The Local Shwartzman Reaction in the Rabbit Immunopathologic Findings in the Skin Jerry M. Bergstein, MD
Four albino rabbits received two intradermal injections of endotoxin followed in 24 hours by an intravenous injection of endotoxin. All animals developed the local Shwartzman reaction, characterized by hemorrhagic necrosis over each intradermal injection site. Light microscopy of the Shwartzman lesion showed intense inflammation and vascular thrombosis. Frozen sections of the skin lesion revealed marked vascular deposition of fibrin with lesser amounts of IgG, IgM, C,, and albumin. Light microscopy was normal and immunofluorescence was negative in skin obtained prior to the initial injection of endotoxin and skin adjacent to the Shwartzman lesion. These findings suggest nonspecific trapping of serum proteins within vascular thrombi and indicate that immune mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction. (Am J Pathol 86:117-122. 1977)
THE PATHOGENESIS OF THE LOCAL SHWARTZMAN RE.CTION remains
to be defined. To evaluate the role of immune mechanisms in the development of the vascular lesion, frozen sections of normal rabbit skin and skin from the Shvartzman lesion were studied for the presence of immunoglobulin, C:3, fibrin, and albumin. Materials and Methods Endotoxn Endotoxin (lipopolysaccharide B from Escherichia coli 0-26: B6) was obtained from Difco Laboratories. Detroit. \Mich. In the production of the local Shwartzman reaction, the preparaton- intradermal injection of endotoxin was 0.20 mg dissolved in 0.2 ml of isotonic saline and the provoking intravenous injection was 0:3 mg in 1.0 ml of saline. HistoIo
Skin was obtained at biopsy or immediately after sacrifice by cervical subluxation. Part of the tissue '-as placed in buffered formalin (pH 7.4). sectioned at 4 ,M and stained for light microscops \ ith hematoxN-lin and eosin stain. The remainder of the tissue was snapfrozen in isopentane precooled in liquid nitrogen and processed for immunofluorescent microscopx- according to methods previously described.' Fluorescein isothiocyanate-conjugated antisera to rabbit IgG, IgM. C.3. fibrin, and albumin were prepared as previously outlined.' The amount of immunofluorescence was arbitrarily graded as negatise. trace. 1+. 2+. and :3+ From the Department of Pediatrics and the Gwynne Hazen Cherrs Memorial Laboratories. U niversitv of California. Los Angeles. School of Medicine. Los Angeles. California Supported bh a general research grant from the Unisersity of California. Los Angeles.
Accepted for publication August 17, 19766 Address reprint requests to Dr Jern- Ml. Bergstein, Department of Pediatrics. Disision of 'ephrolog\. L-CLX Center for the Health Sciences. Los Angeles, CA 90024 117
118
BERGSTEIN
American Journal of Pathology
Experimental Design
The abdomen of four 1-kg albino rabbits was shaved with an electric clipper. Following skin biopsy, each animal received two intradermal injections of endotoxin at widely spaced sites. Twenty-four hours after the intradermal injections of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, skin was obtained from one intradermal injection site and from adjacent normal appearing skin. Twenty-four hours after the intravenous injection of endotoxin, the animals were sacrificed and skin was obtained from the remaining intradermal injection site and from adjacent normal-appearing skin.
