American Journal of Respiratory Cell and Molecular Biology

The clinical manifestations of tuberculosis represent a complex interaction between the causative organism, Mycobacterium tuberculosis, and the human host immune response. Although the disease of consumption was recognized many centuries ago, the modern understanding of what has remained one of the world's great public health problems is usually traced to the work of Robert Koch. Koch assuredly deserves his status as one of the great founding figures of medicine and microbiology, although at least two other individuals made major contributions during the “pre-history” that led to Koch's discovery. One of these men is familiar to most students of medical history; the other is not.

René Théophile Hyacinthe Laennec (1781–1826) was a French physician known for, among other accomplishments, his invention of the stethoscope. However, Laennec also holds an important place in medical history for being among the pioneers of clinical-pathological correlations, and his descriptions of the pulmonary lesions in patients who had died of consumption really mark the beginning of our understanding of the pathology and pathogenesis of tuberculosis. In his landmark work A Treatise on Diseases of the Chest, which was translated into English by John Forbes and published in London in 1821, Laennec provided figures illustrating tuberculous cavities and described in detail the pathologic changes now so familiar as caseous necrosis:

Whatever the form in which the tuberculous matter develops, it begins as a grey, semi-transparent matter that little by little becomes yellow, opaque, and dense. Then it softens, and slowly acquires a liquidity like pus, and, when it is expelled through the airways, it leaves cavities, commonly called ulcers of the lung, that we will designate as tuberculous excavations. (1)

Laennec's careful autopsy studies also established that pathologically similar lesions in different parts of the body were in fact due to the same disease, tuberculosis, although he could not and did not state what was the cause of that illness.

Less well known perhaps, but quite important scientifically (and a true forerunner of Koch) was Jean-Antoine Villemin, a French physician who published a landmark study in 1868 in Paris, entitled Etudes sur la Tuberculosis (Studies on Tuberculosis). Villemin clearly established the infectious nature of tuberculosis. In the seventh study in his series (described in Part 4 of his book), he describes the transmission of tuberculosis from humans to rabbits, from cows to rabbits, from rabbits to rabbits, and so on. The precision of the proof of the transmissible nature of tuberculosis is exemplified in the following description from Villemin's book:

March 6, 1865, we took two rabbits about three weeks old, very healthy… In one of these rabbits we introduced into a little subcutaneous wound behind each ear, two small fragments of tubercle and a small amount of purulent liquid from a tuberculous cavity, removed from the lung and the intestine of a consumptive, who had been dead twenty-three hours.

June 20th, that is, at the end of three months and fourteen days, there was no appreciable change in the health of the animal, [and so] we sacrificed it and noted the following: The lungs are full of large tubercular masses, formed, in an obvious manner, from the agglomeration of numerous granulations . . The other rabbit, who had shared the same conditions of life with the inoculated rabbit … did not show a single tubercle.” (2)

Villimin had certainly established that tuberculosis was a transmissible disease, but he did not identify the causative agent. This of course was left to Robert Koch, who announced his achievement at a meeting of the Physiological Society of Berlin on March 24, 1882. The paper describing his work, “Der Aetiologie der Tuberculose,” was published in Berliner Klinische Wochenschrift on April 10, 1882.

Koch's contributions to the study of tuberculosis, and to medicine and microbiology in general, were enormous. He developed staining techniques for M. tuberculosis, developed culture media in which to grow the organism, demonstrated the mode of transmission of the illness, and based on his understanding of the spread of the disease, recommended isolation of patients with tuberculosis. His approach to the proof that M. tuberculosis was in fact the causative agent of the illness remains the standard to this day for implicating a microorganism as a causative agent of disease:

To prove that tuberculosis is caused by the invasion of bacilli, and that it is a parasitic disease primarily caused by the growth and multiplication of bacilli, it is necessary to isolate the bacilli from the body, to grow them in pure culture, until they are freed from every disease product of the animal organism, and, by introducing isolated bacilli into animals, to reproduce the same morbid condition that is known to follow from inoculation with spontaneously developed tuberculous material [emphasis added]. (3)

Ironically, another significant contribution of Koch was based on what was really a failed attempt to treat tuberculosis. Several years after identifying the microorganism responsible for the disease, Koch reported on a mysterious substance which he felt could cure, or alter least ameliorate the disease. The substance, he later revealed, was made from supernatants of M. tuberculosis grown in broth, and was what we now know as tuberculin. Although to Koch's frustration and probable shame, tuberculin did not cure the disease, he was able to demonstrate for the first time, unwittingly perhaps, cell-mediated immune responses that we now recognize as the driving forces behind the clinical manifestations of tuberculosis. (In addition, a refinement of Koch's preparation of tuberculin remains a key component of tuberculosis diagnostics even today, which he himself predicted: “I assume that the material will be valuable diagnostic measure in the future. It will become possible to diagnose questionable cases of phthisis even in those cases where bacilli cannot be detected in the sputum.” (4))

