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Shwartzman Reaction
The local Shwartzman reaction is a hemorrhagic and necrotizing inflammatory lesion elicited by endotoxin from gram-negative bacteria. Shwartzman1,2 and Hanger3 demonstrated in rabbits that an intradermal injection of a sterile bacteria culture filtrate, containing endotoxin, followed by an intravenous injection of the same filtrate 20 to 24 hours later resulted in a local hemorrhagic and necrotic lesion developing at the site of the intradermal injection (Fig. 1). This reaction is now called the local Shwartzman reaction. It is a two-stage reaction requiring an initial preparatory intradermal injection of endotoxin and an intravenous injection of endotoxin 18 to 24 hours later. The time interval between injections is a crucial determinant for the reaction to occur, since if the intravenous injection of endotoxin is made much earlier or later, a hemorrhagic lesion at the site of the intradermal injection does not occur. It is interesting that the local Shwartzman reaction could be elicited in only 167 of 212 rabbits tested in the early experiments of Shwartzman.2 Some rabbits who still showed erythema at the sites of intradermal injection at the time of intravenous challenge only developed a more pronounced erythema 4 to 5 hours after the intravenous challenge, but no hemorrhage or necrosis. Why some rabbits are resistant to developing a localized Shwartzman reaction is still not known. A generalized Shwartzman reaction can be elicited by two intravenous injections of endotoxin spaced 18 to 24 hours apart. The generalized Shwartzman reaction first described by Sanarelli4 and Apitz5,6 is a model for disseminated intravascular coagulation (DIC). Rabbits receiving a second intravenous injection of endotoxin become obviously ill within 4 to 6 hours after the
injection, having symptoms of increasing weakness and dyspnea.7 The intravenous challenge injection of endotoxin in rabbits causes neutropenia and thrombocytopenia. Circulating neutrophils rapidly decrease during the first 15 minutes after injection with margination of neutrophils in the pulmonary vascular bed accounting for most of the decrease in circulating neutrophils.8,9 A decrease in circulating platelets parallels the decrease in circulating neutrophils. Approximately 4 hours after injection, the number of circulating neutrophils gradually increases. Conjunctival petechial hemorrhages develop shortly before death, 18 to 24 hours after the second injection. Kidneys show bilateral cortical necrosis with numerous fibrin microthrombi in glomerular capillaries. Examination of lungs, liver, and lymph nodes also shows varying degrees of hemorrhage and necrosis. The bilateral cortical necrosis with fibrin microthrombi is the most characteristic anatomic lesion of the generalized Shwartzman reaction.2 Similar to the local Shwartzman reaction, the generalized reaction is a two-step phenomenon requiring both a preparative intravenous injection of endotoxin and a provocative injection. Timing of the second injection is critical, with the optimum interval being 18 to 24 hours after the first injection. Tissue damage in the local Shwartzman reaction appears similar to tissue damage in the generalized reaction. These observations pointed to a common mechanism involved in the pathogenesis of both the local and generalized Shwartzman reactions.6,7 Since the local reaction is easier to manipulate and more predictable than the generalized reaction, the local Shwartzman reaction in animals has been used as a model of the general Shwartzman reaction and DIC in humans.
HISTOPATHOLOGY From the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. Reprint requests: Dr. Brozna, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-3135. 326
An intradermal injection of endotoxin elicits a transient inflammatory reaction at the site of injection characterized initially by hyperemia, increased vascular
Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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JOHN P. BROZNA, M.D., Ph.D
FIG. 1. Thrombohemorrhagic lesions in skin sites that received injections of bacterial filtrate 24 hours before an intravenous challenge. (From Shwartzman.2 Reprinted with permission.)
permeability, and leukocyte infiltration.1'2'10 Initially, there is margination and emigration of neutrophils in small veins and venules with neutrophils then infiltrating into the dermis.11 Using labeled cells and proteins, the accumulation of neutrophils was determined to be maximal 4 hours after the intradermal injection. The accumulation of neutrophils after an intradermal injection of endotoxin does not progress beyond 6 hours after the intradermal injection.12 Maximum accumulation of monocytes occurs at about the time of maximum neutrophil infiltration, but monocytes continue to accumulate for at least 24 hours after injection. At the time of intravenous injection of endotoxin, 18 to 24 hours after the initial intradermal injection, residual polymorphonuclear neutrophils and monocytes are still present at the intradermal site. The small veins and venules show perivascular cuffing with neutrophils. The vessels are patent and there is no evidence of hemorrhage or necrosis.13 Shwartzman2 originally observed that the intensity of the hemorrhagic reaction occurring after the intravenous injection of endotoxin did not correlate with the intensity of the initial inflammatory reaction elicited with the intradermal injection of endotoxin, even in rabbits that did not develop a hemorrhagic lesion after intravenous challenge. Other investigators have observed that the intensity of the thrombohemorrhagic reaction at intradermal injection sites induced by intravenous chal-
327 lenge tended to correlate with the intensity of the initial inflammatory reaction at sites of intradermal injection. 312 Shwartzman estimated the initial inflammatory response by observing qualitatively the magnitude of the erythematous response to the intradermal injection of endotoxin and not by quantitating the intensity of the leukocyte inflammatory cell infiltrate. Recent reports suggesting a correlation between intensity of the initial inflammatory reaction to intensity of the thrombohemorrhagic reaction are based on actual measurements of leukocyte inflammatory infiltrates, which are maximal approximately 4 hours after the intradermal injection of lipopolysaccharide (LPS). It is interesting that a Shwartzman reaction cannot be elicited 4 hours after the preparative injection of LPS, even though at that time dermal inflammation is maximal. A modest thrombohemorrhagic lesion can only be elicited by an intravenous injection of LPS administered at least 12 to 15 hours after the preparative injection. This would suggest that inflammatory mediators released during the early inflammatory reaction require a minimum time interval to prepare the skin for a local Shwartzman reaction. It is unknown which cells in the skin are essential for preparing it for a local Shwartzman reaction or for producing a Shwartzman reaction in response to intravenously administered LPS. Thrombohemorrhagic lesions develop rapidly after intravenous challenge. Within 15 minutes of the intravenous challenge, the small veins and capillaries in the prepared skin sites show microthrombi composed of platelets, fibrin, and varying numbers of neutrophils. Two hours after the injection, perivascular neutrophil infiltrates appear associated with a necrotizing vasculitis involving small veins and venules. Endothelial cells become swollen and red blood cells leak into the surrounding tissue. Within 4 to 5 hours after the intravenous challenge, the severest hemorrhage and necrosis are fully developed.2 The histopathologic lesions are essentially those of vascular injury. Necrosis is severest when the preparatory skin test is made 18 to 24 hours before the intravenous injection and is rare when the intravenous injection is given less than 12 hours or more than 48 hours after the intradermal injection. In contrast to skin sites prepared with an intradermal injection of endotoxin, sections of normal skin obtained before or after the intravenous injection of endotoxin show no histologic abnormalities.13 It is not clear why fibrin microthrombi and aggregates of platelets and neutrophils are found primarily at skin sites previously prepared with endotoxin, and not in the capillaries of skin not prepared by an intradermal injection of endotoxin. This observation suggests that endothelium of capillaries in sites prepared with LPS is more thrombogenic or more sticky for leukocytes and platelets, or both. Hence, it
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would appear that the key to understanding the Shwartzman reaction in animals and purpuric skin diseases in humans is to focus on the endothelial cell and its response to inflammatory mediators.
