The progression of the immune response in the lungs after aerosol infection with Mycobacterium tuberculosis is a complex cellular event dominated by macrophages and lymphocytes. Although the phenotype of lymphocytes participating in this response is becoming increasingly well characterized, the dynamic influx of these cells during the infection and their spatial arrangements within the lung tissue are still poorly understood. This study shows that in the first month after aerosol infection with M. tuberculosis there was a steady increase in the percentages of total CD3 ؉ , CD3 ؉ CD4 ؉ and CD3 ؉ CD8 ؉ cells, with consistently larger numbers of CD3 ؉ CD4 ؉ cells than of CD3 ؉ CD8 ؉ cells. As granuloma formation continued, the granuloma was found to consist of macrophages, CD4, and CD8 T cells, as well as a smaller number of B cells. Whereas CD4 T cells formed organized aggregates, CD8 T cells were fewer and more scattered and tended to be more prominent toward the periphery of the granulomas. The possible ramifications of the juxtapositions of these two major T-cell subsets are discussed.It is estimated that one-third of the population worldwide has been infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (26). Natural infection with M. tuberculosis occurs via the airway, where the bacillus infects the macrophages in the lungs. Vaccines and chemotherapeutic agents against M. tuberculosis infection do exist, but for multiple reasons they are unable to contain the epidemic (18). One of the main reasons for this failure is the lack of understanding of the pathogenesis and immune mechanisms that take place in the lungs during the infection.Acquired immunity generated against M. tuberculosis infection develops slowly in the lungs (3,5,15), with bacterial growth tending to stop as this immunity appears (7, 16). Numerous studies using specific-gene-disrupted mice (4, 25) and immune T-cell transfer (14,17) have demonstrated that immunity to M. tuberculosis infection is dependent on the emergence of specific subpopulations of T cells. It is well known that CD4 T-cell populations are critical for survival of this infection (4, 20), but it is also becoming apparent that other cell populations such as CD8 T cells (11,13,25), ␥␦ T cells (1, 9), and NK cells may also be important, although to date their roles are far less well characterized. What is clear, however, is that the host response to M. tuberculosis infection is dependent on the production of gamma interferon␥ by primed T cells (6, 12, 21), which in turn is dependent on interleukin-12 production by antigen-presenting cells (macrophages and dendritic cells) (8).In the infected lung, these complex interactions take place in the context of a host tissue remodeling response called granuloma formation, and it is the construction of these structures that forms the hallmark of the disease. This process is complex and seems to follow a series of pathologically distinct stages, as we have previously described (22). However, the actual makeup of the gr...

The interleukin-12 and gamma interferon (IFN-␥) pathway of macrophage activation plays a pivotal role in controlling tuberculosis. In the murine model, the generation of supplementary nitric oxide by the induction of the nitric oxide synthase 2 (NOS2) gene product is considered the principal antimicrobial mechanism of IFN-␥-activated macrophages. Using a low-dose aerosol-mediated infection model in the mouse, we have investigated the role of nitric oxide in controlling Mycobacterium tuberculosis in the lung. In contrast to the consequences of a systemic infection, a low dose of bacteria introduced directly into the lungs of mice lacking the NOS2 gene is controlled almost as well as in intact animals. This is in contrast to the rapid progression of disease in mice lacking IFN-␥ or a key member of the IFN signaling pathway, interferon regulatory factor 1. Thus while IFN-␥ is pivotal in early control of bacterial growth in the lung, this control does not completely depend upon the expression of the NOS2 gene. The absence of inducible nitric oxide in the lung does, however, result in increased polymorphonuclear cell involvement and eventual necrosis in the pulmonary granulomas of the infected mice lacking the NOS2 gene.Tuberculosis is principally a disease of the lung and is transmitted by aerosol droplets containing only a few bacteria. These infected droplets must be deposited in the alveoli for infection to be established, and thus the exposure of the bacteria to the immune response is limited during the early stages of infection. This discreet entry into the lung means that innate responses must bear the burden of controlling the infection until the acquired response recognizes that infection has occurred. Currently, only two products of the immune response, gamma interferon (IFN-␥) (7) and tumor necrosis factor alpha (2), have been shown to be crucial to the early control of low-dose aerosol infection with virulent Mycobacterium tuberculosis. These two cytokines are known to activate macrophages to a microbistatic and/or microbicidal state.The exact molecular mechanisms of macrophage activation that limit mycobacteria have not yet been determined. One of the likely mediators of mycobacterial control is the expression of high levels of nitric oxide (28) following induction of the inducible nitric oxide synthase (iNOS) gene (NOS2). This molecule is thought to act in concert with superoxide radicals within acidic phagosomes to generate toxic products capable of limiting the survival and growth of M. tuberculosis. Indeed, studies performed using systemic models of mycobacterial infection confirmed that this molecule is crucial to the containment of systemic M. tuberculosis infection (4, 5, 16). A potential problem with the relevance of these studies to tuberculosis is that the bacteria are introduced intravenously and become lodged within the liver, spleen, and interstitium of the lung, resulting in an extensive, systemic bacterial infection. This level of infection requires the maximum protective responses of the ...

