Immunology, Pathogenesis, Virulence

Chapter 5: Immunology, Pathogenesis, Virulence

by Rogelio Hernández-Pando, Rommel Chacón-Salinas, Jeanet Serafín-López, and Iris Estrada

5.1. Immune response against Mycobacterium tuberculosis

The immune response against tuberculosis (TB) plays a fundamental role in the outcome of M. tuberculosis infection. It is clear that the immune system reacts efficiently in the vast majority of infections. This is particularly evident in the case of TB, where most people infected by the tubercle bacillus (~ 90 %) do not develop the disease throughout their lifetimes. Nevertheless, the risk of developing the disease increases considerably when TB infection co-exists with an alteration in the immune system, such as co-infection with human immunodeficiency virus (HIV).

Also, it is well known that bacille Calmette-Guérin (BCG) vaccination has not been completely efficient in the prevention of pulmonary TB. Thus, the design of vaccines against TB is a field in which much effort has been invested with the aim of fighting this disease. Recently, it has become clear that, in order to develop a more efficient vaccine, a better understanding of the relation between the immune response of the host and the tubercle bacillus is needed.

In view of this, the present chapter provides an updated overview of the cellular and molecular immune mechanisms involved in the development of the disease.

5.1.1. Innate immune response Neutrophil leukocytes

Even though macrophages are considered the main targets for infection by Mycobacterium tuberculosis, it has been recently proposed that other cell populations can also be infected by mycobacteria and therefore may be important in the development of the disease. Neutrophils are found within this group of cells (Figure 5-1). Characteristically, they are among the earliest cells recruited into sites where any noxious agent enters into the body and/or inflammatory signals are triggered. They also have well-characterized microbicidal mechanisms such as those dependent on oxygen and the formation of neutrophil extracellular traps (Urban 2006).

Using the murine experimental model, the role played by neutrophils in TB is controversial. These cells have been detected at the beginning of infection as well as several days after infection (Pedrosa 2000, Fulton 2002) and were thought to have an important role in the control of mycobacterial growth. Indeed, if neutrophils are eliminated before infection, mycobacterial growth increases in the lungs of experimentally infected animals; and conversely, if mice are treated with an agent that increases neutrophils, the bacillary growth rate decreases (Appelberg 1995, Fulton 2002). However, when the microbicidal ability of neutrophils against mycobacteria was analyzed, controversial results were obtained. There are reports of neutrophils being able to kill mycobacteria (Jones 1990) and other reports where this phenomenon was not observed (Denis 1991). Nevertheless, it is believed that the function of neutrophils goes beyond their microbicidal ability. Therefore, these cells are thought to contribute to the control of infection through the production of chemokines (Riedel 1997), the induction of granuloma formation (Ehlers 2003) and the transference of their own microbicidal molecules to infected macrophages (Tan 2006).

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Figure 5-1: Neutrophils ingest Mycobacterium tuberculosis. Human purified neutrophils were incubated with Mycobacterium tuberculosis H37Rv and DNA was stained with SYTOXTM Green. Fluorescent rods on the left are intracellular bacilli. On the other hand, neutrophils have recently been ascribed a role in the development of the pathology, rather than the protection of the host. TB susceptible animals were found to have a larger and longer accumulation of neutrophils in TB lesions compared to TB resistant animals (Eruslanov 2005). This event seems to be influenced by the differential expression of molecules which are chemoattractant to neutrophils (Keller 2006). The different susceptibility of the hosts may explain the discrepancies in the results of these recent studies and those of earlier ones, which suggested a protective role of neutrophils in the control of TB infection. While those early studies showing protection were conducted in mouse strains that were naturally resistant to TB, the later studies mainly focused on the role of these cells in TB susceptible mouse strains. Evidently, a more precise definition of the role played by neutrophils during infection will depend on an evaluation