The Host

In the decades following Koch, several investigators established the nature and importance of host immune responses, and specifically cell-mediated host immune responses, in the pathogenesis of tuberculosis. Experiments by Bail (5), Landesteiner (6), Chase (7), and Lawrence established that immune responses could be elicited by serum-free cells; Lawrence also transferred immune reactivity between patients by transferring leukocytes alone (8). That lymphocytes specifically were the primary component of the cellular immune response was further established by experiments in animals performed by Wesslén (9) and Coe (10). The important role of macrophages as effector cells in host defense against M. tuberculosis was initially identified and elucidated by scientists such as Patterson, Youmans, Bloom, Bennett, David, Lurie, and Suter (1115). These aforementioned researchers and their work set the stage for later studies which have led to our understanding of the important role both of granuloma formation in the containment of infection as well as the complex interplay between effector cells such as lymphocytes, macrophages, and dendritic cells, as mediated by chemokines and cytokines, interferon-γ (IFN-γ) chief among them, in human immune reponses to M. tuberculosis. Overall, our understanding of the pathogenesis of tuberculosis supports a framework of immune responses which include the initial response to inhaled organisms (outright elimination or establishment of a latent state), granuloma formation and establishment of a state of latent infection, and reactivation of latent infection. A large body of epidemiologic evidence supports the notion that 90% of persons with latent tuberculosis infection will never develop clinical illness. In the last several years, the important components of a successful and sustained host response have been described more fully. Given space constraints, the remainder of this review will focus on selected highlights in the advancement of our understanding of tuberculosis pathogenesis in the past several years.

Genetic susceptibility to tuberculosis.

It has long been recognized that certain populations appear to have a high degree of vulnerability to tuberculosis. Extreme susceptibility to the disease has been observed in Eskimo populations in North America, in Yanomami Indians in the Brazilian Amazon, and in black populations in the United States (1618). These observations have led researchers to assume that tuberculosis probably originated in western Europe, creating selection pressure for a measure of innate resistance among the population there. In the ages of exploration and colonization, Europeans moved to the east and south, and tuberculosis was introduced to populations where no innate resistance was present, with devastating results. In addition, it has long been known that a substantial percentage of individuals will recover from tuberculosis without drug treatment. A tragic experience in Lubeck, Germany in 1929, in which 249 infants were accidentally injected with a stock of live, virulent M. tuberculosis, leading to 76 deaths but leaving 173 survivors, also supports the notion of some capability for innate resistance.

A clue to the genetic basis of resistance was provided by Gros and colleagues, who identified a strain of mice with exquisite vulnerability to overwhelming infection with leishmania, salmonella, and certain mycobacteria, particularly M. bovis (1921). Eventually, the genetic basis of this susceptibility was mapped to a locus encoding a gene on murine chromosome 1 initially called Bcg, then renamed nramp1, responsible for the production of a so-called natural resistance–associated macrophage protein. It appears that the protein product of this gene, which in humans is located on 2q35 and has been renamed as NRMP1 or Scl11a1 (solute carrier family 11 member 1), is a transmembrane iron transporter located in the late endosomal compartment of resting macrophages whose exact function remains somewhat uncertain (2224).

Several polymorphisms in the human NRAMP1 gene have been identified, and population-based studies in several regions have been conducted which have identified increased relative risk for moving from latent infection to active disease associated with certain polymorphisms (25). However, the risk attributable to these polymorphisms is relatively small, and it is clear that NRAMP alone explains only a portion of genetic susceptibility to tuberculosis.

Several other genes and gene families, including those for vitamin D receptors and the components of IFN-γ–signaling pathways, have also been studied for their role in susceptibility to tuberculosis (2527). It is clear from these investigations that resistance or susceptibility to tuberculosis is a complex genetic trait, and there is unlikely to be a single dominant genetic factor involved.

Macrophages in defense against tuberculosis.

