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Interactions between Mycobacterium tuberculosis and the immune system by Ben Buxton
or
"How I survive the proverbial firey furnace" by M. Tuberculosis

Mycobacterium tuberculosis has a very puzzling method of pathogenisis. It seems to defy logic by actually using macrophages, the front line defenses of the human immune system, as a safe haven for its reproduction. This paper will discuss the mechanisms of this interaction especially as they relate to topics covered in Bio 307, Immunology.

The paper is structured as follows:
1. TB disease overview (brief).
2. Recognition and phagocytosis of tuberculi.
3. Pathogen survival in the phagolysosome.
4. Turning the tide - elimination of pathgen.

The spread of tuberculosis (TB) is one of the more present crises currently facing the human global community. Untreated cases and multidrug resistant strains often result in death. Though rates of infection declined with the advent of antibiotics, TB infection is again on the rise and currently 26 % (2.7 million) of all avoidable deaths in the world are due to TB (brown.edu, 2000). TB is spread through inhalation of airborne Mycobacterium tuberculosis cells which multiply in macrophages and within the large cystic tubercles they form - liquified caseous tissue surrounded by infected macrophages (Bichun et al., 1996). These inhibit normal lung function and can rupture to spread the pathogen.

Fig. 1: TB infection of macrophages in the broader view of tubercle formation in the lung.
(Permission has been requested for the use of this figure from the site http://virtual.class.uconn.edu/~terry/Spring96/WebTB2/Groups/Group3/Final.html and will be removed if permission is denied.)

Macrophages form a key part of the front line of defense against invading pathogens in the human immune system. They are part of the innate immune response and can respond to a broad range of specific invaders. They act through endocytosis of pathgens combined with lysosomal action to destroy pathogens. Endocytosis is mediated by compliment molecules, antibodies and receptors specific for common bacterial cell wall constituents which allows action against a variety of pathogens . With a view towards energy efficiency, macrophages are not normally activated to full antimicrobial function upon the initial contact with pathogen and this has dire consequences in the case of mycobacterial infection (Janeway et al., 1999). Macrophages must be activated by cytokines released by other leucocytes order to effectively destroy invading M. tuberculosis (Banki et al., 1996).

In this early defensive role, alveolar macrophages ingest inhaled M. tuberculosis cells (as well as other pathogens) on contact as mediated by compliement receptors CR1, 3 and 4 as well as mannose receptors (MR) (Sinai and Joiner, 1997). M. Tuberculosis displays lipoglycan molecues on its cell wall including Lipoarbinomannan (LAM) which, in virulent strains, includes mannose oligosaccharides at the terminus of the molecule (Schlesinger et al., 1994). Schlesinger et. al find that the presence of this mannose terminus inceases attachment of LAM coated microspheres to macrophages and propose that this is evidence of an important mechanism of phagocytosis (1994). Additionally, phagocytosis of mycobcteria is enhanced by addition of the compliment protein C3 from nonimmune serum, and the serum protein mannose-binding protein, which recognizes LAM and lipomannan (Sinai and Joiner, 1997).

LAM, which consists of repeating saccharide units of arabinose and mannose attached to the mycobacterial membrane through a phosphatidylinositol moiety, has additionally been directly implicated in the virulence of M. tuberculosis (Chan et al. 1991). It functions in suppressing interferon-gamma induced macrophage activation (through blocking transcriptional activation of IFN-gamma induced genes), scavenging cytotoxic oxygen free-radicals and inhibiting protein kinase C activity (Chan et al., 1991).

The sequence of events occurring to the newly endocytosed M. tuberculosis containing phagosome is still not fully understood; contradictory research has been reported (Sturgill-Koszycki et al. 1994). It seems clear that the normal pathway leading to the destruction of ingested pathogen must be somehow altered and the standard explanation for many years has been that the mycobacterial phagosome does not fuse with lysosomes (Sinai and Joiner, 1997). Recent research suggests vessicular traffic with the mycobacterial phagosome does take place, but that the phagosome has characteristics of an early endosomal compartment that does not mature or fuse with lysosomes or late endosomal compartments (Sinai and Joiner, 1997). Macrophage vacuoles rapidly acidify under normal circumstances, but research has shown that vacuoles containing mycobacterium are less acidic than neighboring macrophage lysosomes (Sturgill-Koszycki et al. 1994). In fact, experiments with closely related M. avium show a vaculolar pH of 6.3-6.5 for bacterial phagosomes while normal phagolysosomes show a pH of 5.5 or below (Sturgill-Koszycki et al. 1994). This same group found that proton-ATPase vesicles normally fuse with phagosomes soon after their internalization (rather than being present in the membrane at the moment of endocytosis), but that Mycobacterium containing phagosomes prevent fusion with proton-ATPase containing vesicles while allowing the fusion of other vesicles including those bearing LAMP-1, normally a phagosomal marker protein (Sturgill-Koszycki et al. 1994). Subesquent research showed mycobacterial phagosomes to contain a LAMP level intermediate between polystyrene bead containing vacuoles and Legionella phagosomes which nearly lack LAMP (Sinai and Joiner, 1997). In their review of contemporary research, Sinai and Joiner suggest that the mycobacterial phagosome is somehow arrested in the early endosome stage and not simply routed to an alternative pathway of development (1997).

