Anthrax poses a clear and present danger as an agent of biological terrorism. Among suspected weapons of mass infection are a double-handful of viral, bacterial and chemical toxins, namely, anthrax, smallpox, botulinum, Hantaan, Ebola, bubonic plague, ricin and sarin.
Late in 2001, it took less than a teaspoonful of an unfamiliar powder, mailed in sealed envelopes, to kill five U.S. citizens, critically infect eight others and discombobulate the U.S. Senate and postal service. That powdery content had all the earmarks of anthrax spores (Bacillus anthracis).
Those spores can lie dormant in soil or animal debris for long periods, until some stimulus triggers them back to life and deadly infectivity. An antibiotic called ciprofloxacin proved effective in emergency treatment of lethal inhalation anthrax. Although it cleared the infection and killed the bacillus, it didn't neutralize the deadly toxins the germ secreted.
In the ding-dong battle between B. anthracis and human immune defenses, the latest shot is fired in the July 17, 2003, issue of Nature. The paper's title fingers the shooter: "Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin." Its senior author is immunologist Bali Pulendran at Emory University in Atlanta, where he is associate professor of pathology.
"In the first study of its kind," Pulendran told BioWorld Today, "we have shown that anthrax lethal factor impairs the function of dendritic cells and thereby compromises the immune system's ability to fight the microbe. Our findings may have implications for developing more effective anthrax therapies and guiding researchers in better controlling detrimental immune responses."
Three Arrows In Quiver: PA LF, EF Tipped To Kill
In new research, the paper reports that the anthrax bacillus deploys a novel strategy for evading its target's immune reactions. The bug packs an arsenal of toxic factors, topped by "lethal factor" (LF), an enzyme that binds to and degrades a key signaling protein inside human cells. Besides LF, its toxic armamentarium includes "protective antigen" (PA), which enables LF to enter macrophages and other target immune cells, followed by "edema factor" (EF).
Symptoms emerge only after the bacillus has already multiplied inside its prey and begun to produce large amounts of the tripartite toxin - PA, LF and EF, which gang up to kill their human patient. Cutaneous anthrax is rarely fatal, whereas systemic inhalation of bacterial spores into the lungs is what led to the five U.S. deaths. This fatality is usually preceded by inflammation, rampant bacteremia and multisystem dysfunction.
Bali Pulendran and his co-authors report in Nature that the critical virulence factor, anthrax lethal toxin (LT) targets the immune system's pivotal dendritic cells. Their prime function is to recognize foreign particles and bring them to the notice of the adaptive immune system for destruction.
"We demonstrated," Pulendran continued, "that LF impairs dendritic cell function by disrupting the mitogen-activated protein (MAP kinase enzymes) within dendritic cells. Consequently, these cells become lethargic and unable to act normally, thereby preventing the activation of the immune system to attack microbes such as anthrax.
"When a person is infected by a microbe," he commented, "we count on the immune system to begin fighting the foreign substance immediately. When the dendritic cells are compromised, as in our study, with the anthrax lethal factor, the innate immunity [early advance detachment] is unable to stimulate the adaptive [full-force] immune response, thus permitting the microbe to spread unchecked. Our ultimate goal is to apply this novel finding to develop better anthrax treatments and to shape future research into controlling immune responses more appropriately."
To stimulate dendritic cells for in vivo mouse experiments, Pulendran and his co-authors washed those cells in lipopolysaccharide. This potent sugar coats the walls of many pathogens and sets off sure-fire reactions by human immune defenses. These release tumor necrosis factor plus interleukins-1, -6, and -12, but anthrax had the last laugh over these well-armed cytokines. The dendritic cells were severely impaired in LT-treated anthrax toxic factors.
Evasion Is Name Of B. Anthracis Game
"Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules. They secrete greatly diminished amounts of proinflammatory cytokines and do not effectively stimulate antigen-specific T cells in vivo. Furthermore," the paper noted, "injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity. These data," he wrote, "suggest a role for LT in suppressing host immunity during B. anthracis infections and represent an immune evasion strategy where a microbe targets MAP kinases in dendritic cells to disarm the immune response.
"Given these potent effects on dendritic cells in vitro," Pulendran went on, "we determined whether LT-primed dendrites could induce T-cell unresponsiveness in vivo. We used a transgenic mouse model in which the fate of antigen-specific CD4-positive T cells could be tracked in vivo. The model involves mice that express transgenic T-cell receptors that recognize an ovalbumin (OVA) peptide in the context of the class II major histocompatibility complex. We then immunized the reconstituted mice with OVA. Spleen dendritic cells were exposed to PA plus LF and after four hours were injected into the footpads of the mice. In vivo stimulation with dendritic cells plus PA plus LF resulted in a profound diminution of THelper1 and THelper2 cytokines in vivo. Therefore, LT-treated dendritic cells failed to prime antigen-specific CD4+ T cells in vivo."
The Emory investigators' immediate next steps are to test the effects of LP in suppressing other immune models and to look more closely at immune responses several days post infection, when toxic shock-like symptoms begin.
Perhaps paradoxically, the Nature paper concludes by proposing that anthrax, "LT or its derivatives may also represent potential therapeutic tools in the regulation of deleterious immune responses in autoimmunity or transplantation. This new therapeutic approach may be especially feasible, given the recent elucidation of the crystal structure of B. anthracis LF and PA."
Meanwhile, the AP and The New York Times reported July 12 that a nationwide test of a new anthrax detection system at the Postal Service is scheduled to begin July 21. Developed after the 2001 anthrax letter attacks, the "Biohazard Detection System" uses rapid DNA testing to detect microorganisms.