Mycobacteria can be taken up by a variety of receptors on macrophages, including those for complement and surfactant, and as has been described more recently, through several of the toll-like receptors, including TLR2 and TLR4 (28, 29). The relative importance of these various receptors remains somewhat unclear. Killing, or growth inhibition, of mycobacteria by macrophages occurs through activation of macrophages and the production of both reactive oxygen and nitrogen species. The relative contributions of these two molecules to the macrophages response to the mycobacterium is still a somewhat open issue, but in a series of papers Chan, Bloom, Flynn, and Nathan and their coworkers have certainly demonstrated the importance of nitric oxide most convincingly, at least in the mouse model (3033). These investigators have demonstrated that mouse macrophages can kill M. tuberculosis in vitro, but not in the presence of inhibitors of inducible nitric oxide sythnase (iNOS), such as NMMA, an L-arginine analog. In the presence of scavengers of oxygen radical such as catalase or superoxide dismustase, killing of mycobacteia by macrophages is unimpeded (33). Mice in which the iNOS gene has been disrupted are susceptible to overwhelming mycobacterial infection in a manner reminiscent of immunosuppression induced with corticosteroids (31). Finally, mice with relatively stable and asymptomatic infection with M. tuberculosis rapidly develop fatal acute illness when treated with inhibitors of iNOS (31, 32). Disruption of iNOS function in mice leads to death from tuberculosis in weeks as compared with the months it takes for the organism to kill the untreated wild-type murine host (31, 34). In addition, alveolar macrophages from the lungs of patients with active pulmonary tuberculosis express high levels of the iNOS gene (35).

Granuloma formation.

The granuloma is the cardinal feature of the initial host immune response to tuberculosis, and a great deal of effort has been devoted to understanding granuloma formation and its role in host defenses against M. tuberculosis (36, 37). Inhaled mycobacteria enter alveolar macrophages and a complex series of events follows which will result in either elimination of the bacterium completely, containment of the infection in a granuloma for a prolonged period (forever, or until an alteration in immune status results in breakdown of the granuloma and reactivation of latent infection), or immediate progression to active disease with clinical illness. This latter event usually occurs only in the setting of impaired immunity.

Early insights into the formation and role of granulomas in tuberculosis pathogenesis were provided by the work of Lurie and Dannenberg, using a rabbit model, which has both advantages and disadvantages for making observations relevant to human disease (3843). Rabbits are extremely susceptible to infection with M. bovis, more so than with M. tuberculosis. On the other hand, rabbits with tuberculosis develop caseous necrosis and cavity formation which mimic the human situation fairly closely, at least in terms of histopathology. Using this model, Dannenberg correctly surmised that after engulfing mycobacteria, macrophages send signals to other cells which are then recruited to the site of infection, forming the familiar pattern recognized as a granuloma. Necrotic material at the center represents debris from macrophages which have died, probably through apoptotic mechanisms (44).

Recent work by Flynn and Chan has yielded several important insights into the role of granulomas in maintaining the latent state of tuberculosis infection, and has demonstrated the important role of the cytokine tumor necrosis factor-α (TNF-α) in maintaining granuloma function. Using a murine model of latent infection in which mice inoculated via the tail vein with M. tuberculosis remain clinically well until the disease “reactivates” roughly 9 mo later, these investigators first showed that in vivo inhibition of the production of reactive nitrogen species led to reactivation of disease, as mentioned above. They also observed that TNF-α was expressed throughout the period of latent or quiescent infection, suggesting that this cytokine may have a role in maintaining the latent state. In addition, it was known that TNF-deficient mice are susceptible to overwhelming tuberculosis, and that TNF, along with IFN-γ, stimulates production of reactive nitrogen species by macrophages. These investigators then used the low-dose model of persistent murine tuberculosis to further analyze the role of TNF in the immune response during latency. Six months after inoculation with a virulent lab strain of M. tuberculosis, mice were treated with a murine TNF-α–specific IgG-neutralizing antibody. Following administration of the anti-TNF antibody, bacterial burden in the lungs of treated animals increased 10-fold within 20 d, as compared with rats receiving IgG controls, which showed no increase. All mice that had received the anti-TNF IgG died within 120 d of being treated, whereas none of the control mice succumbed during that time. Histopathologic examination of the lungs from anti-TNF–treated mice demonstrated what disorganized granulomas without significant recruitment or migration of inflammatory cells to the granuloma. In contrast, control animals showed well-demarcated granulomas with conspicuous lymphoid aggregates.

This work, demonstrating the critical role of TNF in maintaining granuloma structure and function in mice, has received striking confirmation in humans. Keane and colleagues reported 70 cases of tuberculosis which developed after treatment with infliximab, a humanized monoclonal antibody against TNF (45). Of note, most of these cases developed within 12 wk after beginning administration of infliximab, most were in patients from countries with a low prevalence of tuberculosis, and disseminated disease was common. Since that initial report, many similar cases have been reported both in patients receiving infliximab as well as the somewhat less potent TNF receptor antagonist etanercept (46). The recognition of the important role of TNF in maintaining the latent state has led to the development of guidelines for screening and treatment for latent tuberculosis infection in patients receiving anti-TNF therapy (47).