The precise mechanism by which phagosome-lysosome fusion is prevented has not been elucidated but research suggests that ammonia produced by M. tuberculosis may play a large role (Sinai and Joiner, 1997). Since ammonia would rapidly diffuse out of the lysosome, other weak bases produced by M. tuberculosis may play a role in preventing normal acidification - a process necessary for the recruitment of various coat proteins which may be required to mediate future fusion events (Sinai and Joiner, 1997)

The immune system mounts a complex response to M. tuberculosis, and one that is sometimes successful in stopping the spread of M. tuberculosis. Two cytokines secreted in response to the pathogen are of particular importance: IFN-gamma and TNF. IFN-gamma improves H₂O₂ Production by Macrophages, while TNF can enhance phagocytic and mycobactericidal activity of macrophages (Barnes et al., 1990). Investigation has shown that patients with tuberculosis pleuritis, a form of TB which the body normally recovers from without administration of therapy, show IFN-gamma and TNF concentrations 5-30 times higher than blood concentrations (Barnes et al., 1990). Furthermore, exogenous IFN-gamma was found to be essential for control of M. tuberculosis in infected mice of the gko strain with disrupted IFN-gamma genes (Flynn et al., 1995).

The improved H₂O₂ production found by Barnes et al. may prove a moot point as later research found the action of reactive nitrogen intermediates (RNI) to be the principle effector mechanism of M. tuberculosis killing, at least in murine macrophages (Chan et al., 1992). Effector L-argninine-dependant RNI molecules include NO, NO₂ and HNO₂ and the pathway has been implicated in killing activity by macrophages against Toxoplasma gondii, Chlamydia psittaci, Bacillus Calmette-Guerin, Leishmania donovani and schistosoma mansoni(Chan et al., 1992). This group found that murine macrophages activated by IFN-gamma and LPS or TNF-alpha mounted effective responses to phagocytosed M. tuberculosis in a manner independant from the action of reactive oxygen intermediates, suggesting that the production of RNI is principally responsible for killing and growth inhibiting ingested pathogen (Chan et al., 1992).

IL-12, secreted by B-cells and macrophages, is another important cytokine employed against M. tuberculosis. It induces the production of IFN-gamma from T and NK cells, enhancing their proliferation in the process, while CD8 T cells and NK cells are simultaneously down-regulated (Flynn et al., 1995). Administration of exogenous IL-12 has been shown to increase resistance of BALB/c mice (which are particularly susceptible to M. tuberculosis infection), though only in the presence of IFN-gamma (Flynn et al., 1995). One is tempted to suggest that IL-12 exerts its effect through the action of IFN-gamma, but results of IL-12 treatment could not be replicated by administration of exogenous IFN-gamma (Flynn et al., 1995). Additionally, one research team found that exogenous IL-12 did not increase production of IFN-gamma in infected mice (Flynn et al., 1995). Clearly, we do not yet have the full picture of IL-12 cytocidal action.

Work Cited:

Banki, A., Jenei, P. M., Richards, G. M. Mycobacterium tuberculosis and its host cell, the macrophage. 2000. <http://www.sp.uconn.edu/~terry/Spring96/WebTB2/Groups/Group5/Final.html> Accessed 2000, Apr 13.

Barnes PF; Fong SJ; Brennan PJ; Twomey PE; Mazumder A; Modlin RL. 1990. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J. Immunol. 145(1): 149-54

Bichun, L.A., Pedrosa, M.M., Trippel, S.J. 1996 Molecular Biology of mycobacterium tuberculosis. <http://www.sp.uconn.edu/~terry/Spring96/WebTB2/Groups/Group14/Final.html> Accessed 2000 Apr 20.

Brown.edu. TB genome. 2000. <http://www.brown.edu/Research/TB-HIV_Lab/tb/tbintro.html> Accessed 2000 Apr 20.

Chan, J., X-D. Fan, S.W. Hunter, P.j. Brennan, and B.R. Bloom. 1991. Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis with macrophages. Infect. Immun. 59: 1755-1761

Chan J; Xing Y; Magliozzo RS; Bloom BR. 1992 Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J. Exp. Med. 175(4): 1111-22

Flynn JL; Goldstein MM; Triebold KJ; Sypek J; Wolf S; Bloom BR 1995. IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection. J. Immunol. 155(5): 2515-24

Janeway, Charles A., Travers, Paul, Walport, Mark, Capra, J. Donald. 1999. Immunobiology: the immune system in health and disease, 4th Edition. London: Current Biology. 116-135

Schlesinger, L.S., S.R. Hull, and T.M. Kaufman. 1994. Binding of the Terminal mannosyl Units of Lipoarabinomannan from a Virulent Strain of Mycobacterium tuberculosis to Human Macrophages. Journal of Immunology. 152: 4070-78

Joiner, A.P., Joiner, K.A. 1997. Safe Haven: the Cell Biology of Nonfusogenic Pathogen Vacuoles. Annu. Rev. Microbiol. 51: 415-62

Sturgill-Koszycki S; Schlesinger PH; Chakraborty P; Haddix PL; Collins HL; Fok AK; Allen RD; Gluck SL; Heuser J; Russell DG. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263(5147): 678-81

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Direct correspondence to: bebuxton@davidson.edu