Cutaneous Vascular Fibrinolytic Activity in the Local Shwartzman Reaction Jerry M. Bergstein, MD
A fibrin slide test was utilized to study cutaneous vascular plasminogen activator activity in normal rabbit, rat, monkey, and human skin and in rabbit skin following development of the local Shwartzman reaction and after substituting e-aminocaproic acid for the preparatory injection of endotoxin in the local Shwartzman reaction. Cutaneous vascular plasminogen activator activity was also studied in rats after attempted induction of the local Shwartzman reaction following inhibition of fibrinolysis with (-aminocaproic acid and/or pregnancy. Plasminogen activator activity was detected in vessels of normal rat, monkey, and human skin but was absent in skin from normal rabbits and rabbits with the local Shwartzman reaction. Intradermal injection of e -aminocaproic acid failed to prepare for the local Shwartzman reaction. In the rat, which has greater cutaneous vascular plasminogen activator activity than the rabbit, inhibition of vascular activator with E-aminocaproic acid and/or pregnancy failed to prepare for the local Shwartzman reaction. These studies indicate that although the markedly diminished level of cutaneous vascular plasminogen activator in the rabbit may be important in the pathogenesis of the local Shwartzan reaction, factors other than inhibition of fibrinolysis are also necessary for preparation of the reaction. (Am J Pathol 88:655-662, 1977)
INHIBITION OF GLO\IERULLAR PLAS\MINOGEN- ACTIVATOR activity
seems important in the pathogenesis of the generalized Shwvartzman reaction.1'2 The role of cutaneous vascular fibrinolvtic (plasminogen activator) activity in the local Shwartzman reaction remains to be defined. Using a fibrin slide technique, I have studied cutaneous vascular fibrinolvtic activity in normal rabbit, rat, monkey, and human skin and in rabbit skin following development of the local Shwartzman reaction and after substituting E-aminocaproic acid (EACA) for the preparatory injection of endotoxin in the local Shwartzman reaction. To further evaluate the role of skin vascular lvsis in preparation for the local Shwartzman reaction, rats (which have greater skin lvtic activity than rabbits) were studied after attempted induction of the local Shwartzman reaction following inhibition of lvsis with EACA and/or pregnancy. From the Department of Pediatrics and the Gwynne Hazen Cherry \lemorial Laboratories. Unisersitv of Califomia. Los Angeles. School of Medicine. Los Angeles. California Supported by a general research grant from the U niversity of Califomia. Los Angeles Accepted for publication April .30. 1977 Address reprint reqluests to Dr Jerry Bergstein. Department of Pediatrics. James Whitcomb Riley Hospital for Children. Indiana Unisersity School of \Medicine. 1100 West Michigan Street. Indianapolis. IN 4620(2
655
656
BERGSTEIN
American Journal of Pathology
Materials and Methods Endotoxin
Endotoxin (lipopolysaccharide B from Escherichia coli 026:B6) was obtained from Difco Laboratories, Detroit, Mich. In the production of the local Shwartzman reaction in rabbits, the preparatory intradermal injection of endotoxin was 0.2 mg dissolved in 0.2 ml of isotonic saline and the provoking intravenous injection was 0.3 mg in 1.0 ml of saline. In rats, the preparatory injection of endotoxin was 1.0 mg dissolved in 0.2 ml of isotonic saline and the provoking injection was 1.0 mg in 1.0 ml of saline. The dose of endotoxin in rats was several-fold greater than the amount required to produce the generalized Shwartzman reaction in pregnant rats and approaches the LD50 for these animals.34 In certain rat experiments, the preparatory injection of endotoxin (1.0 mg) was dissolved in 0.2 ml EACA. E-Aminocaproic Acid
-Aminocaproic acid (250 mg/ml) was obtained from Lederle Laboratories, Pearl River, N.Y., and was administered by intravenous injection or gavage feeding; rapid absorption, resulting in sustained blood levels, is obtained by either method of administra-
tion.5
Histology
Skin was obtained at biopsy or immediately after sacrifice by cervical subluxation. Part of the tissue was placed in buffered formalin (pH 7.4), sectioned at 4 ,u, and stained for light microscopy with hematoxylin and eosin stain. The remainder of the tissue was snapfrozen in isopentane precooled in liquid nitrogen and processed for immunofluorescent microscopy and fibrin slides according to methods previously described.67 Fluorescein isothiocyanate-conjugated antiserum to rabbit fibrinogen was prepared as previously outlined.6 Plasminogen-rich and plasminogen-free fibrinogen for fibrin slides were prepared by the methods of Brakman.8 Because of prolonged incubation periods, the volume of fibrinogen solution was increased to 0.8 ml. Fibrin slides containing two frozen sections from each tissue specimen were fixed in buffered formalin after 20, 40, 60, 120, 180, and 240 minutes of incubation. The slides were evaluated by light microscopy. The presence of lytic activity was denoted by the appearance of a clear area within the fibrin film overlying the tissue section by 240 minutes of incubation. Experimental Design
