Comments for:
Abstract
Bacillus anthracis lethal toxin (LT) is the major virulence factor of anthrax and reproduces most of the laboratory manifestations of the disease in animals. We studied LT toxicity in BALB/cJ and C57BL/6J mice. BALB/cJ mice became terminally ill earlier and with higher frequency than C57BL/6J mice. Timed histopathological analysis identified bone marrow, spleen, and liver as major affected organs in both mouse strains. LT induced extensive hypoxia. Crisis was due to extensive liver necrosis accompanied by pleural edema. There was no evidence of disseminated intravascular coagulation or renal dysfunction. Instead, analyses revealed hepatic dysfunction, hypoalbuminemia, and vascular/oxygenation insufficiency. Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, eotaxin, FasL, and IL-1β. No changes in TNF-α occurred. The C57BL/6J mice did not mount a similar cytokine response. These factors were not induced in vitro by LT treatment of toxin-sensitive macrophages. The evidence presented shows that LT kills mice through a TNF-α–independent, FasL-independent, noninflammatory mechanism that involves hypoxic tissue injury but does not require macrophage sensitivity to toxin.
Authors
Mahtab Moayeri, Diana Haines, Howard A. Young, Stephen H. Leppla
Response to Chojkier
Submitter: Stephen Leppla | sleppla@niaid.nih.gov
National Institute of Allergy and Infectious Diseases
Published December 15, 2003
We are in agreement with the comments made by Dr. Chojkier. Our manuscript may have misled some readers to conclude that liver necrosis is thought to be the primary cause of death in most mice. In fact, centrilobular liver necrosis, as well as necrosis in marrow and dental pulp, along with pleural and peritoneal fluid accumulation, are all likely indications of vascular collapse and the resulting hypoxia in LT-treated mice. The AST and ALT increases likely represent the beginning of problems in the liver, but we agree that liver failure is not the cause of death in most mice – and is simply a result of the circulatory collapse accompanied by many other events relating to this collapse. We also believe that the extent of effusions in the pleural cavity, in most cases, were insufficient to be the sole cause of death. However, it is possible that edema in the alveolar spaces not evident in histological preparations contributes to death. Additionally, we believe the toxin affects endothelial and epithelial cells at a molecular level in a manner not discernable by light microscopy, but clearly resulting in alteration of function. The mechanisms by which mice die are clearly linked to circulatory collapse, but it is not known how this event is initiated. The primary conclusion of our study was that LT- toxicity is not due to a TNF-alpha or cytokine and inflammatory- mediated classic shock, as previously assumed, but rather that this toxin induces vascular collapse through a novel mechanism which is independent of macrophage lysis and cytokine release.
Anthrax lethal toxin and liver injury
Submitter: Mario Chojkier | mchojkier@ucsd.edu
University of California, San Diego and San Diego VA Healthcare Center
Published December 13, 2003
I have read with great interest the valuable study by Moayeri and coworkers (1). However, I disagree with one of their main conclusions that death induced by anthrax lethal toxin [LT] may be due to extensive hypoxic liver necrosis. I believe that the degree of liver injury was not a major contributor to death in these animals. For example, at the time when mortality of BALBc mice was ~ 50% (Fig 1d), the degree of liver necrosis, judging by the levels of serum alanine aminotransferase (ALT), its most reliable indicator, was rather modest (Fig 6a) and inconsistent with the diagnosis of fulminant liver failure (2;3). Specifically, ischemic and hypoxic fulminant liver failure results in markedly raised ALT values (4;5). Serum ALT levels below 300 U/l, as measured in the vast majority of mice (Fig 6a and 6b), are not associated with a fulminant progression of acute liver injury in either animal models or patients (4- 9). Moreover, the ALT values in non-treated mice were higher (up to ~ 100 U/l) (Fig 6) than those in normal individuals, further blunting the relative ALT increase to about three-fold or less, in LT-treated mice. In this context, the FDA considers a three-fold increase in ALT from upper limits of normal values a threshold for potential hepatotoxicity of new drugs (10). Some approved medications have even exceeded twenty-fold the normal ALT values in premarketing clinical trials, without any associated liver failure (11). Fulminant liver failure usually develops in animals and patients when serum ALT levels reach thousands U/l (2;3;7-9). In addition, the rapid decrease in serum albumin levels, with the subsequent decrease in oncotic pressure, that followed LT injections cannot be a reflection of impaired hepatic synthesis due to fulminant liver failure, since the half-life of albumin is ~ 21 days (12). Therefore, proteins with a half-life of ~ 6 h, such as clotting factors, are used to assess the severity and prognosis of acute liver failure (2;4). The low serum albumin induced by LT was most likely a consequence of extensive transudation, at least in part into peritoneal and pleural fluids, and/or possibly, degradation (12). In spite of the misinterpretation of the severity of the liver injury, I believe that the comprehensive pathological findings and the well-documented hypoxic events will be helpful in elucidating the mechanisms by which LT induces death.
References
1. Moayeri,M., Haines,D., Young,H.A., and Leppla,S.H. 2003. Bacillus anthracis lethal toxin induces TNF-alpha–independent hypoxia-mediated toxicity in mice. J.Clin.Invest. 112:670-682.
2. Lee,W.M. 1993. Acute liver failure. N.Engl.J.Med. 329:1862-1872.
3. Ostapowicz,G., Fontana,R., Schiodt,F., Larson,A., Davern,T., Han,S., McCashland,T., Shakil,A., Hay,J., Hynan,L. et al. 2002. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med. 137:947-954.
4. Stolz,A. and Kaplowitz,N. 1990. Biochemical Tests for Liver Disease. In Hepatology: A Textbook of Liver Disease. D.Zakim and Boyer,T.D., editors. W.B. Saunders Company, 637-667.
5. Baker,A.L. 1992. Liver Chemistry Tests. In Liver and Biliary Diseases. N.Kaplowitz, editor. Williams & Wilkins, 182-194.
6. Scholmerich,J., DeLuca,M., and Chojkier,M. 1984. Bioluminescence assays for bile acids in the detection and follow-up of experimental liver injury. Hepatology 4:639-643.
7. Chojkier,M. and Fierer,J. 1985. D-Galactosamine hepatotoxicity is associated with endotoxin-sensitivity and mediated by lymphoreticular cells in mice. Gastroenterology 88:115-121.
8. Leonis,M., Toney-Earley,K., Degen,S., and Waltz,S. 2002. Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure. Hepatology 36:1053-1060.
9. Wolf,D., Hallmann,R., Sass,G., Sixt,M., Kusters,S., Fregien,B., Trautwein,C., and Tiegs,G. 2001. TNF-alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1--relevance for concanavalin A-induced hepatitis. J.Immunol. 166:1300-1307.
10. http://www.fda.gov/cder/livertox/clinical.pdf
11. Watkins,P.B., Zimmerman,H.J., Knapp,M.J., Gracon,S.I., and Lewis,K.W. 1994. Hepatotoxic effects of tacrine administration in patients with Alzheimer's disease. JAMA 271:1023-1024.
12. West,J.B. 1990. Physiological Basis of Medical Practice. Williams & Wilkins, Baltimore.
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