Possible Mechanisms Involved in the Local Shwartzman Reaction Classically, gram-negative endotoxin has been used to prepare a skin site for a local Shwartzman reaction. Other agents that have been used successfully as prepa ratory agents for a local Shwartzman reaction include vaccinia virus,14 isolated leukocyte granules,15 and tu berculin in animals immunized with bacille CalmetteGuerin. 1617 Some agents such as turpentine, which induce inflammation in the skin, cannot prepare it for a local Shwartzman reaction. Hence, it would seem that not all agents that can induce an inflammatory reaction in the skin can prepare a skin site for a local Shwartzman reaction. Inhibitors of proteolytic enzymes can prevent the local Shwartzman reaction, presumably by blocking vascular injury and the inflammatory reaction induced by leukocyte proteolytic enzymes. 15,18 Intravenous injec tion of trypsin inhibitor (500 µg of either soybean or pancreatic trypsin inhibitor) in rabbits 15 minutes before the provocative injection of endotoxin inhibits the for mation of thrombohemorrhagic lesions at the site of previously injected endotoxin 24 hours earlier.18 Other similar studies demonstrated that depletion of neutrophils in rabbits with nitrogen mustard suppresses the local Shwartzman reaction.19 These data suggest a role for neutrophils and neutrophil proteases in the pathogenesis of the local Shwartzman reaction. However, nitrogen mustard in addition to depleting neutrophils is toxic for endothelial cells and also causes a decrease in peripheral blood monocytes.20 Hence, it is not possible to exclude other cell types in the pathogenesis of a Shwartzman reaction. The thrombohemorrhagic lesions of a local Shwartzman reaction are manifestations of small vessel thrombosis and acute necrotizing vasculitis. Antiproteases presumably block the acute vascular injury medi ated by neutrophils. Since a detailed histopathologic picture of prepared skin sites in animals treated with protease inhibitors has not been described, it is not known what effect antiproteases have on the formation of leukocyte, platelet, or fibrin microthrombi in prepared skin sites after an intravenous injection of LPS. It would be interesting to determine whether small microthrombi form at prepared skin sites after a provocative intrave nous injection of LPS in animals treated with antipro teases. Since no hemorrhagic lesions develop in animals treated with protease inhibitors, the presence of micro
thrombi at prepared skin sites in dermal vessels would imply that thrombi are not present for a sufficient time to induce ischemic injury leading to hemorrhage. In purpu ric skin lesions associated with protein C deficiency, there is an early reversible lesion characterized by fibrin microthrombi in dermal vessels.21 Administration of plasma containing protein C blocks the formation of hemorrhagic lesions, presumably by facilitating removal of microthrombi and preventing the formation of new thrombi. It remains to be determined whether the local Shwartzman reaction in animals treated with protease inhibitors is a model for the early reversible skin lesions in patients with deficient protein C. It is likely that neutrophils contribute to vascular injury, resulting in hemorrhage in the Shwartzman reac tion, but neutrophils do not appear to contribute directly to the thrombotic process. Since peripheral blood monocytes22-24 and endothelial cells 25,26 produce tissue factor after exposure to small amounts of endotoxin, it is thought that monocytes or endothelial cells, or both, modulate thrombosis at sites of inflammation. In partic ular, since monocytes produce a number of potent inflammatory mediators that can activate other cells as well as induce tissue injury (Table 1), monocytes may play a key role in modulating tissue injury and preparing skin sites for a local Shwartzman reaction. It is thought that a crucial prerequisite for a preparatory agent is the induction of an inflammatory reaction that is associated with activation of leukocytes and the release of specific inflammatory agents such as interleukin-1 (IL-1), tissue necrosis factor (TNF) or interferon-gamma (IF-γ). 10,17 Cultured endothelial cells, when activated by inflamma tory mediators such as endotoxin or IL-1, express tissue factor procoagulant activity that is maximal 8 to 12 hours after activation (Fig. 2). The procoagulant activity then gradually declines to baseline levels 24 to 48 hours after LPS stimulation. In Figure 2 tissue factor activity was measured using purified Factors VII, X, and a chromoge-
TABLE 1. Inflammatory Mediators Produced by Activated Macrophages Interleukin-1 Tumor necrosis factor Tissue factor procoagulant Interferons Transforming growth factors Proteases Toxic oxygen metabolites Chemotactic factors Neutrophil activating factors Plasminogen activator inhibitor
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FIG. 2. Human umbilical cord endothelial cells were cultured in the presence of media ( ) or media containing endotoxin (10 ng/ml) (●). Tissue factor procoagulant activity was measured at different time intervals after LPS stimula tion in a chromogenic assay using purified Factors X and VII and a synthetic substrate for Factor Xa. Tissue factor activity is expressed as [(A405/min2/105 cells] × 106] ± SD.
nic Factor Xa substrate. Using this method tissue factor procoagulant activity is proportional to ([A405/min2/105 cells] × 105).24 Endothelial cells lining vascular surfaces are in a position to play an important role in regulating intravascular coagulation. It was once thought that endothelial cells were metabolically inert and did not have an active role in regulating coagulation. During the past decade, it has become apparent that endothelial cells are metabolically active and synthesize numerous agents that can modulate both coagulation and inflammatory reactions at the endothelial cell membrane surface (Table 2). Normally, endothelial cells present a nonthrombogenic membrane surface to coagulation factors in plasma, but when stimulated by endotoxin they become thrombogenic.25-27 Endotoxin induces endothelial cells, as well as monocytes, to synthesize and express tissue factor, the most potent procoagulant known on the plasma membrane surface. Tissue factor activates the extrinsic pathway of coagulation by forming a complex with Factor VIIa to activate Factor X to Xa. Endotoxin also induces suppression of endothelial cell thrombo-
TABLE 2. Functional Responses of Endothelial Cells Expression of tissue factor procoagulant Expression of thrombomodulin Modulation of protein S-protein C anticoagulant system Release of platelet-derived growth factor Generation of coagulation proteases Release of interleukin-1 Synthesis of prostacyclin
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modulin, which is an important cofactor in the protein C thrombomodulin anticoagulant system.27,28 Thrombinactivated protein C is a serine protease and a potent inhibitor of Factor Va and Factor VIIIa of the coagulation cascade.29 The protein C thrombomodulin anticoagulant system functions under in vivo conditions to suppress thrombosis and is thought to be essential for maintaining homeostasis and ensuring normal flow of blood. It has been shown that protein C can prevent the coagulopathies and lethal effects of LPS in the baboon.30 Baboons injected with LPS develop lethal DIC, whereas, baboons injected with protein C and LPS were protected. Similarly, baboons injected with antibodies to protein C developed DIC with lower amounts of LPS. The protective effects of protein C might be explained in part by protein C anticoagulant properties and by protein C inhibition of endothelial cell endocytosis of thrombomodulin-thrombin complexes,31 which would oppose the thrombogenic effects of endotoxin and other inflammatory cytokines. It is possible that endotoxin induces endothelial cells in vivo to be thrombogenic by inducing an increase in tissue factor procoagulant and a decrease in thrombomodulin. IL-1 and TNF, which are other inflammatory mediators released from leukocytes at inflammatory foci, have been shown to induce an increase in endothelial cell tissue factor32'33 and to cause a decrease in endothelial cell thrombomodulin.34 Usually, anticoagulant mechanisms predominate on the endothelial membrane surface, but when perturbed by endotoxin, IL-1, or TNF, the balance is shifted toward thrombosis.