Group 1 contained 7 normal 1-kg albino rabbits, 2 normal 230-g Sprague-Dawley rats, and 1 normal monkey (Macaca mulatta), which were sacrificed. The abdomen was immediately shaved with an electric clipper, and skin specimens were removed. Shaved abdominal skin was also obtained from 2 humans. Group 2 included 4 1-kg albino rabbits whose abdomens were shaved. Following skin biopsy, each animal received an intradermal injection of endotoxin. Twenty-four hours after the intradermal injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed, and skin was obtained from the intra.dermal injection site. Group 3 contained 4 1-kg albino rabbits. After the abdomen was shaved, each animal was given two intradermal injections (0.2 ml) of EACA. One intradermal site was biopsied 1 hour after injection. Twenty-four hours after the intradermal injections, each rabbit was given an intravenous injection of endotoxin. Six hours later, the animals were sacrificed, and skin was obtained from the remaining intradermal injection site. Group 4 contained 4 Sprague-Dawley rats whose abdomens were shaved. After skin
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biopsy, each animal was given 750 mg of EACA intravenously, followed in 30 minutes by an intradermal injection of endotoxin. An identical amount of EACA was given intravenouslv to each animal 6, 12, 18 and 24 hours following the initial injection of EACA. Twentv-four hours after the initial injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradermal injection site. Group 5 included 6 240-g pregnant (20 davs of gestation) Sprague-Dawlev rats; the abdomens of the rats were shaved and a skin biopsy was obtained. Each animal was then given an intradermal injection of endotoxin followed in 24 hours by an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradernal injection site. Group 6 contained 6 pregnant (20 days of gestation) rats whose abdomens were shaved. After skin biopsy, each animal was given 750 mg EACA by gavage, followed in 30 minutes by an intradermal injection of endotoxin. An identical amount of EACA was given (by gavage) to each animal 6, 12, 18, and 24 hours after the first administration of EACA. Twentv-four hours after the initial injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradermal injection site. Group 7 contained 4 pregnant (20 da's of gestation) rats whose experimental protocol was similar to animals in Group 6 except that the initial intradermal dose of endotoxin was dissolved in EACA. Results
Using plasminogen-rich fibrin slides, vascular fibrinolytic activity was detected (Table 1) in frozen sections of all normal rat, monkey, and human skin (Figure 1) but was absent in sections of all normal rabbit skin (Group 1). In species with vascular fibrinolvtic activitv, Iysis was present at 20 minutes incubation time; bv 240 minutes of incubation time, lysis of the fibrin film overlving the entire tissue section was noted. On plasminogen-free fibrin slides, vascular Iysis was absent in skin from all species. Light microscopy was normal, immunofluorescent microscopv was negative, and vascular fibrinolytic activity was absent in the initial skin biopsy from all Group 2 rabbits. Six hours after the provoking injection of endotoxin, all Group 2 animals had evidence of the local Shwartzman reaction, as characterized by 2-cm-square areas of hemorrhagic necrosis over each intradermal injection site. Light microscopy of the lesion revealed edema of the dermis with an intense inflammatory infiltrate consisting primarily of polvmorphonuclear leukocytes. Thrombi were present Table 1-Vascular Fibrinolytic Activity in Normal Skin (Group 1)
Species Rabbit Rat Monkey Human
No. of animals
No. of animals with absent vascular tysis
7 2 1 2
7 0 0 0
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American Journal of Pathology
in the majority of small blood vessels. Immunofluorescent studies of frozen sections from the skin lesion of all 4 rabbits revealed heavy staining of vessels for fibrin. Vascular fibrinolytic activity was absent in skin from the Shwartzman lesion of all 4 animals. No cutaneous lesions were detected in Group 3 animals. An occasional small area of granulocyte and round cell infiltrate was found by light microscopy in skin obtained from 1 rabbit 1 hour after intradermal injection of EACA and from another rabbit at the time of sacrifice. Light microscopic findings were normal on the remaining tissue specimens from all Group 3 animals. Immunofluorescent microscopy was negative and cutaneous vascular fibrinolysis was absent in skin from all Group 3 rabbits (Table 2). Studies were then performed in animals (rats) with cutaneous vascular fibrinolytic activity to further evaluate the role of vascular fibrinolysis in the development of the local Shwartzman reaction (Table 2). Light microscopy was normal, immunofluorescent microscopy for fibrin was negative, and vascular fibrinolytic activity was present in the initial skin biopsy from all Group 4 rats. No gross lesions were seen at the intradermal injection site up to the time of sacrifice of Group 4 animals (normal rats; inhibition of fibrinolysis by systemic EACA). Although vascular fibrinolytic activity was absent in skin obtained at the time of sacrifice, light microscopic findings were normal and vascular thrombi were not detected by immunofluorescence. Light microscopy was normal, immunofluorescence negative, and vascular fibrinolytic activity present in the initial skin biopsy from the pregnant rats in Group 5 (inhibition of fibrinolysis by pregnancy). No gross skin lesions were seen at the intradermal injection site after the provoking injection of endotoxin up to the time of sacrifice. Except for a rare granulocyte in the dermis of 2 animals, light microscopic findings were Table 2-Vascular Fibrin Deposition and Fibrinolytic Activity in Skin of Animals Prepared for the Local Shwartzman Reaction by Inhibition of Fibrinolysis No. of Mechanism of No. of inhibition animals with animals with absent No. of of fibrinolysis vascular lysis vascular fibrin animals Group Species 4 3 Rabbit EACA (intradermal) 0 4 4 Rat EACA (systemic) 4 4 0 0 5 Rat 0 6 Pregnancy 6 Rat 5 6 0 Pregnancy EACA (systermic) 7 Rat 4 4 0 Pregnancy EACA (systemic) EACA (intradermal)