of the kinetics and magnitude of the response that these cells have in the early stages of the disease. Mast cells Mast cells are effector cells with a relevant role in allergic reactions (Woodbury 1984, Miller 1996, Galli 1999, Williams 2000); and are also critical for the development of a T helper 2 (Th2) response (Galli 1999, Metcalfe 1997). They are found in the mucosa of the respiratory, gastrointestinal, and urinary tracts and can also be observed in the vicinity of blood and lymph vessels. These cells express a receptor with high affinity for IgE (FceRI) and therefore this immunoglobulin is bound to their membrane. Upon the union of the antigen to the active sites of FceRI-bound IgE, mast cells liberate several molecules, including preformed mediators and mediators synthesized de novo (Metzger 1992, Turner 1999, Williams 2000). Among the preformed mediators contained in mast cell granules are histamine, tryptase, chymase, carboxypeptidase, and heparin, while mediators synthesized de novo include leukotriene C4, prostaglandin D2, platelet-activating factor (PAF), tumor necrosis factor alpha (TNF-a), transforming growth factor (TGF-b), fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), and interleukins IL-4, IL-5 and IL-8 (William 2000, Turner 1999, Sayama 2002). Besides this interaction between IgE and the antigen, other agents are able to induce the activation of mast cells and the liberation of cytokines and other mediators. For instance, microbial products (Di Nardo 2003, Feger 2002) stimulate mast cells via two members of the toll-like receptor (TLR) family, TLR-2 and TLR-4 (Supajatura 2002, Sabroe 2002, McCurdy 2003). The locations where mast cells are usually found are common gateways for infectious agents and there is evidence of these cells being excellent mediators of the inflammatory response (Williams 2000, Metcalfe 1997). At least in bacterial infections by Klebsiella pneumoniae and Escherichia coli, mast cells are required for the triggering of innate immunity (Malaviya 1996, Malaviya 2001). In addition, due to their strategic distribution within the lung, mast cells have a fundamental role in the defense of the host against mycobacteria. An early study showed an increased number of mast cells and their degranulation in the lungs of animals experimentally infected with M. tuberculosis (Ratnam 1977). The presence of mast cells has also been described in the duodenum and the ileum of cows infected with Mycobacterium paratuberculosis, a microorganism that causes granulomatous enteropathic lesions (Lepper 1988). Muñoz et al (2003) demonstrated that there is an interaction between mast cells and M. tuberculosis through the CD48 molecule. This interaction triggers the release of preformed mediators, such as histamine and b-hexosamidase, and the liberation of de novo synthesized cytokines, such as IL-6 and TNF-a, which are involved respectively in the activation of neutrophils and the maintenance of the integrity of the granuloma (Muñoz 2003, Law 1996, Adams 1995). The secretory proteins Mycobacterium tuberculosis secreted antigen (MTSA-10) and 6-kiloDalton (kDa) early secretory antigenic target (ESAT-6) contribute to the activation not only of macrophages and dendritic cells but also of mast cells for the liberation of their pro-inflammatory mediators (Muñoz 2003, Trajkovic 2004). Macrophages The macrophage is the paradigmatic cell with regard to M. tuberculosis infection. Indeed, alveolar macrophages have been shown to play an essential role in the elimination of particles that enter the organism through the airways; and have long been considered the first cell population to interact with the tubercle bacillus. More macrophages are recruited afterwards from the bloodstream, and are in charge of maintaining the infection in the host (Dannenberg 1991, Dannenberg 1994). The initial interactions of the bacilli with the macrophage take place through cellular receptors, such as receptors for Fc, complement (Schlesinger 1990), mannose (Schlesinger 1993), surfactant protein (Zimmerli 1996), CD14 (Peterson 1995), and CD43 (Randhawa 2005). Though it is unknown if the bacteria interact with one or more of these receptors during in vivo infection, the results of in vitro experiments suggest that the macrophage response depends on the type of receptor with which the bacteria interact. Their interaction with Fc receptors increases the production of reactive oxygen intermediates and allows the fusion of the bacteria-containing phagosomes with lysosomes (Armstrong 1975). On the other hand, interaction of the bacteria with the complement