Role of Cytokines in the Local Shwartzman Reaction Although the provocative intravenous injection of LPS can be substituted by a number of agents, including immune-complexes,8 vaccinia virus,15 zymosan, kaolin, and agar,13'35 until recently it had been difficult to substitute for the preparative intradermal injection of endotoxin in the local Shwartzman reaction. The prerequisites for preparing a skin site for a subsequent thrombohemorrhagic reaction elicited with an intravenous injection of LPS are unknown. However, the ability to elicit a local inflammatory reaction with infiltrating inflammatory cells at the site of injection appears to be an important property of a preparatory agent. Recently, it was shown that IL-1 can induce an acute inflammatory response in the skin very similar to the inflammatory reaction elicited with LPS. 36 It was shown that an intradermal injection of IL-1 can prepare a skin site in rabbits for a local Shwartzman reaction, provoked by an intravenous injection of LPS. In addition, TNF was found to act synergistically with IL-1 to prepare skin sites
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for a local Shwartzman reaction. The combination of IL-1 and TNF was more potent than either agent alone in preparing a site for a Shwartzman reaction.17 Clearly, endothelial cells at the site of LPS injection are altered, so that 18 to 24 hours after the initial dermal injection of LPS a provocative intravenous injection of LPS elicits a local Shwartzman reaction by causing leukocytes to adhere specifically to the endothelium of small vessels in the prepared skin site. Therefore it would appear that inflammatory agents that can prepare skin sites for a Shwartzman reaction cause the membrane surface of endothelial cells to become "sticky" for leukocytes. Numerous cytokines, including IL-1, TNF, and IF-γ, are released from inflammatory cells at sites of inflammation that affect the endothelial cell plasma membrane (Table 3). 17,35,36 In particular, monocytes at sites of inflammation produce a large number of biolog ically active molecules, including IL-1, 3 7 , 3 8 TNF, 39 and IF-7. 40 IL-1 is an important immunoregulatory molecule that has many diverse effects on nonspecific host defense mechanisms. Monocytes37 and keratinocytes41 are pos sible sources of IL-1 in skin sites affected by immuno logic or other inflammatory reactions. IL-1 is chemotactic for neutrophils in vitro and can induce leukocytes to adhere to endothelial cells. Leukocyte adherence to endothelial cells is a pivotal event in the inflammatory response and a prerequisite for leukocyte-dependent vascular injury. 38,42,43 Numerous recent in vitro experiments have shown that IL-1, TNF, LPS, and IF-7 can cause human umbil ical cord endothelial cells to become more adhesive for isolated peripheral blood leukocytes. 43-47 Recent studies have identified specific inducible endothelial cell recep tors involved in leukocyte adherence to endothelial cells. These include intercellular adherence molecule-1 (ICAM-1)46 and endothelial-leukocyte adhesion mole cule-1 (ELAM-1),47 which can be induced by IL-1, LPS, or TNF treatment of endothelial cells. These adhesion molecules are thought to be ligands for the family of heterodimeric leukocyte cell surface proteins LFA-1, MO-1, p150 43,44 or other undefined adhesion molecules on leukocytes. Expression of leukocyte adherence-pro TABLE 3. Functional Effects of Interleukin-1 on Endothelial Cells
moting proteins can also be increased by IL-1, TNF, or LPS treatment of leukocytes. The leukocyte adherence-promoting proteins consist of three distinct molecules with different α-subunits and a common ß-chain subunit. The individual α-subunits are designated CD1 la for LFA-1, CD11b for MO-1, and CD11c for p150 and the common α-subunit CD18. 48 Several recent studies using monoclonal antibodies di rected against individual subunits of the leukocyte adhe sion-promoting proteins and to the endothelial cell ad hesion proteins ICAM-1 and ELAM-1 indicated that CD11/CD18 receptors are important for migration of leukocytes across small vessels. Intravenous injection of monoclonal antibodies directed against CD18 blocked the development of tissue injury and the development of leukocytosis in the cerebrospinal fluid of animals chal lenged intracisternally with living bacteria or endo toxin.49 In other studies it was shown that adherence of human neutrophils to human umbilical vein endothelial cells was dependent on ICAM-1 on endothelial cells and on CD11/CD18 glycoproteins on neutrophils.43 It was reported that unstimulated neutrophils exhibit LFA1-dependent attachment to ICAM-1, whereas neutro phils stimulated with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine exhibited enhanced bind ing to ICAM-1 by a MO-1 dependent process. From in vivo and in vitro studies, it is clear that endothelial cells and neutrophils have distinct surface adhesion molecules that are regulated independently and can be induced by a number of cytokines found in sites of inflammation, including IL-1, TNF, LPS, and IF-7. The interaction of leukocyte and endothelial cell adhesion molecules is complex and probably important in initiating inflamma tory reactions. The CD11/CD18 receptor complex is expressed in the unstimulated neutrophil and can be up-regulated by cytokine stimulation.50 Unstimulated endothelial cells express low levels of ICAM-1, which can also be up-regulated by cytokines,-whereas ELAM-1 is not expressed in unstimulated endothelial cells but can be induced by inflammatory cytokines. 42,45,51 These observations indicate that leukocyte adherence to endo thelial cells increases after leukocytes or endothelial cells are activated by cytokines. Hence, preparation of skin sites for a local Shwartzman reaction might be dependent on cytokines released from endotoxin-activated mono cytes at sites of induced inflammation, leading to alter ations of endothelial cell membrane surfaces.
Increased tissue factor procoagulant expression Suppressed thrombomodulin-protein C anticoagulant system Increased plasminogen activator inhibitor Decreased plasminogen activator Increased expression of ICAM-1 adhesion molecule Increased expression of ELAM-1 adhesion molecule Increased platelet activating factor
Possible Role for Endothelial Cell Adhesion Molecules in the Local Shwartzman Reaction Intradermal injection of skin sites with LPS elicits an inflammatory reaction that prepares the site for a local Shwartzman reaction provoked 18 to 24 hours later by an
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SUMMARY The local and generalized Shwartzman reactions are models of thrombohemorrhagic skin necrosis and DIC, respectively. An intravenous preparatory injection of endotoxin followed by an intradermal injection of endotoxin 24 hours later elicits a thrombohemorrhagic lesion only at the site of intradermal injection of endotoxin in the local Shwartzman reaction. Two intravenous injections of endotoxin spaced 24 hours apart induce a systemic generalized Shwartzman reaction characterized by coagulopathy, petechial hemorrhages, microthrombi, and decreased circulating platelets similar to DIC. Of particular interest is the observation that thrombohemorrhagic lesions of the Shwartzman reaction only develop at sites of intradermal injections of endotoxin. Microthrombi composed of platelets and leukocytes only adhere or accumulate in dermal vessels after an intrader-
mal injection of endotoxin. Prior to the endotoxin injection, biopsies of skin show normal vessels without microthrombi or significant inflammation. Since endothelial cells line the small vessels in the dermis, where a Shwartzman reaction appears to be initiated, it is likely that endothelial cells are important for initiating a local Shwartzman reaction. IL-1 and TNF can substitute for the intradermal injection of endotoxin in the local Shwartzman reaction, induce endothelial cells to become thrombogenic, and can induce the expression of cell adhesion molecules on endothelial cells making endothelial cells more sticky for leukocytes. These observations suggest that endothelial cells play a central role in the local Shwartzman reaction and may be important in understanding diseases associated with thrombohemorrhagic skin necrosis.
