Bacillus anthracis lethal toxin induces cell-type-specific cytotoxicity in human lung cell lines.

AIMS: Inhalational anthrax is caused by the entry of Bacillus anthracis spores into the lung. Inhaled spores are phagocytosed by alveolar macrophages. Bacilli then escape from the macrophage and spread to other cells, initiating a systemic anthrax infection. Based on the pathological studies of primate and human inhalational anthrax cases, it appears that lung tissue injury is a lethal consequence of the disease. Although the cytotoxicity of anthrax lethal toxin to macrophages is well known, it is not clear how anthrax toxin affects the various lung cell types. METHODS AND RESULTS: Using model cell lines representing different physiological compartments of the lung, we have investigated the cytotoxic effects of anthrax lethal toxin. The cell response was evaluated through MTT metabolism, neutral red uptake, initiation of apoptosis, and expression and binding activity of anthrax toxin receptors. We found that a human small airway epithelial cell line, HSAEC, was susceptible to anthrax lethal toxin. The other cell lines, A549, MRC-5, H358 and SKLU-1, displayed resistance to anthrax lethal toxin-mediated toxicity, although the expression of anthrax toxin receptors was detected in all the cell lines tested. CONCLUSIONS: Our results indicate that cell-type-specific toxicity may be induced by anthrax lethal toxin in human lung tissues and does not correlate with anthrax toxin receptor expression levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This work suggests that cell-type-specific cytotoxicity of anthrax toxin in lung cells may cause subsequent lung disease progression. It may explain the initial pathogenic step of inhalational anthrax.

Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages.

The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax. Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor alpha (TNF-alpha). In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-alpha in mouse peritoneal macrophages. We found that treatment with DHEA significantly inhibited the TNF-alpha production caused by anthrax lethal toxin. Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-alpha to the extracellular medium as compared to the control. However, combined use of DHEA and MLT also inhibited TNF-alpha release, but not more than single therapies. These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin.

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity.

The lethal toxin of Bacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin.

Involvement of phospholipase A2 activation in anthrax lethal toxin-induced cytotoxicity.

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.

Sex differences in dizocilpine (MK-801) neurotoxicity in rats.

The sex differences in the clinical signs and the distribution of astrocytic glial fibrillary acidic protein (GFAP) induced by an N-methyl-d-aspartate antagonist, dizocilpine (MK-801), were examined. A single intraperitoneal injection of MK-801 (5 mg/kg body weight) caused a prolonged recumbency (35-40 h), leading to a severe loss of body weight in female rats, in contrast to a light effect in males, independent of age. Early salivation or lacrimation was also severe in females and delayed bloody lacrimation was observed in females only. The pretreatment with 17ß-estradiol (0.1 or 1.0 mg/kg body weight) made early signs worse in both sexes, but a remarkable mortality (20-40%) was observed in females only. The treatment with MK-801 greatly enhanced GFAP expression in retrospenial cortex of both sexes with a higher enhancement in females. The MK-801-induced expression of GFAP was further increased by the pretreatment with 17ß-estradiol (1 mg/kg body weight) in females. Overall, the expression of GFAP in the retrospenial cortex of rats treated with MK-801 appeared to be higher in females than males, somewhat in parallel with more severe clinical signs in females. The results indicate the higher sensitivity of female rats to MK-801 neurotixicity, and the possible involvement of 17ß-estradiol in the sex differences of the sensitivity.

Organophosphate-induced brain injuries: delayed apoptosis mediated by nitric oxide.

The features of organophosphate-induced brain injuries were investigated. Rats were poisoned intraperitoneally with 9 mg/kg (1.8 LD(50)) of diisopropylfluorophosphate. Pyridostigmine bromide (0.1 mg/kg) and atropine methylnitrate (20 mg/kg), which are centrally inactive, were pre-treated intramuscularly to reduce the mortality and eliminate peripheral signs. Diisopropylfluorophosphate induced severe limbic seizures, and early necrotic and delayed apoptotic brain injuries. The necrotic brain injury was observed to be maximal as early as 1 h after diisopropylfluorophosphate treatment predominently in hippocampus and piriform/entorhinal cortices, showing a spongiform change (malacia) of neuropils in severe cases. In contrast, typical apoptotic (TUNEL-positive) cells started to appear at 12 h in thalamus, and a mixed type in amygdala. Separately, nitrite/nitrate content in cerebrospinal fluid was found to significantly increase after 2 h, reaching a maximal level at 6 h. Pre-treatment with l-N(G)-nitroarginine, an inhibitor of nitric oxide synthase, reduced nitrite/nitrate content and, noteworthy, attenuated only apoptotic brain injury in all four brain regions without affecting seizure intensity and necrotic injury. Taken together, the delayed apoptotic injury of brain induced by diisopropylfluorophosphate poisoning in rats might be mediated in part through nitric oxide production.

Apoptosis as a mechanism of 2-chloroethylethyl sulfide-induced cytotoxicity.

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.

A role of nitric oxide in organophosphate-induced convulsions.

The effects of nitric oxide-regulating compounds on convulsions and mortality of rats administered i.p. with diisopropylfluorophosphate was investigated. l-N(G)-nitroarginine methyl ester, a nitric oxide synthase inhibitor possessing an anticholinergic action, markedly attenuated the intensity of convulsions and significantly reduced the mortality rate. A similar result was obtained with anticholinergic procyclidine, an N-methyl-d-aspartate receptor antagonist. Noteworthy, l-N(G)-nitroarginine, another inhibitor of nitric oxide synthase, significantly attenuated the seizure intensity when administered in combination with atropine sulfate (5 mg/kg), though either l-N(G)-nitroarginine or atropine sulfate was inactive alone. It is suggested that nitric oxide may be a proconvulsant or a convulsion-promoting factor in anticholinesterase poisoning, and both the reduction of nitric oxide level and blockade of cholinergic systems may be required for more effective protection of seizures.

Effects of calmodulin antagonists and anesthetics on the skin lesions induced by 2-chloroethylethyl sulfide.

The effects of calmodulin antagonists and anesthetics on the skin lesions induced by an alkylating vesicant, 2-chloroethylethyl sulfide, were investigated using female hairless mice. 2-Chloroethylethyl sulfide, topically applied (0.6 microliter/5 mm in diameter) on the back skin of hairless mice, induced mild to moderate petechiae on the 1st day, and ulcers with a thick scab after 3 days. The healing process started after 6 days, resulting in shedding of scabs on 9.52 days. Water-soluble ointment bases showed some beneficial effects, whereas oily bases made the skin lesions worse. Trifluoperazine (0.5-1%) and thioridazine (2%), potent calmodulin antagonists, in Pluronic F-127 base substantially prevented the development of 2-chloroethylethyl sulfide-induced skin lesions. A similar effect was achieved with pentamidine (10%), another type of calmodulin antagonist, but not with ketoconazole, a weak calmodulin antagonist. In addition, anesthetics, such as lidocaine and pentobarbital, showed some protection, although at high concentrations (> 5%). As judged by the microscopic appearance, trifluoperazine successfully reduced the hemorrhage and the infiltration of inflammatory cells in early skin lesions, and the formation of thick scabs, which leads to granulomatous scar tissue in late lesions. These results suggest that some calmodulin antagonists and anesthetics in water-soluble bases might be a choice for the treatment of 2-chloroethylethyl sulfide-induced skin burns.