receptor 3 (CR3) prevents the respiratory burst (LeCabec 2000) and blocks the maturation of phagosomes harboring the bacteria, thus preventing fusion with lysosomes (Sturgill-Koszycki 1996). The interactions of mycobacteria with members of the Toll-like receptor family have been studied for some years. TLR-2 (Brightbill 1999) and TLR-4 (Jeans 1999) are activated by several M. tuberculosis components. Among others, the 19-kDa lipoprotein and lipoarabinomanann (LAM) activate macrophages through TLR-2, promoting the production of IL-12 and inducible nitric oxide synthase (iNOS) (Brightbill 1999). Regardless of the receptor with which the bacteria interact, it has been observed that the cellular cholesterol present in the macrophage cell membrane is an essential molecule for the internalization of the bacteria (Gatfield 2000). It is believed that cellular cholesterol works as a direct anchorage point for the bacterium and stabilizes its interaction with the macrophage membrane. Afterwards, the bacterium is efficiently internalized (Pieters 2001). Once the bacteria enter the macrophage, they generally locate themselves in the mycobacterial phagosome (Armstrong 1971, Armstrong 1975). This structure derives from the plasma membrane and presents some cell surface receptors (Russell 1996, Hasan 1997). In contrast to normal phagocytosis, during which the phagosomal content is degraded upon fusion with lysosomes, the mycobacteria block this process (Armstrong 1971, Armstrong 1975). This inhibition depends on an active process induced by viable mycobacteria, since dead bacilli can be easily found in lysosomal compartments (Armstrong 1971, Armstrong 1975). Besides having a different morphology, the vacuoles in which the bacteria reside present “early” endosomal compartment markers instead of the characteristic “late” endosomes (Hasan 1997, Clemens 1996, Baker 1997). In addition, these mycobacterial phagosomes retain “early” markers, such as Rab5 and Rab14 GTPases, and do not acquire the “late” Rab7 molecule; a finding which is also consistent with a blockage of the maturation process from early to late endosome (Via 1997, Kyei 2006). Another characteristic of the mycobacterial phagosome is its limited acidification (Crowle 1991). Normally, material transported through an endosomal route finds an acidic medium due to the action of the vesicular proton-pump adenosine triphosphatase (V-ATPase) in the late endosome. It is suggested that such reduced acidification is the result of a low or zero concentration of V-ATPase in the mycobacterial phagosome (Sturgill-Koszycki 1994). A more recently described property is that this mycobacterial phagosome can not physically associate with iNOS (Miller 2004). The inability of the mycobacterial phagosome to mature has been attributed to the active retention of a protein present in phagosomes, known as tryptophan aspartate coat protein (TACO), which was elegantly demonstrated by Ferrari et al. When these authors infected TACO-deficient cells, the maturation of mycobacterial phagosomes was not arrested and therefore these cells were able to eliminate bacilli by fusion of phagosomes with lysosomes (Ferrari 1999). It is also worth noting that TACO binds itself to the plasmatic membrane of macrophages through cholesterol, which also plays an essential role in mycobacterial uptake by macrophages. These events show both molecules to be importantly associated in the mycobacterial mechanisms for survival (Gatfield 2000). The inhibition of phagosome maturation by mycobacteria may be reverted by cytokines, such as interferon-gamma (IFN-g) and TNF-a, which also stimulate microbicidal mechanisms, including the production of reactive oxygen and nitrogen intermediates (Flesch 1990, Chan 1992). The protective role of nitrogen intermediates has been demonstrated in different murine models (MacMicking 1997, Flynn 1998), and a similar function has been suggested for these molecules in human TB (Nicholson 1996). In contrast, the role played by the reactive oxygen intermediates during infection has not been completely explained, though it is known that hydrogen peroxide produced by macrophages activated by cytokines has a mycobactericidal activity (Walter 1981). Also, it has been found that the tubercle bacillus presents molecules, such as LAM and phenolic glycolipid I, which work as oxygen radical scavenger molecules (Chan 1989, Chan 1991). Dendritic cells Dendritic cells are clearly involved in the protective immune response to M. tuberculosis infection. As explained above, when M. tuberculosis bacilli are inhaled and phagocytosed by the