic shock or infusions of TNF induce vascular leakage, which accounts in part for the hemodynamic collapse characteristic of these states. This vascular leakiness can be due either to the direct effects of TNF on the endothelium, or to indirect effects mediated by activated neutrophils. Tumor necrosis factor in vitro causes endothelial cell retraction and increased permeability by a direct mechanism that, it has been suggested, involves a pertussis toxinsensitive G protein.24 Human recombinant TNF produces levels of pulmonary edema in neutropenic sheep equivalent to that seen in normal animals.25 Tumor necrosis factor, however, also causes aggregation and activation of neutrophils, and these, when adherent to the endothelium, may be responsible for the majority of the vascular leakiness that is associated with shock. It has been suggested that cytokine-treated endothelial cells are more susceptible both to direct lysis and to lysis by activated neutrophils.2627 The high plasma elastase levels documented in this study in animals with septic shock suggest a high degree of neutrophil activation, not seen in animals with traumatic shock. Finally although these studies are consistent with the possibility that ELAM-1 itself may contnbute to leukocyte adhesion and eventual organ damage in septic shock, the extent of this contribution will require the use of inhibitory agents such as blocking antibodies or ELAM-1 soluble forms in vivo. The results of such studies will be of great interest.
Note Added in Proof Furthermore, we observed that in cynomolgous monkeys treated with a lethal dose of endotoxin, ELAM-1 expression was also not restricted to post-capillary venules, but was widely expressed on the vasculature of the main organs including heart and brain (Engelberts 1, Samyo SK, Leeuwenberg JFM, van der Linden CJ, Buurman WA: A role for ELAM-1 in the pathogenesis of MOF during septic shock. J Surg Res (In press).
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