Differential susceptibility of macrophage cell lines to Bacillus anthracis–Vollum 1B

Abstract

Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mϕs) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mϕs. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mϕ cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mϕs are sensitive to LeTx whereas IC-21 Mϕs are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mϕs demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mϕs with LeTx + Vollum 1B (V1B) spores for >15 h and assayed for Mϕ cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (≈50% mortality) was observed in RAW264.7 and J774A.1 Mϕs at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mϕs demonstrated 100% mortality. In contrast, IC-21 Mϕs, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mϕ cell lines to the LeTx + V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mϕs and BA spores.