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normal in the tissue. At the time of sacrifice, cutaneous vascular fibrinolytic activity was present and no vascular thrombi were seen by fluorescence. In the initial skin biopsy from pregnant rats in Group 6, light microscopic findings were normal, immunofluorescence was negative, and vascular fibrinolytic activity was present. No gross lesions were detected at the intradermal injection site up to the time of sacrifice of Group 6 animals (inhibition of fibrinolysis by pregnancy and systemic EACA). Except for a rare granulocyte in the dermis of 2 animals, light microscopy of the skin obtained at sacrifice was normal. Cutaneous vascular fibrinolytic activity was absent in skin from 5 of 6 rats. No vascular thrombi were detected by immunofluorescence. In Group 7 pregnant rats, the initial skin biopsy was normal by light and immunofluorescent microscopy. Vascular fibrinolysis was present in all specimens. Although fibrinolysis was inhibited by pregnancy plus the systemic and local administration of EACA, no gross lesions resembling the local Shwartzrnan reaction were noted following the provoking injection of endotoxin. Light microscopy of skin obtained at the time of sacrifice was normal. Although vascular fibrinolytic activity was absent in these specimens, no vascular thrombi were detected by immunofluorescence. Our studies of fibrinolysis in normal rat and human skin agree with those of other investigators."'4 That cutaneous vascular fibrinolytic activity was present in normal rat, monkey, and human skin studied with plasminogen-rich fibrin slides but was absent with plasminogen-free fibrin slides indicates that Iysis was related to plasminogen activator activity. The unique sensitivity of the rabbit to the generalized Shwartzman reaction may be related to the low level of renal cortical plasminogen activator.'5 In this study, no detectable cutaneous vascular plasminogen activator activity was found in normal rabbit skin. Lytic activity was found in normal rat, monkey, and human skin. Although we cannot be certain that vascular plasminogen activator activity is completely absent from rabbit skin, it seems likely that the level of activator is much less than the other species studied. Thus, the absent or low level of cutaneous vascular plasminogen activator may be an important factor in the pathogenesis of the local Shwartzman reaction in the rabbit. Inhibition of fibrinolysis plays an important role in the development of the generalized Shwartzman reaction. Indeed, in the generalized Shwartzman reaction, EACA may substitute for the preparatory '6 and
660
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provoking 17 injections of endotoxin. However, the precise role of inhibition of fibrinolysis remains to be defined. McKay et al. have devised an experimental model which reproduces the coagulation and histologic alterations induced by endotoxin in the generalized Shwartzman reaction."8 These alterations were reproduced by infusion of ellagic acid (Hageman factor activation), EACA (inhibition of fibrinolysis), and norepinephrine (stimulation of a-receptor sites in the kidney) but did not occur in the absence of any one compound. We have found that, in the generalized Shwartzman reaction, a preparatory injection of endotoxin 1 or Thorotrast 19 depletes glomerular fibrinolytic activity. However, we were unable to demonstrate inhibition of glomerular fibrinolysis following cortisone preparation.19 We have also found that inhibition of glomerular fibrinolytic activity with polymyxin B fails to prepare for the generalized Shwartzman reaction.20 The majority of the above studies seem to indicate that inhibition of fibrinolysis is only one factor involved in preparation for the generalized Shwartzman reaction. To determine the role of cutaneous vascular fibrinolytic activity in preparation for the local Shwartzman reaction, inhibition of vascular fibrinolysis was evaluated in a species (rat) with brisk lytic activity. Cutaneous vascular fibrinolysis was inhibited with systemic and intradermal administration of EACA in normal and pregnant rats (the increased susceptibility of the pregnant rat near term to the development of the generalized Shwartzman reaction following a single injection of endotoxin appears to be due to inhibition of the fibrinolytic system 21). Although cutaneous vascular fibrinolytic activity was inhibited, no evidence of the local Shwartzman reaction was detected in rats given two doses of endotoxin. Additionally, EACA failed to substitute for the preparatory injection of endotoxin in the rabbit. These results are consistent with most observations noted in the generalized Shwartzman reaction and suggest that factors other than diminished fibrinolytic activity are also necessary in preparation for the local Shwartzman reaction. References 1. Bergstein JM, Michael AF Jr: Renal cortical fibrinolytic activity in the rabbit following one or two doses of endotoxin. Thromb Diath Haemorrh 29:27-32, 1973 2. Bergstein JM: Platelet inhibition of renal cortical fibrinolytic activity in the rabbit. Lab Invest 35:171-178, 1976 3. Bergstein JM, Hoyer JR, Michael AF Jr: Glomerular fibrinolytic activity following endotoxin-induced glomerular fibrin deposition in the pregnant rat. Am J Pathol 75:195-202, 1974 4. Wong T-C: A study on the generalized Shwartzman reaction in pregnant rats induced by bacterial endotoxin. Am J Obstet Gynecol 84:786-797, 1962 5. McNicol GP, Fletcher AP, Alkjaersig N, Sherry S: The absorption, distribution, and
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6.