REFERENCES 1. Shwartzman G: A new phenomenon of local skin reactivity to B. typhosus culture filtrate. Proc Soc Exp Biol Med 25:560-561, 1928. 2. Shwartzman G: Phenomenon of Local Tissue Reactivity and its Immunological, Pathological and Clinical Significance. Paul B. Hoeber, New York, 1937. 3. Hanger FM: Effect of intravenous bacterial filtrates on skin tests and local infections. Proc Soc Exp Biol Med 25:775-777, 1928. 4. Sanarelli G: De la pathogenie du cholera (neuvieme memoire). Le cholera experimental. Ann Inst Pasteur 38:11-72, 1924. 5. Apitz K: Die Wirkung bakterieller Kulturfiltrate nach Umstimmung des gesamten Endothels beim Kaninchen. Virchows Arch Pathol Anat 293:1-33, 1934. 6. Apitz K: A study of the generalized Shwartzman phenomenon. J Immunol 29:255-266, 1935. 7. Thomas L, RA Good: Studies on the generalized Shwartzman reaction. I. General observations concerning the phenomenon. J Exp Med 96:605-624, 1952. 8. Stetson CA: Similarities in the mechanisms determining the Arthus and Shwartzman phenomenon. J Exp Med 94:347-358, 1951. 9. Cybulsky MI, HZ Movat: Experimental bacterial pneumonia in rabbits: Polymorphonuclear leukocyte margination and sequestration in the lungs of rabbits and quantitation and kinetics of Cr-labelled polymorphonuclear leukocytes in E. Coli induced lung lesions. Exp Lung Res 4:47-66, 1982. 10. Movat HZ, CE Burrowes: The local Shwartzman reaction: Endotoxin-mediated inflammatory and thrombo-hemorrhagic lesions. In: Berry LJ (Ed): Handbook of Endotoxins, vol 3, Elsevier Science Publishers, New York, 1985, pp 260-302. 11. Ebert RH, D Koch-Weser: In vivo observations of the Shwartzman phenomenon. Trans Am Clin Climatol Assoc 70:103-114, 1958. 12. Movat HZ, BJ Jeyens, MM Kopaniakk, S Wasi: Quantitation of the development and progression of the local Shwartzman reaction. In: Agarwal MK, Ed: Bacterial Endotoxins: Unity or Diversity of Host Response. Elsevier/North Holland, Amsterdam, 1980, pp 179-201. 13. Stetson CA: Studies on the mechanism of the Shwartzman phenomenon. Certain factors involved in the production of the local hemorrhagic necrosis. J Exp Med 93:489-511, 1951. 14. Koplik LH: Shwartzman phenomenon in vaccina virus lesions. Am J Pathol 11:842-843, 1935.
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intravenous injection of LPS. Remarkable is the observation that adjacent skin sites not prepared by previous injections of endotoxin are essentially unaffected by the intravenous injection of endotoxin. This suggests that the endothelia lining of vessels in skin sites prepared for a local Shwartzman reaction is altered, so that leukocytes, activated by an intravenous injection of endotoxin, preferentially adhere to the vessels at prepared sites. Endothelial cell adhesion molecules could be induced by the dermal inflammatory reaction, making the endothelium at prepared skin sites more adhesive for circulating neutrophils. Neutrophils activated by an intravenous injection of LPS express increased leukocyte adhesion molecules and would preferentially adhere to endothelium expressing increased endothelial cell adhesion molecules. In addition to the micro vasculature of prepared skin sites being more sticky for leukocytes, it is likely that cytokine-activated endothelium is thrombogenic due to increased expression of tissue factor procoagulant and decreased expression of thrombomodulin on endothelial cells. Hence, increased adherence of leukocytes to activated endothelial cells, which express tissue factor procoagulant, could injure endothelial cells and initiate the thrombohemorrhagic lesions so characteristic of the Shwartzman reaction. A recent report indicates that monoclonal antibodies against leukocyte adhesion molecules reduced inflammation and tissue damage in bacterial meningitis in rabbits.50 When adhesion molecules are crucial for initiating a local Shwartzman reaction, monoclonal antibodies directed against specific adhesion molecules could have a potential therapeutic role in treating neutrophil-mediated tissue injury in Shwartzman-like inflammatory lesions, such as immune complex-induced vasculitis or purpuric skin diseases.
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35. Good RA, L Thomas: Studies on the generalized Schwartzman reaction. II. The production of bilateral cortical necrosis of the kidneys by a single injection of bacterial toxin in a rabbit previously treated with Thorotrast or trypan blue. J Exp Med 96:625-641, 1952. 36. Beck G, GS Habicht, JL Benach, F Miller: Interleukin-1: A common endogenous mediator of inflammation and the local Shwartzman reaction. J Immunol 136:3025-3031, 1986. 37. March CJ, B Mosley, A Larsen, DP Cerretti, G Braedt, V Price, S Gillis, CS Henney, SR Kronheim, K Grabstein, PJU Conlon, TP Hopp, D Casman: Cloning, sequence and expression of two distinct human interleukin-1 complementary DNA's. Nature 315:614-647, 1985. 38. Dinarello CA: Interleukin-1. Rev Infect Dis 6:51-95, 1984. 39. Old, LJTumor necrosis factor (TNF). Science 230:630-632 1985. 40. Le J, JX Lin, D Henriksel-Destefanco, J Vilcek: Bacterial LPSinduced interferon-γ production: Roles of interleukin-1 and interleukin-2. J Immunol 136:4525-4530, 1986. 41. Lisby G, C Avnstorp, G Wantzin: Interleukin-1, a new mediator in dermatology. Int J Dermatol 26:8-13, 1987. 42. Bevilacaqua MP, SJ Pober, ME Wheeler, D Mendrick, RS Cotran, MA Gimbrone: Interleukin-1 acts on vascular endothelial cells to increase their adhesitivity for blood leukocytes. (Abst.) Fed Proc 44:1494, 1985. 43. Smith CW, SD Marlin, R Rothlein, C Toman, DC Anderson, P Speros: Cooperative interactions of LFA-1 and MAC-1 with intercellular adhesion molecule 1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J Clin Invest 83:2008-2107, 1989. 44. Dobrina A, BR Schwartz, TM Carlos, HD Ochs, PG Beatty, JM Harlan: CD11/CD18—independent neutrophil adherence to induc ible endothelial-leukocyte adhesion molecules (ELAM) in vitro. Immunology 67:502-508, 1989. 45. Dustin ML, R Rothlein, AH Bhan, CA Dinarello,CA Springer: Induction by IL-1 and interferon-a tissue distribution, biochemis try and function of a natural adherence molecule (ICAM-1). J Immunol 137:245-254, 1986. 46. Dustin ML, T Springer: Lymphocytes function-associated antigen1 (LFA-1) interaction with intercellular adhesion molecule (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol 107:321-331, 1988. 47. Bevilacqua MP, S Stengelin, MA Gimbrone, B Seed: ELAM. Science 243:1160-1165, 1989. 48. Kishimoto TK, RS Larson, AL Corbi, ML Dustin, DE Staunton, TA Springer: The leukocyte integrins. Immunol 46:149-182, 1989. 49. Tuomanen EI, K Saukronen, S Sande, C Cioffe, SD Wright: Reduction of inflammation, tissue damage and mortality in bac terial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes. J Exp Med 170:959-969, 1989. 50. Patarroyo M, MW Makgoba: Leukocyte adhesion to cells: Molec ular basis, physiological relevance and abnormalities. Scand J Immunol 30:129-164, 1989. 51. Luscinskas FW, AF Brock, MA Arnaout, MA Gimbrone: Endo thelial-leukocyte adhesion molecule-1 dependent and leukocyte (CD11/18)-dependent mechanisms contribute to polymorphonu clear leukocyte adhesion to cytokine-activated human vascular endothelium. J Immunol 142:2257-2263, 1989.