We have developed a rapid new in vivo method for screening experimental drugs for their activity against Mycobacterium tuberculosis by using the gamma interferon gene-disrupted (GKO) C57BL/6 mouse. Due to the rapid growth of the infection, statistical differences indicating positive efficacy of active compounds can be seen after only 8 days of treatment. To validate this model, several fluoroquinolones, including ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin, were tested in parallel.Therapy for tuberculosis is arduous due to its long duration and multidrug regimens. Therefore, there is an increasing demand for the development of new compounds and screening of existing compound libraries in order to treat tuberculosis. As part of the overall research effort at the National Institutes of Health, the National Institute of Allergy and Infectious Diseases has established a screening program at several institutions to efficiently screen large numbers of compounds for possible activity against Mycobacterium tuberculosis. The compounds are received as part of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), which provides no-cost screening of compounds from industrial and academic sources. To date, over 60,000 compounds have been provided to the program. About 0.3% of the compounds which performed well during the extensive initial testing in in vitro assays are subsequently tested in mouse models (more information on the program and assays used can be found at www .taacf.org). In order to screen a larger number of compounds against tuberculosis in animals, we have developed a rapid new in vivo method.Mouse models have been extensively used for preclinical assessment of experimental compounds against tuberculosis (2, 6, 7, 9, 10). In intravenous infection models, Swiss outbred mice are infected with M. tuberculosis with ϳ5 ϫ 10 6 bacteria and treated once daily with various dosages for 28 days starting on the day after infection (4,11,12,14,15). More recently, we have developed a second model in which inbred C57BL/6 mice are infected with 50 to 100 CFU by low-dose aerosol and treated daily for 35 to 45 days starting 20 days postinfection when the bacterial load reached its peak (13). In both models, the drug regimen lasts 1 to 1.5 months of daily therapy. In an attempt to shorten this process, we have developed a novel short-term in vivo model for the rapid screening of experimental compounds.This model uses the highly susceptible gamma interferon gene-disrupted (GKO) C57BL/6 mouse (3, 5, 8, 18) and was performed as follows. Eight-to ten-week-old female specificpathogen-free C57BL/6-Ifngtm1ts (GKO) mice (Jackson Laboratories, Bar Harbor, Maine) were exposed to a low-dose aerosol infection with M. tuberculosis in a Glas-Col inhalation exposure system as previously described (13). The virulent M. tuberculosis strain Erdman (TMCC 107) has been used as the standard strain for drug testing in animal models in this laboratory (13). One day postinfection, three mice were sacrificed to ...