7. 8. 9.
10. 11.
12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
excretion of epsilon-aminocaproic acid following oral or intravenous administration to man. J Lab Clin Med 59:15-24, 1962 Bergstein JNM, Michael AF: Generalized Shwartzman reaction in the rabbit: Immunopathologic findings in the kidney. Arch Pathol 97:2.30-231, 1974 Bergstein JNM, Michael AF Jr: Cortical fibrinol%tic activity in normal and diseased human kidneys. J Lab Clin NMed 79:701-709, 1972 Brakman P: Fibrinolysis. Amsterdam, Scheltema and Holkema, 1967 Tympanidis K, Astrup T: Fibrinolytic activity in injured rat skin. Exp NMol Pathol 16:101-108, 1972 Smokovitis A, Kok P, Astrup T: Tissue repair in rats in presence of locally applied tissue plasminogen activator. Exp Mol Pathol 22:109-117, 1975 Todd AS: Some topographical observations on fibrinolysis. J Clin Pathol 17:324-327, 1964 Tumer RH, Rvan TJ: Fibrinolvtic activity in human skin. Trans St Johns Hosp Dermatol Soc 55:212-217, 1969 Sun NCJ, Conn DL, Schroeter AL, Kazmier FJ: Skin fibrinolvtic activity in cutaneous and systemic vasculitis. Mavo Clin Proc 51:216-22'2, 1976 Cunliffe WJ, Dodman B, Holmes RL, Forster RA: Local fibrinolv-tic activity in patients with cutaneous vasculitis. Br J Dermatol 84:420-423, 1971 Epstein MD, Beller FK, Douglas GW: Kidney tissue activator of fibrinolvsis in relation to pregnancy. Obstet Gvnecol 32:494-504, 1968 Lipinski B, Worowski K, jeljaszewicz J, Niewiarowski S, Rejniak L: Participation of soluble fibrin monomer complexes and platelet factor 4 in the generalized Shwartzman reaction. Thromb Diath Haemorrh 20:285-295, 1968 Lee L: Reticuloendothelial clearance of circulating fibrin in the pathogenesis of the generalized Shwartzman reaction. J Exp Med 115:1065-1082, 1962 McKay DG, NMlller-Berghaus G, Cruse V: Activation of Hageman factor by ellagic acid and the generalized Shwartzman reaction. Am J Pathol 54:393-420, 1969 Bergstein JNM, NMichael AF Jr: The effect of Thorotrast and cortisone on renal cortical fibrinolvtic activity in the rabbit. Am J Pathol 71:113-118, 1973 Bergstein JM, NMichael AF Jr: Effect of polvmyxin B and endotoxin on renal cortical fibrinolvsis. Proc Soc Exp Biol NMed 144:369-372, 1973 NMlller-Berghaus G, Obst R: Induction of the generalized Shwartzman reaction in pregnant and nonpregnant rats by colchicine. Am J Pathol 69:131-138, 1972
Ackwledmlients The author thanks Dr. Boguslaw Lipinski for his helpful suggestions. Linda Shevlin for technical assistance, and Rochelle Beddard for preparing the manuscript.
I,,,
Figure 1-Fibrin slide of normal human skin demonstrating clear zone of fibrinolytic activity (arrows) around a dermal blood vessel (arrowhead) (Incubation time 20 minutes; Harris hematoxylin stain, x 100).