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Shwartzman Reaction
The local Shwartzman reaction is a hemorrhagic and necrotizing inflammatory lesion elicited by endotoxin from gram-negative bacteria. Shwartzman1,2 and Hanger3 demonstrated in rabbits that an intradermal injection of a sterile bacteria culture filtrate, containing endotoxin, followed by an intravenous injection of the same filtrate 20 to 24 hours later resulted in a local hemorrhagic and necrotic lesion developing at the site of the intradermal injection (Fig. 1). This reaction is now called the local Shwartzman reaction. It is a two-stage reaction requiring an initial preparatory intradermal injection of endotoxin and an intravenous injection of endotoxin 18 to 24 hours later. The time interval between injections is a crucial determinant for the reaction to occur, since if the intravenous injection of endotoxin is made much earlier or later, a hemorrhagic lesion at the site of the intradermal injection does not occur. It is interesting that the local Shwartzman reaction could be elicited in only 167 of 212 rabbits tested in the early experiments of Shwartzman.2 Some rabbits who still showed erythema at the sites of intradermal injection at the time of intravenous challenge only developed a more pronounced erythema 4 to 5 hours after the intravenous challenge, but no hemorrhage or necrosis. Why some rabbits are resistant to developing a localized Shwartzman reaction is still not known. A generalized Shwartzman reaction can be elicited by two intravenous injections of endotoxin spaced 18 to 24 hours apart. The generalized Shwartzman reaction first described by Sanarelli4 and Apitz5,6 is a model for disseminated intravascular coagulation (DIC). Rabbits receiving a second intravenous injection of endotoxin become obviously ill within 4 to 6 hours after the
injection, having symptoms of increasing weakness and dyspnea.7 The intravenous challenge injection of endotoxin in rabbits causes neutropenia and thrombocytopenia. Circulating neutrophils rapidly decrease during the first 15 minutes after injection with margination of neutrophils in the pulmonary vascular bed accounting for most of the decrease in circulating neutrophils.8,9 A decrease in circulating platelets parallels the decrease in circulating neutrophils. Approximately 4 hours after injection, the number of circulating neutrophils gradually increases. Conjunctival petechial hemorrhages develop shortly before death, 18 to 24 hours after the second injection. Kidneys show bilateral cortical necrosis with numerous fibrin microthrombi in glomerular capillaries. Examination of lungs, liver, and lymph nodes also shows varying degrees of hemorrhage and necrosis. The bilateral cortical necrosis with fibrin microthrombi is the most characteristic anatomic lesion of the generalized Shwartzman reaction.2 Similar to the local Shwartzman reaction, the generalized reaction is a two-step phenomenon requiring both a preparative intravenous injection of endotoxin and a provocative injection. Timing of the second injection is critical, with the optimum interval being 18 to 24 hours after the first injection. Tissue damage in the local Shwartzman reaction appears similar to tissue damage in the generalized reaction. These observations pointed to a common mechanism involved in the pathogenesis of both the local and generalized Shwartzman reactions.6,7 Since the local reaction is easier to manipulate and more predictable than the generalized reaction, the local Shwartzman reaction in animals has been used as a model of the general Shwartzman reaction and DIC in humans.
HISTOPATHOLOGY From the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. Reprint requests: Dr. Brozna, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-3135. 326
An intradermal injection of endotoxin elicits a transient inflammatory reaction at the site of injection characterized initially by hyperemia, increased vascular
Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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JOHN P. BROZNA, M.D., Ph.D
FIG. 1. Thrombohemorrhagic lesions in skin sites that received injections of bacterial filtrate 24 hours before an intravenous challenge. (From Shwartzman.2 Reprinted with permission.)
permeability, and leukocyte infiltration.1'2'10 Initially, there is margination and emigration of neutrophils in small veins and venules with neutrophils then infiltrating into the dermis.11 Using labeled cells and proteins, the accumulation of neutrophils was determined to be maximal 4 hours after the intradermal injection. The accumulation of neutrophils after an intradermal injection of endotoxin does not progress beyond 6 hours after the intradermal injection.12 Maximum accumulation of monocytes occurs at about the time of maximum neutrophil infiltration, but monocytes continue to accumulate for at least 24 hours after injection. At the time of intravenous injection of endotoxin, 18 to 24 hours after the initial intradermal injection, residual polymorphonuclear neutrophils and monocytes are still present at the intradermal site. The small veins and venules show perivascular cuffing with neutrophils. The vessels are patent and there is no evidence of hemorrhage or necrosis.13 Shwartzman2 originally observed that the intensity of the hemorrhagic reaction occurring after the intravenous injection of endotoxin did not correlate with the intensity of the initial inflammatory reaction elicited with the intradermal injection of endotoxin, even in rabbits that did not develop a hemorrhagic lesion after intravenous challenge. Other investigators have observed that the intensity of the thrombohemorrhagic reaction at intradermal injection sites induced by intravenous chal-
327 lenge tended to correlate with the intensity of the initial inflammatory reaction at sites of intradermal injection. 312 Shwartzman estimated the initial inflammatory response by observing qualitatively the magnitude of the erythematous response to the intradermal injection of endotoxin and not by quantitating the intensity of the leukocyte inflammatory cell infiltrate. Recent reports suggesting a correlation between intensity of the initial inflammatory reaction to intensity of the thrombohemorrhagic reaction are based on actual measurements of leukocyte inflammatory infiltrates, which are maximal approximately 4 hours after the intradermal injection of lipopolysaccharide (LPS). It is interesting that a Shwartzman reaction cannot be elicited 4 hours after the preparative injection of LPS, even though at that time dermal inflammation is maximal. A modest thrombohemorrhagic lesion can only be elicited by an intravenous injection of LPS administered at least 12 to 15 hours after the preparative injection. This would suggest that inflammatory mediators released during the early inflammatory reaction require a minimum time interval to prepare the skin for a local Shwartzman reaction. It is unknown which cells in the skin are essential for preparing it for a local Shwartzman reaction or for producing a Shwartzman reaction in response to intravenously administered LPS. Thrombohemorrhagic lesions develop rapidly after intravenous challenge. Within 15 minutes of the intravenous challenge, the small veins and capillaries in the prepared skin sites show microthrombi composed of platelets, fibrin, and varying numbers of neutrophils. Two hours after the injection, perivascular neutrophil infiltrates appear associated with a necrotizing vasculitis involving small veins and venules. Endothelial cells become swollen and red blood cells leak into the surrounding tissue. Within 4 to 5 hours after the intravenous challenge, the severest hemorrhage and necrosis are fully developed.2 The histopathologic lesions are essentially those of vascular injury. Necrosis is severest when the preparatory skin test is made 18 to 24 hours before the intravenous injection and is rare when the intravenous injection is given less than 12 hours or more than 48 hours after the intradermal injection. In contrast to skin sites prepared with an intradermal injection of endotoxin, sections of normal skin obtained before or after the intravenous injection of endotoxin show no histologic abnormalities.13 It is not clear why fibrin microthrombi and aggregates of platelets and neutrophils are found primarily at skin sites previously prepared with endotoxin, and not in the capillaries of skin not prepared by an intradermal injection of endotoxin. This observation suggests that endothelium of capillaries in sites prepared with LPS is more thrombogenic or more sticky for leukocytes and platelets, or both. Hence, it
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would appear that the key to understanding the Shwartzman reaction in animals and purpuric skin diseases in humans is to focus on the endothelial cell and its response to inflammatory mediators.