pulmonary macrophages, they remain, and even replicate, within the cell phagosome. Dendritic cells recruited from blood, and probably also from lung tissues, may play a role in protective immunity since they are found in increased numbers in TB lesions (Sturgill-Koszycki 1994, Pedroza-González 2004, García-Romo 2004). Dendritic cells recognize, capture and process antigens, thus being able to present them in the context of major histocompatibility complex (MHC) molecules, as well as through CD1 (Banchereau 1998, Gumperz 2001). Dendritic cells bind antigens via C-type lectin receptors and Fcg/Fce receptors, and internalize them by endocytosis (Engering 1997, Fanger 1996, Jiang 1995). M. tuberculosis endocytosis is carried out through known C-type lectin receptors, such as dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin (DC-SIGN) (Geijtenbeek 2003, Tailleux 2003). This molecule interacts with mannose capped-LAM, a component of the mycobacterial cell wall (Geijtenbeek 2003, Figdor 2002). In addition, peripheral blood dendritic cells and immature dendritic cells derived from monocytes express TLR-2 and TLR-4 (Jarrossay 2001, Kadowaki 2001), two Toll-like receptors with which mycobacteria seem to interact. Thus, it can be assumed that a protective host response may be induced through these signals. Additional signals generated by the association of mannose capped-LAM to DC-SIGN induce IL-10 production (Geijtenbeek 2003), while the union of a 19 kDa M. tuberculosis lipoprotein to TLR-2 induces production of IL-12, TNF-a, and IL-6 (Means 2001, Means 1999, Underhill 1999). Once the antigens have been captured and internalized, dendritic cells become mature (indicated by phenotypical and functional changes) and efficiently migrate to peripheral lymph nodes. There is evidence of in vivo M. tuberculosis and BCG transport from lung tissues to the lymph nodes inside infected dendritic cells (Dieu 1998). This migration of infected dendritic cells requires the expression of the chemokine receptor 7 (CCR7) on their surface, which makes them sensitive to chemokines (CC) CCL19 and CCL21 (Dieu 1998, Gunn 1998, Kriehuber 2001, Bhatt 2004). It is important to mention that maturation of dendritic cells is not only accompanied by an increased synthesis of MHC class I and II, but also by the expression of co-stimulating molecules, such as CD80 and CD86 (Turley 2000), and the production of IL-12 (Steinmann 2001). The internalization of M. tuberculosis into human and murine dendritic cells has been observed in several in vitro (Bodnar 2001, Fortsh 2000, Giacomini 2001, Hanekom 2002, Henderson 1997, Inaba 1993) and in vivo (Jiao 2002, Pedroza-González 2004, García-Romo 2004) studies. Reportedly, when dendritic cells derived from monocytes are infected with M. tuberculosis, their ability to present lipidic antigens is impaired and thus the expression of CD1 decreases (Stenger 1998). Components of the mycobacterial cell wall were also shown to inhibit the phenotypical maturation of dendritic cells induced by lipopolysaccharides. Different lineages of M. tuberculosis may vary in the degree by which they affect the dendritic cells. In particular, the enhanced virulence ascribed to Beijing strains might well be related to their inability to stimulate dendritic cell maturation (Lopez 2003, Ebner 2001). In a protective immune response, dendritic cells induce maturation of T cells towards a T helper 1 (Th1) profile by secreting cytokines, such as IL-12, IL-18, IL-23, and probably IFN-a and b, but not IFN-g (Wozniak 2006, Kadowaki 2001, Kalinski 1999, Thurnher 1997). Th1 cells expand in response to the BCG antigens presented by the dendritic cells in the lymphoid nodules and migrate toward infection sites, such as the lung tissue, where they liberate IFN-g, thus activating local macrophages that control bacilli replication (Humphreys 2006). Natural killer cells Natural killer cells play a very important role in the development of the innate immune response. Their main function has been associated with the development of cytotoxicity to target cells and they are among the first cell populations to produce IFN-g during the immune response. For a long time, the study of this cell population was focused on their role in viral and tumoral diseases. More recently, however, increasing interest has arisen in their eventual function in several bacterial infections. The number of natural killer cells was shown to increase in the lungs of C57BL/6 mice during the first 21 days after aerosol infection with M. tuberculosis complex strains. This cell expansion was associated with an