In this study different inbred strains of mice appeared to control and contain a low dose aerosol infection with Mycobacterium tuberculosis in a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection with M. tuberculosis may reflect an inability to localize antigen-specific lymphocytes within the lung.Disease caused by Mycobacterium tuberculosis is often not due to primary infection but instead is caused by reactivation of a latent or dormant infection that the patient may have carried for many years (20). It is unclear however, how the host initially expresses resistance in the lung and why this resistance is eventually lost, thus allowing bacterial regrowth. In this regard, the mouse is a useful model of specific resistance to M. tuberculosis infection. After low or moderate doses of bacilli are delivered by either intravenous or aerogenic routes, an apparently stable chronic disease state is established after a few weeks in the infected organs of the animal (3, 24). This chronic infection continues for a prolonged period in the C57BL/6 mouse strain until influenced by immunosenescence (23). It is currently speculated that during the chronic phase of infection, the bacteria remain in some form of latent state.That this assumption may be wrong, however, is suggested by recent evidence indicating that chronic M. tuberculosis infection in the lung is in fact a dynamic event (27), at least in terms of granuloma pathology, which changes dramatically over the life span of the animal. In addition, studies directed toward understanding the role of the Bcg gene (Nramp1) have shown that certain inbred strains of mice clearly differ in their ability to survive a chronic M. tuberculosis infection (16, 18), in confirmation of much earlier reports of this phenomenon (15,26).The results of the current study confirm and extend these findings by showing that certain strains of mice, although able to initially control a low-dose aerosol infection with M. tuberculosis, eventually succumb during the chronic phase of disease. The current study shows that this early mortality was associated with an increased bacterial burden within the lung and occurred prior to events that could be attributed to immunosenesence (23). Comp...

An effective new vaccine for the control of tuberculosis is badly needed. While current research attempts to improve vaccination are concentrating on new prophylactic or immunotherapeutic vaccines, virtually no emphasis has been placed on boosting individuals already inoculated with Mycobacterium bovis BCG. It is shown here that mice vaccinated with BCG gradually lose their capacity to resist an aerosol challenge infection as they age. However, if these mice are inoculated with the 30-kDa mycolyl transferase A protein in midlife, after challenge when aged they express levels of protection in the lungs comparable to those of young mice, associated with minimal pathological damage.Currently available epidemiologic data indicate that disease caused by the facultative intracellular bacterial parasite Mycobacterium tuberculosis remains a serious global problem, with around 8 million new cases per year, and there is recent disturbing evidence that the death rate may be increasing (4,7,14,15). For several decades the Mycobacterium bovis-derived bacillus Calmette-Guérin (BCG) has been the only widely used vaccine for tuberculosis, and accumulating data from clinical trials and subsequent meta-analysis (5, 18) have tended to reveal its general ineffectiveness in adults (including those vaccinated with BCG as young children). As a result, many laboratories are now involved in a major effort to develop a new vaccine (12, 13), with virtually all efforts directed towards discovering new candidate vaccines that can be used in a prophylactic or immunotherapeutic mode (1,8,11).The mechanism underlying the gradual loss of effectiveness of BCG as the (neonatally inoculated) individual reaches 10 to 15 years of age is poorly understood. One possible assumption is that memory immunity generated by BCG has disappeared and the individual is now equivalent to a naive host who can be vaccinated with a new candidate vaccine designed to induce primary immunity. An alternate possibility is that memory immunity slowly declines but can be recovered by boosting if a candidate antigen that can be specifically recognized by this immunity is reintroduced. The results of this study support the latter contention.Several laboratories have shown that proteins found in the culture filtrates of M. tuberculosis are highly immunogenic and have promise as candidate vaccines (12). A component of this pool of proteins is the mycolyl transferase A (Ag85A) (3), and we have previously demonstrated that the largest proportion of CD4 T cells accumulating in the lungs of memory-immune mice after challenge infection recognize this antigen (6). Thus, to determine if Ag85A has the potential to boost existing memory immunity in BCG-vaccinated mice, these animals were boosted twice at 9 and 15 months of age and then challenged with virulent M. tuberculosis when elderly. As shown here, this procedure restored the capacity of these mice to express resistance to this infection.Female C57BL/6J mice were purchased from Jackson Laboratories, Bar Harbor, Maine. They were k...