Results
Light microscopy was normal and immunofluorescent microscopy was negative in the initial skin biopsy from all rabbits. Six hours after the intravenous injection of endotoxin, all 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 (Figure 1) of the lesion revealed marked edema of the dermis with an intense inflammatory infiltrate consisting primarily of polymorphonuclear leukocytes. Thrombi were present in the majority of small blood vessels; erythrocytes were seen throughout the interstices of the dermis. Immunofluorescent studies of frozen sections from the skin lesion of all 4 rabbits revealed 2+ to 3+ staining of vessels for fibrin (Figure 2A). Fibrin was also detected around vessels and in the interstices of the dermis. In contrast, IgG (Figure 2B), IgM (Figure 2C), C3 (Figure 2D), and albumin (Figure 2E) were found in vessels in only trace to 1+ intensity. Light microscopy was normal in normal-appearing skin adjacent to the Shwartzman lesion. Frozen sections of normal-appearing skin from 2 rabbits showed occasional vascular fibrin deposition in 1+ intensity; stains for IgG, IgM, C3, and albumin were negative. Twenty-four hours after the provoking injection of endotoxin, all animals showed an extension of the zone of cutaneous hemorrhagic necrosis. In comparison to the biopsy at 6 hours, light microscopy revealed greater hemorrhage and necrosis. Background staining of frozen sections with all fluorescein-tagged antisera was increased over tissue obtained at 6 hours. However, immunofluorescent studies of the 6 and 24 hour specimens were similar. Light microscopy was normal and immunofluorescent microscopy was negative in normal appearing skin adjacent to the Shwartzman lesion. Discussion
The role of immune mechanisms in endotoxin-induced pathologic alterations remains to be defined. Fong and Good have suggested that depletion of C3 and the terminal complement components with cobra venom factor prevents the generalized Shwartzman reaction.2 Recent studies
Vol. 86, No. 1 January 1977
LOCAL SHWARTZMAN REACTION
119
indicate that C3 and the terminal complement components may not play a significant role in the development of endotoxin-induced shock,3'4 intravascular coagulation,4'5 endothelial injury,6 or the generalized Shwartzman reaction.5'7 Complement depletion has also been suggested to prevent the local Shwartzman reaction.2'8 The gross and light microscopic changes seen in skin from our animals with the local Shwartzman reaction resemble those described bv other investigators.9 In comparison to immunopathologic findings in the kidney of animals with the generalized Shwartzman reaction 1 (glomerular deposition of IgG, IgM, C3, fibrin, and albumin), frozen sections of skin from animals with the local Shwartzman reaction revealed only heavy staining for fibrin. The weak staining for IgG, IgM, C3, and albumin suggests nonspecific trapping of serum proteins within vascular thrombi and indicates that immunologic mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction. References 1. Bergstein J\1. Michael AF: Generalized Shwartzman reaction in the rabbit: Immunopathologic findings in the kidney. Arch Pathol 97:230-231, 1974 2. Fong JSC, Good RA: Prevention of the localized and generalized Shwartzman reactions bv an anticomplementarv agent, cobra venom factor. J Exp M1ed 134:642-635, 1971 3. Kitzmiller JL, Lucas WE, Yelenoskv PF: The role of complement in feline endotoxin shock. Am J Obstet Gynecol 112:414-421, 1972 4. Ulevitch RJ, Cochrane CG, Henson PM, Morrison DC, Doe WF: NMediation systems in bacterial lipopolvsaccharide-induced hypotension and disseminated intravascular coagulation. I. The role of complement. J Exp NMed 142:1570-1590, 1975 5. Mrlller-Berghaus G, Lohmann E: The role of complement in endotoxin-induced disseminated intravascular coagulation: Studies in congenitallv C6-deficient rabbits. Br j Haematol 28:403-418, 1974 6. Evensen SA, Pickering RJ, Batbouta J, Shepro D: Endothelial injury induced by bacterial endotoxin: Effect of complement depletion. Eur J Clin Invest 5:463-469, 1975 Bergstein J\1, Michael AF Jr: Failure of cobra venom factor to prevent the generalized Shwartzman reaction and loss of renal cortical fibrinolv tic activity. Am J Pathol 74:19-30, 1974 8. Polak L, Turk JL: Suppression of the haemorrhagic component of the Shwartzman reaction by anti-complement serum. Nature 223:738-739, 1969 9. Stetson CA, Good RA: Studies on the mechanism of the Shwartzman phenomenon: Evidence for the participation of polvmorphonuclear leucocv-tes in the phenomenon. J Exp Med 9'3:49-63, 1951
AdmoWdnents The author thanks Linda Shevlin for technical assistance and Rochelle Beddard for preparing the manuscript.
120
BERGSTEIN
American Journal of Pathology
[Illustrations follow]
Fge I-Skin obtained from rabbit 6 hours after provoking injection of endotoxin, demonstrating marked edema, inflammation, hemorrhage, and vascular thrombosis (H&E, x250).
A
B
E
Figure 2-Immunofluorescent studies of frozen sections of skin obtained from rabbit 6 hours after provoking injection of endotoxin, demonstrating (arrow) vascular deposition of fibrin (A, x 400), IgG (B, x 250), IgM (C, x 400), C3 (D, x 250), and albumin (E, x 400).