increased expression of activation and maturation markers, and IFN-g production. However, the depletion of natural killer cells had no influence on the lung’s bacterial load, indicating that although these cells become activated during the early response in pulmonary TB, they are not essential for host resistance (Junqueira-Kipnis 2003). Natural killer cells also play an important role in human TB by regulating different aspects of the immune response. Human natural killer cells have been shown to have an enhanced cytotoxicity for macrophages infected with M. tuberculosis. They also optimize the ability of CD8+ T lymphocytes to produce IFN-g and to lyse M. tuberculosis infected cells, thus joining innate to adaptive immune responses (Vankayalapati 2002, Vankayalapati 2004). CD1d-restricted natural killer T cells These are a unique subset of human natural killer T cells characterized by the expression of an invariant V alpha 24 T cell receptor that recognizes the nonclassical antigen-presenting molecule CD1d. The activity of CD1d-restricted killer cells is notably enhanced by the marine glycolipid alpha-galactosylceramide derived from sponges. Once activated by alpha-galactosylceramide, CD1d-restricted natural killer T cells contribute to human host defense against M. tuberculosis infection. Human monocyte-derived macrophages expressing CD1d can induce effector functions of natural killer T cells against cells infected with M. tuberculosis when activated with alpha-galactosylceramide. These functions include IFN-? secretion, proliferation, lytic activity, and anti-mycobacterial activity; this latter via the antimicrobial peptide granulysin, which damages the mycobacterial surface. There is further support of the potential interaction of natural killer T cells with CD1d-expressing cells at the site of disease, since CD1d can be readily detected in granulomas of TB patients (Gansert 2003). Such a role has not been proved in M. tuberculosis infected mice. Rather, natural killer T cells have been shown to play a detrimental role, at least in the late phase of mouse experimental infection (Sugawara 2002). Epithelial cells Alveolar macrophages have been considered for a long time to be the first cell population to interact with M. tuberculosis. However, the number of epithelial cells in the alveoli is 30 times higher than the number of macrophages and thus, the likelihood that they are the first cells exposed to the infecting bacilli is similarly higher. The first indication of the involvement of epithelial cells in M. tuberculosis infection was derived from a study where the presence of mycobacterial DNA was detected in necropsy specimens from people who had died from diseases other than TB. In that study, M. tuberculosis DNA was detected in macrophages, type II pneumocytes, fibroblasts, and endothelial cells (Hernandez-Pando 2000). In addition, several in vitro studies have characterized the interaction between epithelial cells and M. tuberculosis. These cells can host M. tuberculosis bacilli and allow their replication (Bermudez 1996). Moreover, epithelial cells are able to establish an initial pro-inflammatory environment by secreting IL-8 (Wickremashinge 1999) and inducing the production of nitric oxide (NO) (Roy 2004). Obviously, in vivo experiments are necessary to better understand the role played by alveolar epithelial cells in M. tuberculosis infection. Defensins A conspicuous element of the innate immune response against microorganisms is a group of small endogenous antimicrobial peptides known as defensins (Diamond 1998). These cationic peptides, consisting of approximately 30 to 50 amino acids, are present in myeloid and epithelial cells of all animal species. They were shown to display antibacterial (Gabay 1989, Ganz 1985, Selsted 1987), antifungal (Selsted 1985), and antiviral (Daher 1986) activities. These molecules are classified as alpha, beta, and theta defensins based on the position of cysteine residues and the number of disulfur bonds (Bals 2000, Hoover 2000, Lehrer 1993). In phagocytic cells, defensins represent the main microorganism destruction components independent of oxygen metabolism (Miyakawa 1996, Ogata 1992). Allegedly, these peptides break the membrane of several microorganisms and some of them are even able to pass through the cytoplasmic membrane and enter the infected cell (Ganz 2003, Rivas-Santiago 2006). Defensins were first described in guinea pig and rabbit neutrophils (Zeya 1963, Zeya 1966). There is no report of human monocytes and macrophages having defensins, although neutrophils have been reported to have four