A fibrin slide test was utilized to study cutaneous vascular plasminogen activator activity in normal rabbit, rat, monkey, and human skin and in rabbit skin following development of the local Shwartzman reaction and after substituting e-aminocaproic acid for the preparatory injection of endotoxin in the local Shwartzman reaction. Cutaneous vascular plasminogen activator activity was also studied in rats after attempted induction of the local Shwartzman reaction following inhibition of fibrinolysis with (-aminocaproic acid and/or pregnancy. Plasminogen activator activity was detected in vessels of normal rat, monkey, and human skin but was absent in skin from normal rabbits and rabbits with the local Shwartzman reaction. Intradermal injection of e -aminocaproic acid failed to prepare for the local Shwartzman reaction. In the rat, which has greater cutaneous vascular plasminogen activator activity than the rabbit, inhibition of vascular activator with E-aminocaproic acid and/or pregnancy failed to prepare for the local Shwartzman reaction. These studies indicate that although the markedly diminished level of cutaneous vascular plasminogen activator in the rabbit may be important in the pathogenesis of the local Shwartzan reaction, factors other than inhibition of fibrinolysis are also necessary for preparation of the reaction. (Am J Pathol 88:655-662, 1977)
INHIBITION OF GLO\IERULLAR PLAS\MINOGEN- ACTIVATOR activity
seems important in the pathogenesis of the generalized Shwvartzman reaction.1'2 The role of cutaneous vascular fibrinolvtic (plasminogen activator) activity in the local Shwartzman reaction remains to be defined. Using a fibrin slide technique, I have studied cutaneous vascular fibrinolvtic activity in normal rabbit, rat, monkey, and human skin and in rabbit skin following development of the local Shwartzman reaction and after substituting E-aminocaproic acid (EACA) for the preparatory injection of endotoxin in the local Shwartzman reaction. To further evaluate the role of skin vascular lvsis in preparation for the local Shwartzman reaction, rats (which have greater skin lvtic activity than rabbits) were studied after attempted induction of the local Shwartzman reaction following inhibition of lvsis with EACA and/or pregnancy. From the Department of Pediatrics and the Gwynne Hazen Cherry \lemorial Laboratories. Unisersitv of Califomia. Los Angeles. School of Medicine. Los Angeles. California Supported by a general research grant from the U niversity of Califomia. Los Angeles Accepted for publication April .30. 1977 Address reprint reqluests to Dr Jerry Bergstein. Department of Pediatrics. James Whitcomb Riley Hospital for Children. Indiana Unisersity School of \Medicine. 1100 West Michigan Street. Indianapolis. IN 4620(2
655
656
BERGSTEIN
American Journal of Pathology
Materials and Methods Endotoxin
Endotoxin (lipopolysaccharide B from Escherichia coli 026:B6) was obtained from Difco Laboratories, Detroit, Mich. In the production of the local Shwartzman reaction in rabbits, the preparatory intradermal injection of endotoxin was 0.2 mg dissolved in 0.2 ml of isotonic saline and the provoking intravenous injection was 0.3 mg in 1.0 ml of saline. In rats, the preparatory injection of endotoxin was 1.0 mg dissolved in 0.2 ml of isotonic saline and the provoking injection was 1.0 mg in 1.0 ml of saline. The dose of endotoxin in rats was several-fold greater than the amount required to produce the generalized Shwartzman reaction in pregnant rats and approaches the LD50 for these animals.34 In certain rat experiments, the preparatory injection of endotoxin (1.0 mg) was dissolved in 0.2 ml EACA. E-Aminocaproic Acid
-Aminocaproic acid (250 mg/ml) was obtained from Lederle Laboratories, Pearl River, N.Y., and was administered by intravenous injection or gavage feeding; rapid absorption, resulting in sustained blood levels, is obtained by either method of administra-
tion.5
Histology
Skin was obtained at biopsy or immediately after sacrifice by cervical subluxation. Part of the tissue was placed in buffered formalin (pH 7.4), sectioned at 4 ,u, and stained for light microscopy with hematoxylin and eosin stain. The remainder of the tissue was snapfrozen in isopentane precooled in liquid nitrogen and processed for immunofluorescent microscopy and fibrin slides according to methods previously described.67 Fluorescein isothiocyanate-conjugated antiserum to rabbit fibrinogen was prepared as previously outlined.6 Plasminogen-rich and plasminogen-free fibrinogen for fibrin slides were prepared by the methods of Brakman.8 Because of prolonged incubation periods, the volume of fibrinogen solution was increased to 0.8 ml. Fibrin slides containing two frozen sections from each tissue specimen were fixed in buffered formalin after 20, 40, 60, 120, 180, and 240 minutes of incubation. The slides were evaluated by light microscopy. The presence of lytic activity was denoted by the appearance of a clear area within the fibrin film overlying the tissue section by 240 minutes of incubation. Experimental Design