T cells in the host immune response to tuberculosis.

The critical role of T lymphocytes in tuberculosis host defense and pathogenesis has been brought into sharp focus by the worldwide epidemic of HIV/AIDS. In regions where HIV infection is common, such as sub-Saharan Africa, tuberculosis rates have increased rapidly. In patients with latent infection, HIV infection, particularly in the absence of antiretroviral therapy, is the leading risk factor for the development of active disease, occurring at a rate as high as 10% per year, as compared with 10% across a lifetime in otherwise healthy, HIV-negative persons. Patients with HIV infection who develop tuberculosis have different clinical presentations and have higher mortality early in the course of therapy than those with tuberculosis without HIV infection (4858). Although treatment with antiretroviral agents can restore some of the immune responses beneficial in tuberculosis (59), these therapies are not widely available in places where HIV/TB co-infection is most common.

Following the demonstration in mice that CD4+ T-lymphocytes can become polarized and phenotypically distinguishable into so-called TH1 and TH2 cells on the basis of patterns of cytokine secretion (with IFN-γ as the prototypical TH1 cytokine, and interleukin [IL]-4, IL-5, and IL-10 as TH2 cytokines), Modlin and colleagues published an important study in patients with leprosy which suggested that T cell polarization could substantially affect the clinical manifestations of chronic mycobacterial infections (60). Patients with necrotic, clinically advanced, heavily infected lepromatous leprosy lesions had skin biopsies that contained largely TH2-type CD4+ cells. Skin biopsies from so-called tuberculoid leprosy lesions, which are paucibacillary and do not demonstrate extensive necrosis, were infiltrated by TH1-type CD4+ lymphocytes.

Since that report, many groups have attempted to demonstrate that host immune responses in tuberculosis could be similarly polarized and could explain at least some of the clinical manifestations of disease. Condos and coworkers demonstrated that a TH1-predominant host immune response, as defined by a marked alveolar lymphocytosis of IFN-γ–secreting cells detected by bronchoalveolar lavage from radiographically abnormal lung segments in patients with tuberculosis, was associated with milder clinical disease, as defined by lack of cavity formation and a low bacterial burden (61). In contrast, patients with more clinically advanced disease, as defined by cavitary lesions and sputum smear positivity, did not have evidence of a TH1 response. This study suggested an association between the ability to mount a TH1-type response in the setting of active disease and a more benign clinical course, consistent perhaps with the observation from the pre-antibiotic era that a significant percentage of patients with tuberculosis in fact recovered. The same investigators extended their observation by administering IFN-γ by aerosol to patients with symptomatic, refractory multidrug-resistant tuberculosis and demonstrating clinical improvement (62).

Balancing the beneficial effects of a TH1-type cytokine response, and likely contributing importantly to reactivation of latent infection, is the elaboration of transforming growth factor (TGF)-β by circulating blood monocytes. Both in vitro and in vivo, this cytokine is capable of suppressing both IFN-γ production and granuloma function (6365).

The Microorganism

The pathogenesis of tuberculosis involves a dynamic interaction between host and pathogen. From the time of Koch's identification of the causative agent of tuberculosis until relatively recently, progress had been relatively slow in understanding the basic biology of M. tuberculosis. However, key developments in several areas have accelerated the pace of progress recently. These include notably the opening up of the entire field of mycobacterial genetics, made possible by the work of Jacobs and colleagues by using plasmids capable of transfecting M. tuberculosis; the development of the entire field of molecular epidemiology, centered on RFLP analysis of isolates and made possible by the identification of the IS6110 insertion element in the M. tuberculosis genome; and finally the sequencing of the entire M. tuberculosis genome, accomplished by investigators at the Pasteur Institute.

Mycobacterial Genetics.

In 1987, Jacobs, Tuckman, and Bloom reported the construction of recombinant shuttle plasmids, which are chimaeras containing mycobacteriophage DNA into which an Escherichia coli cosmid was inserted (66). These plasmids replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permitted for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG, the vaccine strain of M. bovis. This advance created a powerful new tool for the study of mycobacterial biology, and has led to the identification of putative virulence factors, immunogenic determinants, drug targets, and novel vaccine candidates. An example of this was work by Glickman and Jacobs that determined the genetic basis for cording, the formation of strand-like clumps of mycobacteria in culture, a phenotype long associated with virulence (an association noted by Koch himself). Cording behavior was found to be dependent on normal function of the pcaA gene, responsible for a step in the synthesis of a-mycolic acids, important components of the M. tuberculosis cell wall (67). When the gene was mutated, and used to infect mice, in a model which is usually lethal, the organisms did not persist or kill the animals, indicating that the pcaA gene product is indeed a virulence factor.