Possible Mechanisms Involved in the Local Shwartzman Reaction Classically, gram-negative endotoxin has been used to prepare a skin site for a local Shwartzman reaction. Other agents that have been used successfully as prepa ratory agents for a local Shwartzman reaction include vaccinia virus,14 isolated leukocyte granules,15 and tu berculin in animals immunized with bacille CalmetteGuerin. 1617 Some agents such as turpentine, which induce inflammation in the skin, cannot prepare it for a local Shwartzman reaction. Hence, it would seem that not all agents that can induce an inflammatory reaction in the skin can prepare a skin site for a local Shwartzman reaction. Inhibitors of proteolytic enzymes can prevent the local Shwartzman reaction, presumably by blocking vascular injury and the inflammatory reaction induced by leukocyte proteolytic enzymes. 15,18 Intravenous injec tion of trypsin inhibitor (500 µg of either soybean or pancreatic trypsin inhibitor) in rabbits 15 minutes before the provocative injection of endotoxin inhibits the for mation of thrombohemorrhagic lesions at the site of previously injected endotoxin 24 hours earlier.18 Other similar studies demonstrated that depletion of neutrophils in rabbits with nitrogen mustard suppresses the local Shwartzman reaction.19 These data suggest a role for neutrophils and neutrophil proteases in the pathogenesis of the local Shwartzman reaction. However, nitrogen mustard in addition to depleting neutrophils is toxic for endothelial cells and also causes a decrease in peripheral blood monocytes.20 Hence, it is not possible to exclude other cell types in the pathogenesis of a Shwartzman reaction. The thrombohemorrhagic lesions of a local Shwartzman reaction are manifestations of small vessel thrombosis and acute necrotizing vasculitis. Antiproteases presumably block the acute vascular injury medi ated by neutrophils. Since a detailed histopathologic picture of prepared skin sites in animals treated with protease inhibitors has not been described, it is not known what effect antiproteases have on the formation of leukocyte, platelet, or fibrin microthrombi in prepared skin sites after an intravenous injection of LPS. It would be interesting to determine whether small microthrombi form at prepared skin sites after a provocative intrave nous injection of LPS in animals treated with antipro teases. Since no hemorrhagic lesions develop in animals treated with protease inhibitors, the presence of micro
thrombi at prepared skin sites in dermal vessels would imply that thrombi are not present for a sufficient time to induce ischemic injury leading to hemorrhage. In purpu ric skin lesions associated with protein C deficiency, there is an early reversible lesion characterized by fibrin microthrombi in dermal vessels.21 Administration of plasma containing protein C blocks the formation of hemorrhagic lesions, presumably by facilitating removal of microthrombi and preventing the formation of new thrombi. It remains to be determined whether the local Shwartzman reaction in animals treated with protease inhibitors is a model for the early reversible skin lesions in patients with deficient protein C. It is likely that neutrophils contribute to vascular injury, resulting in hemorrhage in the Shwartzman reac tion, but neutrophils do not appear to contribute directly to the thrombotic process. Since peripheral blood monocytes22-24 and endothelial cells 25,26 produce tissue factor after exposure to small amounts of endotoxin, it is thought that monocytes or endothelial cells, or both, modulate thrombosis at sites of inflammation. In partic ular, since monocytes produce a number of potent inflammatory mediators that can activate other cells as well as induce tissue injury (Table 1), monocytes may play a key role in modulating tissue injury and preparing skin sites for a local Shwartzman reaction. It is thought that a crucial prerequisite for a preparatory agent is the induction of an inflammatory reaction that is associated with activation of leukocytes and the release of specific inflammatory agents such as interleukin-1 (IL-1), tissue necrosis factor (TNF) or interferon-gamma (IF-γ). 10,17 Cultured endothelial cells, when activated by inflamma tory mediators such as endotoxin or IL-1, express tissue factor procoagulant activity that is maximal 8 to 12 hours after activation (Fig. 2). The procoagulant activity then gradually declines to baseline levels 24 to 48 hours after LPS stimulation. In Figure 2 tissue factor activity was measured using purified Factors VII, X, and a chromoge-
TABLE 1. Inflammatory Mediators Produced by Activated Macrophages Interleukin-1 Tumor necrosis factor Tissue factor procoagulant Interferons Transforming growth factors Proteases Toxic oxygen metabolites Chemotactic factors Neutrophil activating factors Plasminogen activator inhibitor
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FIG. 2. Human umbilical cord endothelial cells were cultured in the presence of media ( ) or media containing endotoxin (10 ng/ml) (●). Tissue factor procoagulant activity was measured at different time intervals after LPS stimula tion in a chromogenic assay using purified Factors X and VII and a synthetic substrate for Factor Xa. Tissue factor activity is expressed as [(A405/min2/105 cells] × 106] ± SD.
nic Factor Xa substrate. Using this method tissue factor procoagulant activity is proportional to ([A405/min2/105 cells] × 105).24 Endothelial cells lining vascular surfaces are in a position to play an important role in regulating intravascular coagulation. It was once thought that endothelial cells were metabolically inert and did not have an active role in regulating coagulation. During the past decade, it has become apparent that endothelial cells are metabolically active and synthesize numerous agents that can modulate both coagulation and inflammatory reactions at the endothelial cell membrane surface (Table 2). Normally, endothelial cells present a nonthrombogenic membrane surface to coagulation factors in plasma, but when stimulated by endotoxin they become thrombogenic.25-27 Endotoxin induces endothelial cells, as well as monocytes, to synthesize and express tissue factor, the most potent procoagulant known on the plasma membrane surface. Tissue factor activates the extrinsic pathway of coagulation by forming a complex with Factor VIIa to activate Factor X to Xa. Endotoxin also induces suppression of endothelial cell thrombo-
TABLE 2. Functional Responses of Endothelial Cells Expression of tissue factor procoagulant Expression of thrombomodulin Modulation of protein S-protein C anticoagulant system Release of platelet-derived growth factor Generation of coagulation proteases Release of interleukin-1 Synthesis of prostacyclin
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modulin, which is an important cofactor in the protein C thrombomodulin anticoagulant system.27,28 Thrombinactivated protein C is a serine protease and a potent inhibitor of Factor Va and Factor VIIIa of the coagulation cascade.29 The protein C thrombomodulin anticoagulant system functions under in vivo conditions to suppress thrombosis and is thought to be essential for maintaining homeostasis and ensuring normal flow of blood. It has been shown that protein C can prevent the coagulopathies and lethal effects of LPS in the baboon.30 Baboons injected with LPS develop lethal DIC, whereas, baboons injected with protein C and LPS were protected. Similarly, baboons injected with antibodies to protein C developed DIC with lower amounts of LPS. The protective effects of protein C might be explained in part by protein C anticoagulant properties and by protein C inhibition of endothelial cell endocytosis of thrombomodulin-thrombin complexes,31 which would oppose the thrombogenic effects of endotoxin and other inflammatory cytokines. It is possible that endotoxin induces endothelial cells in vivo to be thrombogenic by inducing an increase in tissue factor procoagulant and a decrease in thrombomodulin. IL-1 and TNF, which are other inflammatory mediators released from leukocytes at inflammatory foci, have been shown to induce an increase in endothelial cell tissue factor32'33 and to cause a decrease in endothelial cell thrombomodulin.34 Usually, anticoagulant mechanisms predominate on the endothelial membrane surface, but when perturbed by endotoxin, IL-1, or TNF, the balance is shifted toward thrombosis.