Group 1 contained 7 normal 1-kg albino rabbits, 2 normal 230-g Sprague-Dawley rats, and 1 normal monkey (Macaca mulatta), which were sacrificed. The abdomen was immediately shaved with an electric clipper, and skin specimens were removed. Shaved abdominal skin was also obtained from 2 humans. Group 2 included 4 1-kg albino rabbits whose abdomens were shaved. Following skin biopsy, each animal received an intradermal injection of endotoxin. Twenty-four hours after the intradermal injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed, and skin was obtained from the intra.dermal injection site. Group 3 contained 4 1-kg albino rabbits. After the abdomen was shaved, each animal was given two intradermal injections (0.2 ml) of EACA. One intradermal site was biopsied 1 hour after injection. Twenty-four hours after the intradermal injections, each rabbit was given an intravenous injection of endotoxin. Six hours later, the animals were sacrificed, and skin was obtained from the remaining intradermal injection site. Group 4 contained 4 Sprague-Dawley rats whose abdomens were shaved. After skin
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biopsy, each animal was given 750 mg of EACA intravenously, followed in 30 minutes by an intradermal injection of endotoxin. An identical amount of EACA was given intravenouslv to each animal 6, 12, 18 and 24 hours following the initial injection of EACA. Twentv-four hours after the initial injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradermal injection site. Group 5 included 6 240-g pregnant (20 davs of gestation) Sprague-Dawlev rats; the abdomens of the rats were shaved and a skin biopsy was obtained. Each animal was then given an intradermal injection of endotoxin followed in 24 hours by an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradernal injection site. Group 6 contained 6 pregnant (20 days of gestation) rats whose abdomens were shaved. After skin biopsy, each animal was given 750 mg EACA by gavage, followed in 30 minutes by an intradermal injection of endotoxin. An identical amount of EACA was given (by gavage) to each animal 6, 12, 18, and 24 hours after the first administration of EACA. Twentv-four hours after the initial injection of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, the animals were sacrificed and skin was obtained from the intradermal injection site. Group 7 contained 4 pregnant (20 da's of gestation) rats whose experimental protocol was similar to animals in Group 6 except that the initial intradermal dose of endotoxin was dissolved in EACA. Results
Using plasminogen-rich fibrin slides, vascular fibrinolytic activity was detected (Table 1) in frozen sections of all normal rat, monkey, and human skin (Figure 1) but was absent in sections of all normal rabbit skin (Group 1). In species with vascular fibrinolvtic activitv, Iysis was present at 20 minutes incubation time; bv 240 minutes of incubation time, lysis of the fibrin film overlving the entire tissue section was noted. On plasminogen-free fibrin slides, vascular Iysis was absent in skin from all species. Light microscopy was normal, immunofluorescent microscopv was negative, and vascular fibrinolytic activity was absent in the initial skin biopsy from all Group 2 rabbits. Six hours after the provoking injection of endotoxin, all Group 2 animals had evidence of the local Shwartzman reaction, as characterized by 2-cm-square areas of hemorrhagic necrosis over each intradermal injection site. Light microscopy of the lesion revealed edema of the dermis with an intense inflammatory infiltrate consisting primarily of polvmorphonuclear leukocytes. Thrombi were present Table 1-Vascular Fibrinolytic Activity in Normal Skin (Group 1)
Species Rabbit Rat Monkey Human
No. of animals
No. of animals with absent vascular tysis
7 2 1 2
7 0 0 0
658
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in the majority of small blood vessels. Immunofluorescent studies of frozen sections from the skin lesion of all 4 rabbits revealed heavy staining of vessels for fibrin. Vascular fibrinolytic activity was absent in skin from the Shwartzman lesion of all 4 animals. No cutaneous lesions were detected in Group 3 animals. An occasional small area of granulocyte and round cell infiltrate was found by light microscopy in skin obtained from 1 rabbit 1 hour after intradermal injection of EACA and from another rabbit at the time of sacrifice. Light microscopic findings were normal on the remaining tissue specimens from all Group 3 animals. Immunofluorescent microscopy was negative and cutaneous vascular fibrinolysis was absent in skin from all Group 3 rabbits (Table 2). Studies were then performed in animals (rats) with cutaneous vascular fibrinolytic activity to further evaluate the role of vascular fibrinolysis in the development of the local