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Human alveolar epithelial barrier function is impaired by bacillus anthracis lethal toxin

: Langer, Marybeth; Booth, John L.; Patel, Vineet I.; Duggan, Elizabeth S.; Braun, Armin; Ramani, Vijay; Awasthi, Shanjana; Silasi-Mansat, Robert; Lupu, Florea; Coggeshall, Kenneth M.; Metcalf, Jordan P.

American Journal of Respiratory and Critical Care Medicine 185 (2012), Abstract A3271
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2012, San Francisco/Calif.>
Fraunhofer ITEM ()

RATIONALE: The lung is the site of entry for Bacillus anthracis (Ba) in inhalation anthrax, the deadliest form of the disease. Ba produces virulence toxins that are necessary for clinical disease in higher animal models, including lethal toxin (LT). Prior to studies previously reported from our laboratory, it was thought that human alveolar macrophages (AM) were the primary target of the Ba LT because mouse macrophages are killed by apoptosis through LT-induced MAP kinase kinase (MEK) cleavage. We have reported that human AM are not a target of LT, as they do not contain receptors for the binding component of the toxin, protective antigen (PA), and do not undergo MEK cleavage or undergo apoptosis in response to the toxin. We sought to determine whether the cells that line the functional respiratory units of the lung, alveolar epithelial cells (AEC), are a target of the B. anthracis virulence factor LT.
METHODS: We measured the effect of Ba LT on primary human AEC MEK cleavage by immunoblot, barrier function by measurement of transepithelial resistance (TER), and cell viability by propidium iodide staining and intrinsic dehydrogenase activity. The effect of LT exposure on AEC subcellular morphology was also assessed by electron microscopy (EM).
RESULTS: In contrast to our findings for AM, human AEC were sensitive to LT. Epithelial barrier function was significantly impaired as measured by a 30% drop in TER. Consistent with these results, AEC, unlike AM expressed significant amounts of the anthrax toxin receptor TEM8/ANTXR1. The receptor was functional as LT exposure resulted in cleavage of multiple MEK's, as assessed by immunoblot, and as assessed by measurement of binding of PA to AEC. Surprisingly, despite these effects, LT exposure did not affect AEC viability as determined by propidium iodide staining or intrinsic dehydrogenase activity. LT exposure did, however, impair formation of tight junctions as assessed by EM.
CONCLUSIONS: These findings show that in contrast to AM, human AEC are a target of the B. anthracis virulence factor LT. Compromise of alveolar epithelial barrier function by LT may play a major role in the pathogenesis of inhalation anthrax by facilitating the dissemination of B. anthracis from the lung in early phases of illness, and by promoting barrier disruption and subsequent lung edema that occurs in terminal phases of the disease.