Role of Cytokines in the Local Shwartzman Reaction Although the provocative intravenous injection of LPS can be substituted by a number of agents, including immune-complexes,8 vaccinia virus,15 zymosan, kaolin, and agar,13'35 until recently it had been difficult to substitute for the preparative intradermal injection of endotoxin in the local Shwartzman reaction. The prerequisites for preparing a skin site for a subsequent thrombohemorrhagic reaction elicited with an intravenous injection of LPS are unknown. However, the ability to elicit a local inflammatory reaction with infiltrating inflammatory cells at the site of injection appears to be an important property of a preparatory agent. Recently, it was shown that IL-1 can induce an acute inflammatory response in the skin very similar to the inflammatory reaction elicited with LPS. 36 It was shown that an intradermal injection of IL-1 can prepare a skin site in rabbits for a local Shwartzman reaction, provoked by an intravenous injection of LPS. In addition, TNF was found to act synergistically with IL-1 to prepare skin sites
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for a local Shwartzman reaction. The combination of IL-1 and TNF was more potent than either agent alone in preparing a site for a Shwartzman reaction.17 Clearly, endothelial cells at the site of LPS injection are altered, so that 18 to 24 hours after the initial dermal injection of LPS a provocative intravenous injection of LPS elicits a local Shwartzman reaction by causing leukocytes to adhere specifically to the endothelium of small vessels in the prepared skin site. Therefore it would appear that inflammatory agents that can prepare skin sites for a Shwartzman reaction cause the membrane surface of endothelial cells to become "sticky" for leukocytes. Numerous cytokines, including IL-1, TNF, and IF-γ, are released from inflammatory cells at sites of inflammation that affect the endothelial cell plasma membrane (Table 3). 17,35,36 In particular, monocytes at sites of inflammation produce a large number of biolog ically active molecules, including IL-1, 3 7 , 3 8 TNF, 39 and IF-7. 40 IL-1 is an important immunoregulatory molecule that has many diverse effects on nonspecific host defense mechanisms. Monocytes37 and keratinocytes41 are pos sible sources of IL-1 in skin sites affected by immuno logic or other inflammatory reactions. IL-1 is chemotactic for neutrophils in vitro and can induce leukocytes to adhere to endothelial cells. Leukocyte adherence to endothelial cells is a pivotal event in the inflammatory response and a prerequisite for leukocyte-dependent vascular injury. 38,42,43 Numerous recent in vitro experiments have shown that IL-1, TNF, LPS, and IF-7 can cause human umbil ical cord endothelial cells to become more adhesive for isolated peripheral blood leukocytes. 43-47 Recent studies have identified specific inducible endothelial cell recep tors involved in leukocyte adherence to endothelial cells. These include intercellular adherence molecule-1 (ICAM-1)46 and endothelial-leukocyte adhesion mole cule-1 (ELAM-1),47 which can be induced by IL-1, LPS, or TNF treatment of endothelial cells. These adhesion molecules are thought to be ligands for the family of heterodimeric leukocyte cell surface proteins LFA-1, MO-1, p150 43,44 or other undefined adhesion molecules on leukocytes. Expression of leukocyte adherence-pro TABLE 3. Functional Effects of Interleukin-1 on Endothelial Cells
moting proteins can also be increased by IL-1, TNF, or LPS treatment of leukocytes. The leukocyte adherence-promoting proteins consist of three distinct molecules with different α-subunits and a common ß-chain subunit. The individual α-subunits are designated CD1 la for LFA-1, CD11b for MO-1, and CD11c for p150 and the common α-subunit CD18. 48 Several recent studies using monoclonal antibodies di rected against individual subunits of the leukocyte adhe sion-promoting proteins and to the endothelial cell ad hesion proteins ICAM-1 and ELAM-1 indicated that CD11/CD18 receptors are important for migration of leukocytes across small vessels. Intravenous injection of monoclonal antibodies directed against CD18 blocked the development of tissue injury and the development of leukocytosis in the cerebrospinal fluid of animals chal lenged intracisternally with living bacteria or endo toxin.49 In other studies it was shown that adherence of human neutrophils to human umbilical vein endothelial cells was dependent on ICAM-1 on endothelial cells and on CD11/CD18 glycoproteins on neutrophils.43 It was reported that unstimulated neutrophils exhibit LFA1-dependent attachment to ICAM-1, whereas neutro phils stimulated with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine exhibited enhanced bind ing to ICAM-1 by a MO-1 dependent process. From in vivo and in vitro studies, it is clear that endothelial cells and neutrophils have distinct surface adhesion molecules that are regulated independently and can be induced by a number of cytokines found in sites of inflammation, including IL-1, TNF, LPS, and IF-7. The interaction of leukocyte and endothelial cell adhesion molecules is complex and probably important in initiating inflamma tory reactions. The CD11/CD18 receptor complex is expressed in the unstimulated neutrophil and can be up-regulated by cytokine stimulation.50 Unstimulated endothelial cells express low levels of ICAM-1, which can also be up-regulated by cytokines,-whereas ELAM-1 is not expressed in unstimulated endothelial cells but can be induced by inflammatory cytokines. 42,45,51 These observations indicate that leukocyte adherence to endo thelial cells increases after leukocytes or endothelial cells are activated by cytokines. Hence, preparation of skin sites for a local Shwartzman reaction might be dependent on cytokines released from endotoxin-activated mono cytes at sites of induced inflammation, leading to alter ations of endothelial cell membrane surfaces.
Increased tissue factor procoagulant expression Suppressed thrombomodulin-protein C anticoagulant system Increased plasminogen activator inhibitor Decreased plasminogen activator Increased expression of ICAM-1 adhesion molecule Increased expression of ELAM-1 adhesion molecule Increased platelet activating factor
Possible Role for Endothelial Cell Adhesion Molecules in the Local Shwartzman Reaction Intradermal injection of skin sites with LPS elicits an inflammatory reaction that prepares the site for a local Shwartzman reaction provoked 18 to 24 hours later by an
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SUMMARY The local and generalized Shwartzman reactions are models of thrombohemorrhagic skin necrosis and DIC, respectively. An intravenous preparatory injection of endotoxin followed by an intradermal injection of endotoxin 24 hours later elicits a thrombohemorrhagic lesion only at the site of intradermal injection of endotoxin in the local Shwartzman reaction. Two intravenous injections of endotoxin spaced 24 hours apart induce a systemic generalized Shwartzman reaction characterized by coagulopathy, petechial hemorrhages, microthrombi, and decreased circulating platelets similar to DIC. Of particular interest is the observation that thrombohemorrhagic lesions of the Shwartzman reaction only develop at sites of intradermal injections of endotoxin. Microthrombi composed of platelets and leukocytes only adhere or accumulate in dermal vessels after an intrader-
mal injection of endotoxin. Prior to the endotoxin injection, biopsies of skin show normal vessels without microthrombi or significant inflammation. Since endothelial cells line the small vessels in the dermis, where a Shwartzman reaction appears to be initiated, it is likely that endothelial cells are important for initiating a local Shwartzman reaction. IL-1 and TNF can substitute for the intradermal injection of endotoxin in the local Shwartzman reaction, induce endothelial cells to become thrombogenic, and can induce the expression of cell adhesion molecules on endothelial cells making endothelial cells more sticky for leukocytes. These observations suggest that endothelial cells play a central role in the local Shwartzman reaction and may be important in understanding diseases associated with thrombohemorrhagic skin necrosis.