Four albino rabbits received two intradermal injections of endotoxin followed in 24 hours by an intravenous injection of endotoxin. All animals developed the local Shwartzman reaction, characterized by hemorrhagic necrosis over each intradermal injection site. Light microscopy of the Shwartzman lesion showed intense inflammation and vascular thrombosis. Frozen sections of the skin lesion revealed marked vascular deposition of fibrin with lesser amounts of IgG, IgM, C,, and albumin. Light microscopy was normal and immunofluorescence was negative in skin obtained prior to the initial injection of endotoxin and skin adjacent to the Shwartzman lesion. These findings suggest nonspecific trapping of serum proteins within vascular thrombi and indicate that immune mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction. (Am J Pathol 86:117-122. 1977)
THE PATHOGENESIS OF THE LOCAL SHWARTZMAN RE.CTION remains
to be defined. To evaluate the role of immune mechanisms in the development of the vascular lesion, frozen sections of normal rabbit skin and skin from the Shvartzman lesion were studied for the presence of immunoglobulin, C:3, fibrin, and albumin. Materials and Methods Endotoxn Endotoxin (lipopolysaccharide B from Escherichia coli 0-26: B6) was obtained from Difco Laboratories. Detroit. \Mich. In the production of the local Shwartzman reaction, the preparaton- intradermal injection of endotoxin was 0.20 mg dissolved in 0.2 ml of isotonic saline and the provoking intravenous injection was 0:3 mg in 1.0 ml of saline. HistoIo
Skin was obtained at biopsy or immediately after sacrifice by cervical subluxation. Part of the tissue '-as placed in buffered formalin (pH 7.4). sectioned at 4 ,M and stained for light microscops \ ith hematoxN-lin and eosin stain. The remainder of the tissue was snapfrozen in isopentane precooled in liquid nitrogen and processed for immunofluorescent microscopx- according to methods previously described.' Fluorescein isothiocyanate-conjugated antisera to rabbit IgG, IgM. C.3. fibrin, and albumin were prepared as previously outlined.' The amount of immunofluorescence was arbitrarily graded as negatise. trace. 1+. 2+. and :3+ From the Department of Pediatrics and the Gwynne Hazen Cherrs Memorial Laboratories. U niversitv of California. Los Angeles. School of Medicine. Los Angeles. California Supported bh a general research grant from the Unisersity of California. Los Angeles.
Accepted for publication August 17, 19766 Address reprint requests to Dr Jern- Ml. Bergstein, Department of Pediatrics. Disision of 'ephrolog\. L-CLX Center for the Health Sciences. Los Angeles, CA 90024 117
118
BERGSTEIN
American Journal of Pathology
Experimental Design
The abdomen of four 1-kg albino rabbits was shaved with an electric clipper. Following skin biopsy, each animal received two intradermal injections of endotoxin at widely spaced sites. Twenty-four hours after the intradermal injections of endotoxin, each animal received an intravenous injection of endotoxin. Six hours later, skin was obtained from one intradermal injection site and from adjacent normal appearing skin. Twenty-four hours after the intravenous injection of endotoxin, the animals were sacrificed and skin was obtained from the remaining intradermal injection site and from adjacent normal-appearing skin.