known human neutrophil defensins peptides (Ganz 1990), of which three (HNP-1, HNP-2 and HNP-3) were found to be active against Mycobacterium avium-intracellulare and M. tuberculosis (Ogata 1992, Miyakawa 1996). In vitro, the human alpha defensins present in human neutrophils directly attracts CD4+/CD45RA+ T cells, CD8+ cells, and dendritic cells. The expression of human beta-defensin 1 is constitutive in epithelial cells but the expression of human beta-defensins 2 and 3 is inducible by IL-1, TNF-a, and by Toll-like receptor recognition of bacteria and fungi (Kaiser 2000, Lehrer 1993, Stolzenberg 1997). Human beta-defensins are also chemoattractants for T CD4+/CD45RO+ cells through receptor CCR6 (Chertov 2000). Mice infected with M. tuberculosis express murine beta defensins mBD-3 and mBD-4 n. In the first stages of infection, the epithelial cells of the respiratory tract express both defensins, which correlates to the early control of bacterial proliferation. However, their expression decreases as the disease progresses. In the latent infection model, mBD-3 and MBD-4 are continuously expressed, but their expression is suppressed if the infection is reactivated (Rivas-Santiago 2006). Genetic expression of human beta-defensin 2 (HBD-2) has been identified in epithelial cells of the skin, lung, trachea, and urogenital system (Bals 1998, Kaiser 2000, Lehrer 1993, Linzmeier 1999, Singh 1998, Stolzenberg 1997). This defensin was also detected in bronchial lavage cells from patients infected with M. avium-intracellulare (Ashitani 2001). Peripheral blood monocytes transfected with human beta-defensin HBD-2 have a better control of M. tuberculosis growth than non-transfected monocytes (Kishik 2001). Human alveolar epithelial cells infected with M. tuberculosis were also found to express human beta-defensin HBD-2 (Rivas-Santiago 2005). M. tuberculosis infected mice that have been treated with the defensin peptide HNP-1 show a reduction of bacterial load in the lungs, liver, and spleen (Sharma 2001). This observation suggests that defensins could represent important components of the innate response mechanisms against M. tuberculosis and could be used as new therapeutic tools. 5.1.2. Acquired immune response against M. tuberculosis In contrast to innate mechanisms, the specific or adaptive immune response requires the specific recognition of foreign antigens. The innate immune system has a profound influence on the type of acquired immune mechanisms generated, and vice versa, the specific immune response executes several of its effector functions via the activation of components of the innate immunity. Specific immune responses can be divided into cell-mediated mechanisms, which include T-cell activation and effector mechanisms, and the humoral immune response, consisting of B-cell maturation and antibody production. Both mechanisms are not mutually exclusive, and T helper cells are required for antibody maturation, isotype switching and memory. B cells also function as antigen presenting cells by activating T cells in a specifically driven manner. In the following pages we will focus on the generation of both humoral and cellular immune responses against M. tuberculosis. M. tuberculosis is the most conspicuous example of an intracellular bacterium that persists for long periods within the host, causing a latent infection, namely a chronic asymptomatic infection without tissue damage. This is best illustrated by the fact that two billion people worldwide are infected with M. tuberculosis, but more than 90 % of them remain healthy and free of clinical disease and the tubercle bacilli remain within them in a state of dormancy. Therefore, although the host cell-mediated immunity is enough to control the progression of disease, it fails to exert sterile eradication and hence, those two billion infected persons suffer the latent form of TB (Collins 2002). As for other intracellular infections, the primary protective immune response is cell mediated rather than antibody mediated. M. tuberculosis resides inside the macrophage and is relatively resistant to microbicidal mechanisms that efficiently eliminate other phagocytosed bacteria. This is due in part to the ability of the tubercle bacilli to hinder macrophage activation by IFN-g and IL-12. Several studies have confirmed the critical importance of these cytokines in both human and mice M. tuberculosis infection. In addition, deficiencies in IL-12 or IFN-g, or their receptors, render the individual more susceptible to mycobacterial infections (Jouanguy 1999, Alcais 2005). For the last 20 years, it has been