Shwartzman reaction (Table 2). Light microscopy was normal, immunofluorescent microscopy for fibrin was negative, and vascular fibrinolytic activity was present in the initial skin biopsy from all Group 4 rats. No gross lesions were seen at the intradermal injection site up to the time of sacrifice of Group 4 animals (normal rats; inhibition of fibrinolysis by systemic EACA). Although vascular fibrinolytic activity was absent in skin obtained at the time of sacrifice, light microscopic findings were normal and vascular thrombi were not detected by immunofluorescence. Light microscopy was normal, immunofluorescence negative, and vascular fibrinolytic activity present in the initial skin biopsy from the pregnant rats in Group 5 (inhibition of fibrinolysis by pregnancy). No gross skin lesions were seen at the intradermal injection site after the provoking injection of endotoxin up to the time of sacrifice. Except for a rare granulocyte in the dermis of 2 animals, light microscopic findings were Table 2-Vascular Fibrin Deposition and Fibrinolytic Activity in Skin of Animals Prepared for the Local Shwartzman Reaction by Inhibition of Fibrinolysis No. of Mechanism of No. of inhibition animals with animals with absent No. of of fibrinolysis vascular lysis vascular fibrin animals Group Species 4 3 Rabbit EACA (intradermal) 0 4 4 Rat EACA (systemic) 4 4 0 0 5 Rat 0 6 Pregnancy 6 Rat 5 6 0 Pregnancy EACA (systermic) 7 Rat 4 4 0 Pregnancy EACA (systemic) EACA (intradermal)
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normal in the tissue. At the time of sacrifice, cutaneous vascular fibrinolytic activity was present and no vascular thrombi were seen by fluorescence. In the initial skin biopsy from pregnant rats in Group 6, light microscopic findings were normal, immunofluorescence was negative, and vascular fibrinolytic activity was present. No gross lesions were detected at the intradermal injection site up to the time of sacrifice of Group 6 animals (inhibition of fibrinolysis by pregnancy and systemic EACA). Except for a rare granulocyte in the dermis of 2 animals, light microscopy of the skin obtained at sacrifice was normal. Cutaneous vascular fibrinolytic activity was absent in skin from 5 of 6 rats. No vascular thrombi were detected by immunofluorescence. In Group 7 pregnant rats, the initial skin biopsy was normal by light and immunofluorescent microscopy. Vascular fibrinolysis was present in all specimens. Although fibrinolysis was inhibited by pregnancy plus the systemic and local administration of EACA, no gross lesions resembling the local Shwartzrnan reaction were noted following the provoking injection of endotoxin. Light microscopy of skin obtained at the time of sacrifice was normal. Although vascular fibrinolytic activity was absent in these specimens, no vascular thrombi were detected by immunofluorescence. Our studies of fibrinolysis in normal rat and human skin agree with those of other investigators."'4 That cutaneous vascular fibrinolytic activity was present in normal rat, monkey, and human skin studied with plasminogen-rich fibrin slides but was absent with plasminogen-free fibrin slides indicates that Iysis was related to plasminogen activator activity. The unique sensitivity of the rabbit to the generalized Shwartzman reaction may be related to the low level of renal cortical plasminogen activator.'5 In this study, no detectable cutaneous vascular plasminogen activator activity was found in normal rabbit skin. Lytic activity was found in normal rat, monkey, and human skin. Although we cannot be certain that vascular plasminogen activator activity is completely absent from rabbit skin, it seems likely that the level of activator is much less than the other species studied. Thus, the absent or low level of cutaneous vascular plasminogen activator may be an important factor in the pathogenesis of the local Shwartzman reaction in the rabbit. Inhibition of fibrinolysis plays an important role in the development of the generalized Shwartzman reaction. Indeed, in the generalized Shwartzman reaction, EACA may substitute for the preparatory '6 and
660
BERGSTEIN
American Journal of Pathology
provoking 17 injections of endotoxin. However, the precise role of inhibition of fibrinolysis remains to be defined. McKay et al. have devised an experimental model which reproduces the coagulation and histologic alterations induced by endotoxin in the generalized Shwartzman reaction."8 These alterations were reproduced by infusion of ellagic acid (Hageman factor activation), EACA (inhibition of fibrinolysis), and norepinephrine (stimulation of a-receptor sites in the kidney) but did not occur in the absence of any one compound. We have found that, in the generalized Shwartzman reaction, a preparatory injection of endotoxin 1 or Thorotrast 19 depletes glomerular fibrinolytic activity. However, we were unable to demonstrate inhibition of glomerular fibrinolysis following