Molecular Epidemiology.

Using a repetitive genetic element found in the M. tuberculosis complex as the basis of restriction fragment length polymorphism (RFLP) analysis of clinical isolates, investigators in several laboratories pioneered the development of tuberculosis molecular epidemiology to study transmission dynamics of tuberculosis in communities (6870). These studies have led to fundamental changes in the understanding of how tuberculosis is spread in communities, and have been useful in identifying outbreaks and previously unsuspected clusters of tuberculosis. Studies in New York and San Francisco done during the tuberculosis epidemic of the late 1980s and early 1990s revealed that as many as 40% of cases of tuberculosis could be linked, through RFLP analysis, to other cases (71, 72). As tuberculosis control efforts aimed at rapid diagnosis and treatment took hold, case rates fell as a direct result of decreased transmission in the community; many of the cases that remain are the result of reactivation of latent transmission in persons originally from high-prevalence countries, and the strategies needed to identify and reduce active disease in these populations will differ from those for active cases (73).

The M. tuberculosis Genome.

The announcement of the complete DNA sequence of M. tuberculosis in 1998 will stand as a signal event in understanding the pathogenesis of this disease (74). The sequencing of the virulent laboratory strain H37Rv revealed the genome to contain 4,411,529 base pairs encoding over 4,000 genes. Notable features of the genome were the high percentage of GC-rich sequences and the large numbers of genes devoted to synthesis of fatty acids. Since the sequencing of the initial lab isolates, additional laboratory strains and clinical isolates have been sequenced in whole or in part, including a strain associated with apparent hypervirulence in a community, and the vaccine strain of M. bovis (75, 76). Comparisons of M. tuberculosis and the vaccine strain of M. bovis have been particularly interesting. As compared with M. tuberculosis, the vaccine strain lacks an entire genomic region, known as RD1, which contains genes for secreted proteins such as ESAT-6 (77); this knowledge has been exploited to develop novel diagnostics for latent tuberculosis infection that can distinguish in theory between true infection and delayed-type hypersensitivity reactions caused by prior vaccination with BCG (78). In addition, comparative genomics of mycobacteria have allowed the construction of evolutionary trees that provide important insights into the historical development of human disease (79). Of greatest interest, of course, is the potential to use genomics to distinguish virulent from avirulent strains, and immunonogenic strain from nonimmunogenic strains. Such information should be extremely valuable in the effort to develop better tuberculosis vaccines.

Tuberculosis remains among the world's great public health challenges, and the advances discussed in this article hold promise for the development of better prevention and treatment of the disease. The 123 yr since the identification of M. tuberculosis by Robert Koch have seen great advances in our understanding of many of the crucial events in disease pathogenesis, but tuberculosis is nowhere near eradication or even control in many areas of the globe. It is worth recalling the words of Rene and Jean Dubos in their prescient book, The White Plague: “Tuberculosis, it has been said, is a disease of incomplete civilization. Vague as this statement appears at first, it underlines the fact that the antituberculosis movement cannot be properly understood if seen only in its medical perspective, for the historical and social backgrounds loom large in the picture. However desirable a goal, the complete elimination of tubercle bacilli is rendered impossible by economic and social factors.”

The author is grateful for the helpful comments of Rany Condos, Joseph Burzynski, and Charles Powell. Excellent discussions of the early scientists who studied tuberculosis can be found in Pioneers in Medicine and Their Impact on Tuberculosis, by Thomas M. Daniel (University of Rochester Press, 2000). Much of the discussion of Laennec, Villemin, and Koch in this article was informed by Daniel's essays. Finally, the most rudimentary Medline search for the term “tuberculosis pathogenesis” yields over 50,000 citations. The author apologizes to the scores of outstanding investigators whose important contributions were not mentioned in this article, either due to lack of space or his own ignorance in not realizing their significance.

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Correspondence and requests for reprints should be addressed to Neil W. Schluger, Division of Pulmonary, Allergy, and Critical Care Medicine, Columbia University College of Physicians and Surgeons, PH-8E, Room 101, 622 West 168th Street, New York, NY 10032. E-mail:

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