Results
Light microscopy was normal and immunofluorescent microscopy was negative in the initial skin biopsy from all rabbits. Six hours after the intravenous injection of endotoxin, all 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 (Figure 1) of the lesion revealed marked edema of the dermis with an intense inflammatory infiltrate consisting primarily of polymorphonuclear leukocytes. Thrombi were present in the majority of small blood vessels; erythrocytes were seen throughout the interstices of the dermis. Immunofluorescent studies of frozen sections from the skin lesion of all 4 rabbits revealed 2+ to 3+ staining of vessels for fibrin (Figure 2A). Fibrin was also detected around vessels and in the interstices of the dermis. In contrast, IgG (Figure 2B), IgM (Figure 2C), C3 (Figure 2D), and albumin (Figure 2E) were found in vessels in only trace to 1+ intensity. Light microscopy was normal in normal-appearing skin adjacent to the Shwartzman lesion. Frozen sections of normal-appearing skin from 2 rabbits showed occasional vascular fibrin deposition in 1+ intensity; stains for IgG, IgM, C3, and albumin were negative. Twenty-four hours after the provoking injection of endotoxin, all animals showed an extension of the zone of cutaneous hemorrhagic necrosis. In comparison to the biopsy at 6 hours, light microscopy revealed greater hemorrhage and necrosis. Background staining of frozen sections with all fluorescein-tagged antisera was increased over tissue obtained at 6 hours. However, immunofluorescent studies of the 6 and 24 hour specimens were similar. Light microscopy was normal and immunofluorescent microscopy was negative in normal appearing skin adjacent to the Shwartzman lesion. Discussion
The role of immune mechanisms in endotoxin-induced pathologic alterations remains to be defined. Fong and Good have suggested that depletion of C3 and the terminal complement components with cobra venom factor prevents the generalized Shwartzman reaction.2 Recent studies
Vol. 86, No. 1 January 1977
LOCAL SHWARTZMAN REACTION
119
indicate that C3 and the terminal complement components may not play a significant role in the development of endotoxin-induced shock,3'4 intravascular coagulation,4'5 endothelial injury,6 or the generalized Shwartzman reaction.5'7 Complement depletion has also been suggested to prevent the local Shwartzman reaction.2'8 The gross and light microscopic changes seen in skin from our animals with the local Shwartzman reaction resemble those described bv other investigators.9 In comparison to immunopathologic findings in the kidney of animals with the generalized Shwartzman reaction 1 (glomerular deposition of IgG, IgM, C3, fibrin, and albumin), frozen sections of skin from animals with the local Shwartzman reaction revealed only heavy staining for fibrin. The weak staining for IgG, IgM, C3, and albumin suggests nonspecific trapping of serum proteins within vascular thrombi and indicates that immunologic mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction. References 1. Bergstein J\1. Michael AF: Generalized Shwartzman reaction in the rabbit: Immunopathologic findings in the kidney. Arch Pathol 97:230-231, 1974 2. Fong JSC, Good RA: Prevention of the localized and generalized Shwartzman reactions bv an anticomplementarv agent, cobra venom factor. J Exp M1ed 134:642-635, 1971 3. Kitzmiller JL, Lucas WE, Yelenoskv PF: The role of complement in feline endotoxin shock. Am J Obstet Gynecol 112:414-421, 1972 4. Ulevitch RJ, Cochrane CG, Henson PM, Morrison DC, Doe WF: NMediation systems in bacterial lipopolvsaccharide-induced hypotension and disseminated intravascular coagulation. I. The role of complement. J Exp NMed 142:1570-1590, 1975 5. Mrlller-Berghaus G, Lohmann E: The role of complement in endotoxin-induced disseminated intravascular coagulation: Studies in congenitallv C6-deficient rabbits. Br j Haematol 28:403-418, 1974 6. Evensen SA, Pickering RJ, Batbouta J, Shepro D: Endothelial injury induced by bacterial endotoxin: Effect of complement depletion. Eur J Clin Invest 5:463-469, 1975 Bergstein J\1, Michael AF Jr: Failure of cobra venom factor to prevent the generalized Shwartzman reaction and loss of renal cortical fibrinolv tic activity. Am J Pathol 74:19-30, 1974 8. Polak L, Turk JL: Suppression of the haemorrhagic component of the Shwartzman reaction by anti-complement serum. Nature 223:738-739, 1969 9. Stetson CA, Good RA: Studies on the mechanism of the Shwartzman phenomenon: Evidence for the participation of polvmorphonuclear leucocv-tes in the phenomenon. J Exp Med 9'3:49-63, 1951
AdmoWdnents The author thanks Linda Shevlin for technical assistance and Rochelle Beddard for preparing the manuscript.
120
BERGSTEIN
American Journal of Pathology
[Illustrations follow]
Fge I-Skin obtained from rabbit 6 hours after provoking injection of endotoxin, demonstrating marked edema, inflammation, hemorrhage, and vascular thrombosis (H&E, x250).
A
B
E
Figure 2-Immunofluorescent studies of frozen sections of skin obtained from rabbit 6 hours after provoking injection of endotoxin, demonstrating (arrow) vascular deposition of fibrin (A, x 400), IgG (B, x 250), IgM (C, x 400), C3 (D, x 250), and albumin (E, x 400).