REFERENCES 1. Shwartzman G: A new phenomenon of local skin reactivity to B. typhosus culture filtrate. Proc Soc Exp Biol Med 25:560-561, 1928. 2. Shwartzman G: Phenomenon of Local Tissue Reactivity and its Immunological, Pathological and Clinical Significance. Paul B. Hoeber, New York, 1937. 3. Hanger FM: Effect of intravenous bacterial filtrates on skin tests and local infections. Proc Soc Exp Biol Med 25:775-777, 1928. 4. Sanarelli G: De la pathogenie du cholera (neuvieme memoire). Le cholera experimental. Ann Inst Pasteur 38:11-72, 1924. 5. Apitz K: Die Wirkung bakterieller Kulturfiltrate nach Umstimmung des gesamten Endothels beim Kaninchen. Virchows Arch Pathol Anat 293:1-33, 1934. 6. Apitz K: A study of the generalized Shwartzman phenomenon. J Immunol 29:255-266, 1935. 7. Thomas L, RA Good: Studies on the generalized Shwartzman reaction. I. General observations concerning the phenomenon. J Exp Med 96:605-624, 1952. 8. Stetson CA: Similarities in the mechanisms determining the Arthus and Shwartzman phenomenon. J Exp Med 94:347-358, 1951. 9. Cybulsky MI, HZ Movat: Experimental bacterial pneumonia in rabbits: Polymorphonuclear leukocyte margination and sequestration in the lungs of rabbits and quantitation and kinetics of Cr-labelled polymorphonuclear leukocytes in E. Coli induced lung lesions. Exp Lung Res 4:47-66, 1982. 10. Movat HZ, CE Burrowes: The local Shwartzman reaction: Endotoxin-mediated inflammatory and thrombo-hemorrhagic lesions. In: Berry LJ (Ed): Handbook of Endotoxins, vol 3, Elsevier Science Publishers, New York, 1985, pp 260-302. 11. Ebert RH, D Koch-Weser: In vivo observations of the Shwartzman phenomenon. Trans Am Clin Climatol Assoc 70:103-114, 1958. 12. Movat HZ, BJ Jeyens, MM Kopaniakk, S Wasi: Quantitation of the development and progression of the local Shwartzman reaction. In: Agarwal MK, Ed: Bacterial Endotoxins: Unity or Diversity of Host Response. Elsevier/North Holland, Amsterdam, 1980, pp 179-201. 13. Stetson CA: Studies on the mechanism of the Shwartzman phenomenon. Certain factors involved in the production of the local hemorrhagic necrosis. J Exp Med 93:489-511, 1951. 14. Koplik LH: Shwartzman phenomenon in vaccina virus lesions. Am J Pathol 11:842-843, 1935.
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intravenous injection of LPS. Remarkable is the observation that adjacent skin sites not prepared by previous injections of endotoxin are essentially unaffected by the intravenous injection of endotoxin. This suggests that the endothelia lining of vessels in skin sites prepared for a local Shwartzman reaction is altered, so that leukocytes, activated by an intravenous injection of endotoxin, preferentially adhere to the vessels at prepared sites. Endothelial cell adhesion molecules could be induced by the dermal inflammatory reaction, making the endothelium at prepared skin sites more adhesive for circulating neutrophils. Neutrophils activated by an intravenous injection of LPS express increased leukocyte adhesion molecules and would preferentially adhere to endothelium expressing increased endothelial cell adhesion molecules. In addition to the micro vasculature of prepared skin sites being more sticky for leukocytes, it is likely that cytokine-activated endothelium is thrombogenic due to increased expression of tissue factor procoagulant and decreased expression of thrombomodulin on endothelial cells. Hence, increased adherence of leukocytes to activated endothelial cells, which express tissue factor procoagulant, could injure endothelial cells and initiate the thrombohemorrhagic lesions so characteristic of the Shwartzman reaction. A recent report indicates that monoclonal antibodies against leukocyte adhesion molecules reduced inflammation and tissue damage in bacterial meningitis in rabbits.50 When adhesion molecules are crucial for initiating a local Shwartzman reaction, monoclonal antibodies directed against specific adhesion molecules could have a potential therapeutic role in treating neutrophil-mediated tissue injury in Shwartzman-like inflammatory lesions, such as immune complex-induced vasculitis or purpuric skin diseases.
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35. Good RA, L Thomas: Studies on the generalized Schwartzman reaction. II. The production of bilateral cortical necrosis of the kidneys by a single injection of bacterial toxin in a rabbit previously treated with Thorotrast or trypan blue. J Exp Med 96:625-641, 1952. 36. Beck G, GS Habicht, JL Benach, F Miller: Interleukin-1: A common endogenous mediator of inflammation and the local Shwartzman reaction. J Immunol 136:3025-3031, 1986. 37. March CJ, B Mosley, A Larsen, DP Cerretti, G Braedt, V Price, S Gillis, CS Henney, SR Kronheim, K Grabstein, PJU Conlon, TP Hopp, D Casman: Cloning, sequence and expression of two distinct human interleukin-1 complementary DNA's. Nature 315:614-647, 1985. 38. Dinarello CA: Interleukin-1. Rev Infect Dis 6:51-95, 1984. 39. Old, LJTumor necrosis factor (TNF). Science 230:630-632 1985. 40. Le J, JX Lin, D Henriksel-Destefanco, J Vilcek: Bacterial LPSinduced interferon-γ production: Roles of interleukin-1 and interleukin-2. J Immunol 136:4525-4530, 1986. 41. Lisby G, C Avnstorp, G Wantzin: Interleukin-1, a new mediator in dermatology. Int J Dermatol 26:8-13, 1987. 42. Bevilacaqua MP, SJ Pober, ME Wheeler, D Mendrick, RS Cotran, MA Gimbrone: Interleukin-1 acts on vascular endothelial cells to increase their adhesitivity for blood leukocytes. (Abst.) Fed Proc 44:1494, 1985. 43. Smith CW, SD Marlin, R Rothlein, C Toman, DC Anderson, P Speros: Cooperative interactions of LFA-1 and MAC-1 with intercellular adhesion molecule 1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J Clin Invest 83:2008-2107, 1989. 44. Dobrina A, BR Schwartz, TM Carlos, HD Ochs, PG Beatty, JM Harlan: CD11/CD18—independent neutrophil adherence to induc ible endothelial-leukocyte adhesion molecules (ELAM) in vitro. Immunology 67:502-508, 1989. 45. Dustin ML, R Rothlein, AH Bhan, CA Dinarello,CA Springer: Induction by IL-1 and interferon-a tissue distribution, biochemis try and function of a natural adherence molecule (ICAM-1). J Immunol 137:245-254, 1986. 46. Dustin ML, T Springer: Lymphocytes function-associated antigen1 (LFA-1) interaction with intercellular adhesion molecule (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol 107:321-331, 1988. 47. Bevilacqua MP, S Stengelin, MA Gimbrone, B Seed: ELAM. Science 243:1160-1165, 1989. 48. Kishimoto TK, RS Larson, AL Corbi, ML Dustin, DE Staunton, TA Springer: The leukocyte integrins. Immunol 46:149-182, 1989. 49. Tuomanen EI, K Saukronen, S Sande, C Cioffe, SD Wright: Reduction of inflammation, tissue damage and mortality in bac terial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes. J Exp Med 170:959-969, 1989. 50. Patarroyo M, MW Makgoba: Leukocyte adhesion to cells: Molec ular basis, physiological relevance and abnormalities. Scand J Immunol 30:129-164, 1989. 51. Luscinskas FW, AF Brock, MA Arnaout, MA Gimbrone: Endo thelial-leukocyte adhesion molecule-1 dependent and leukocyte (CD11/18)-dependent mechanisms contribute to polymorphonu clear leukocyte adhesion to cytokine-activated human vascular endothelium. J Immunol 142:2257-2263, 1989.
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