assumed that the induction of a Th1-type immune response affords the host the greatest protective capacity. Despite the fact that there are hundreds of studies published on TB immunity, still there is a lack of information regarding important issues, such as the role of lung antigen presenting cells in vivo during pulmonary TB (Pedroza-González 2004). This type of information would allow a better understanding of the induction of specific immune responses against M. tuberculosis, and therefore the development of tools that could control the disease more effectively. Humoral immune response Because of their intracellular location, it is frequently assumed that tubercle bacilli are not exposed to antibody and therefore this type of immune response is considered to be non-protective. However, during the initial steps of infection, antibodies alone or in conjunction with the proper cytokines may provide important functions, such as prevention of entry of bacteria at mucosal surfaces. Even though the issue remains controversial, the role of antibodies in intracellular bacterial infections has gained renewed attention. Lately, their participation in the control of acute infections, such as chlamydial respiratory infection (Skelding 2006), and chronic infections produced by Actinomycetes, including M. tuberculosis (Salinas-Carmona 2004, Williams 2004, Reljic 2006), was explored. Antibodies can be exploited in two ways in the clinical management and control of TB: as serological diagnostic tools; and as active participants in protection. Serological methods have been regarded for a long time as attractive tools for the rapid diagnosis of TB due to their simplicity, rapidity, and low cost. As early as 1898, Arlöing showed that sera from TB patients could agglutinate tubercle bacilli (cited in Daniel 1987). With the introduction of the enzyme-linked immunosorbent assay in the ’70s, interest was renewed and several groups of investigators committed themselves to finding an optimum antigen for TB serodiagnosis. At that time, complex antigens were used in most cases, such as whole bacteria, culture filtrates, bacterial extracts, tuberculins and their purified derivates (PPD). More recently, individual purified antigens have also been assayed, including proteins, lipopolysaccharides and glycolipids, i.e., Ag 85, 38-kDa protein, LAM or diacylthrehaloses. To date, however, no test has shown sufficiently high sensitivity and specificity values for diagnostic purposes (Al Zahrani 2000, Bothamley 1995, Singh 2003, Raqib 2003, Julián 2004, Lopez-Marin 2003, see also chapter 13). As for their use in protection against TB, antibodies could enhance immunity through many mechanisms including neutralization of toxins, opsonization, complement activation, promotion of cytokine release, antibody-dependent cytotoxicity, and enhanced antigen presentation. In this sense, data from several laboratories indicate that anti-mycobacterial antibodies play an important role in various stages of the host response to TB infection (Costello 1992, Hoft 1999, Hoft 2002, Teitelbaum 1998, Williams 2004, De Valliére 2005). In particular, De Vallière et al. showed that specific antibodies increased the internalization and killing of BCG by neutrophils and monocytes/macrophages. Moreover, antibody-coated BCG bacilli were more effectively processed and presented by dendritic cells for stimulation of CD4+ and CD8+ T-cell responses. This enhanced anti-mycobacterial activity of phagocytes by antibody-coated bacilli is extremely important in the context of mucosal immunity. IgG and IgA antibody classes have been shown to be present in the mucosal secretions of the human lower respiratory tract (Boyton 2002). The specific mycobacterial targets for antibody-mediated enhanced interiorization and/or killing are not known, but surface antigens such as LAM or proteins expressed under stress conditions, such as alpha crystallin protein, may be relevant. In an experiment where 17 recombinant mycobacterial protein antigens, native Ag85 complex, LAM, and M. tuberculosis lysate were used to detect antibody responses induced by BCG vaccination, only LAM-reactive serum IgG responses were significantly increased in both BCG vaccinated individuals and active TB patients. As expected, oral BCG vaccination leads to a significant increase in LAM-reactive secretory IgA (Brown 2003). A new approach toward protection against TB, using passive inoculation with IgA antibodies, was tested in an experimental mouse model of TB lung infection (Williams 2004). Intranasal inoculation of mice with an IgA monoclonal antibod