cortisone preparation.19 We have also found that inhibition of glomerular fibrinolytic activity with polymyxin B fails to prepare for the generalized Shwartzman reaction.20 The majority of the above studies seem to indicate that inhibition of fibrinolysis is only one factor involved in preparation for the generalized Shwartzman reaction. To determine the role of cutaneous vascular fibrinolytic activity in preparation for the local Shwartzman reaction, inhibition of vascular fibrinolysis was evaluated in a species (rat) with brisk lytic activity. Cutaneous vascular fibrinolysis was inhibited with systemic and intradermal administration of EACA in normal and pregnant rats (the increased susceptibility of the pregnant rat near term to the development of the generalized Shwartzman reaction following a single injection of endotoxin appears to be due to inhibition of the fibrinolytic system 21). Although cutaneous vascular fibrinolytic activity was inhibited, no evidence of the local Shwartzman reaction was detected in rats given two doses of endotoxin. Additionally, EACA failed to substitute for the preparatory injection of endotoxin in the rabbit. These results are consistent with most observations noted in the generalized Shwartzman reaction and suggest that factors other than diminished fibrinolytic activity are also necessary in preparation for the local Shwartzman reaction. References 1. Bergstein JM, Michael AF Jr: Renal cortical fibrinolytic activity in the rabbit following one or two doses of endotoxin. Thromb Diath Haemorrh 29:27-32, 1973 2. Bergstein JM: Platelet inhibition of renal cortical fibrinolytic activity in the rabbit. Lab Invest 35:171-178, 1976 3. Bergstein JM, Hoyer JR, Michael AF Jr: Glomerular fibrinolytic activity following endotoxin-induced glomerular fibrin deposition in the pregnant rat. Am J Pathol 75:195-202, 1974 4. Wong T-C: A study on the generalized Shwartzman reaction in pregnant rats induced by bacterial endotoxin. Am J Obstet Gynecol 84:786-797, 1962 5. McNicol GP, Fletcher AP, Alkjaersig N, Sherry S: The absorption, distribution, and
Vol. 88, No. 3
CUTANEOUS VASCULAR FIBRINOLYSIS
661
September 1977
6.
7. 8. 9.
10. 11.
12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
excretion of epsilon-aminocaproic acid following oral or intravenous administration to man. J Lab Clin Med 59:15-24, 1962 Bergstein JNM, Michael AF: Generalized Shwartzman reaction in the rabbit: Immunopathologic findings in the kidney. Arch Pathol 97:2.30-231, 1974 Bergstein JNM, Michael AF Jr: Cortical fibrinol%tic activity in normal and diseased human kidneys. J Lab Clin NMed 79:701-709, 1972 Brakman P: Fibrinolysis. Amsterdam, Scheltema and Holkema, 1967 Tympanidis K, Astrup T: Fibrinolytic activity in injured rat skin. Exp NMol Pathol 16:101-108, 1972 Smokovitis A, Kok P, Astrup T: Tissue repair in rats in presence of locally applied tissue plasminogen activator. Exp Mol Pathol 22:109-117, 1975 Todd AS: Some topographical observations on fibrinolysis. J Clin Pathol 17:324-327, 1964 Tumer RH, Rvan TJ: Fibrinolvtic activity in human skin. Trans St Johns Hosp Dermatol Soc 55:212-217, 1969 Sun NCJ, Conn DL, Schroeter AL, Kazmier FJ: Skin fibrinolvtic activity in cutaneous and systemic vasculitis. Mavo Clin Proc 51:216-22'2, 1976 Cunliffe WJ, Dodman B, Holmes RL, Forster RA: Local fibrinolv-tic activity in patients with cutaneous vasculitis. Br J Dermatol 84:420-423, 1971 Epstein MD, Beller FK, Douglas GW: Kidney tissue activator of fibrinolvsis in relation to pregnancy. Obstet Gvnecol 32:494-504, 1968 Lipinski B, Worowski K, jeljaszewicz J, Niewiarowski S, Rejniak L: Participation of soluble fibrin monomer complexes and platelet factor 4 in the generalized Shwartzman reaction. Thromb Diath Haemorrh 20:285-295, 1968 Lee L: Reticuloendothelial clearance of circulating fibrin in the pathogenesis of the generalized Shwartzman reaction. J Exp Med 115:1065-1082, 1962 McKay DG, NMlller-Berghaus G, Cruse V: Activation of Hageman factor by ellagic acid and the generalized Shwartzman reaction. Am J Pathol 54:393-420, 1969 Bergstein JNM, NMichael AF Jr: The effect of Thorotrast and cortisone on renal cortical fibrinolvtic activity in the rabbit. Am J Pathol 71:113-118, 1973 Bergstein JM, NMichael AF Jr: Effect of polvmyxin B and endotoxin on renal cortical fibrinolvsis. Proc Soc Exp Biol NMed 144:369-372, 1973 NMlller-Berghaus G, Obst R: Induction of the generalized Shwartzman reaction in pregnant and nonpregnant rats by colchicine. Am J Pathol 69:131-138, 1972
Ackwledmlients The author thanks Dr. Boguslaw Lipinski for his helpful suggestions. Linda Shevlin for technical assistance, and Rochelle Beddard for preparing the manuscript.
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Figure 1-Fibrin slide of normal human skin demonstrating clear zone of fibrinolytic activity (arrows) around a dermal blood vessel (arrowhead) (Incubation time 20 minutes; Harris hematoxylin stain, x 100).
