Mycobacterium tuberculosis cell envelope lipids and the host immune response

Cellular Microbiology (2004)

6

(2), 105–116 doi:10.1046/j.1462-5822.2003.00351.x

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKCMICellular Microbiology1462-5814Blackwell Publishing Ltd, 2003

February 2004

6

2105116

Review Article

P. C. Karakousis, W. R. Bishai and S. E. DormanImmune-mediated effects of M. tuberculosis lipids

Received 5 August, 2003; revised 25 September, 2003; accepted 6October, 2003. *For correspondence. E-mail [email protected]; Tel.(+1) 410 502 2717; Fax (+1) 410 955 0740.

Microreview

Mycobacterium tuberculosis

cell envelope lipids and the host immune response

Petros C. Karakousis,

1

William R. Bishai

1,2

andSusan E. Dorman

1

*

1

Center for Tuberculosis Research, Division of Infectious Diseases, Johns Hopkins University School of Medicine, 1503 E. Jefferson St., Room 105, and

2

Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21231, USA.

Introduction


infects about one-third of theworld’s population, causing

ª

3 million deaths annually(Dye

et al

., 1999). The organism is a slow-growing bacillusthat is transmitted by the respiratory route. Although it iscapable of causing disease in most organs, pulmonaryinvolvement is most common. Soon after infection, thebacilli are phagocytosed by alveolar macrophages andsurvive within early phagosomes. Innate immuneresponses directed by macrophages predominate early ininfection. Subsequent recruitment of dendritic cells leadsto cell-mediated responses involving CD4

+

and CD8

+

Tcells and eventual granuloma formation (for a review, seeFenton and Vermeulen, 1996; Flynn and Chan, 2001). Thevast majority of immunocompetent individuals are able tocontain, but not eliminate, the pathogen in pulmonarygranulomas, leading to latent tuberculosis infection(Manabe and Bishai, 2000). In a small minority of cases,through unclear mechanisms, persistent bacilli can reac-tivate to form disease many years to decades after initialinfection.

An interesting feature of mycobacteria is the extraordi-narily high lipid content of the cell envelope, constitutingup to 40% of their dry weight (Anderson, 1940). In additionto conferring unique tinctorial properties, which aid in thediagnosis of infected clinical specimens, the mycobacte-rial cell envelope (see Fig. 1) is important in directinghost–pathogen interactions (Besra and Chatterjee, 1994;Brennan and Nikaido, 1995).

Certain cell envelope-associated lipids, such as phthio-cerol dimycocerosate (PDIM), have received significantattention recently (Camacho

et al

., 1999; Cox

et al

.,1999), because they are considered to be important viru-lence factors. Recent advances in mycobacterial genetictechniques and the availability of the

M. tuberculosis

genome (Cole

et al

., 1998) have provided the opportunityto test directly the virulence of PDIM-deficient mutants.For example, mutation of

mmpL7

(Cox

et al

., 1999), thegene product of which may be involved in PDIM transport,and mutation of genes involved in PDIM synthesis, includ-ing

msl7

and

pks10

(Sirakova

et al

., 2003) and

pks7

(Rousseau

et al

., 2003a), yield strains with attenuatedgrowth in the murine model. Although experimental evi-dence supports the hypothesis that PDIM is important forvirulence, the effects of this molecule on host immuneresponses have not been elucidated.

Other virulence factors have been implicated in theimmunopathogenesis of tuberculosis (TB) and may there-fore represent potential targets for future therapeutic orpreventive interventions. This review will focus on theimmunomodulatory properties of the following cellenvelope-associated lipids of

M. tuberculosis

: (i) lipoara-binomannan; (ii) sulpholipids; (iii) mycolic acid-containingglycolipids; and (iv) the 19 kDa lipoprotein.


cellwall-associated lipids

Lipoarabinomannan (LAM) is a phosphatidylinositol-anchored lipoglycan composed of a mannan core witholigoarabinosyl-containing side-chains with diverse bio-logical activities (Hunter and Brennan, 1990; Chatterjee

et al

., 1991; Chatterjee and Khoo, 1998). LAM is a prom-inent component of the mycobacterial cell envelope,accounting for up to 5 mg g

-

1

bacterial weight (Hunter

et al

., 1986). LAM structure differs according to mycobac-terial species, and three general classes of LAM havebeen described: (i) ManLAM, from the virulent strainsErdman and H37Rv and the avirulent strains H37Ra andBCG (Chatterjee

et al

., 1992a; Prinzis

et al

., 1993), whichis characterized by extensive mannose capping of the

106

P. C. Karakousis, W. R. Bishai and S. E. Dorman

© 2003 Blackwell Publishing Ltd,

Cellular Microbiology

,

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, 105–116

arabinan termini; (ii) phospho-

myo

-inositol-capped LAM(PILAM), found in the rapidly growing mycobacteria

M.smegmatis

and

M. fortuitum

(Nigou

et al

., 2003); and (iii)AraLAM, which was described in the rapidly growing

M.chelonae

and lacks mannosylation in its arabinan termini(Guerardel

et al

., 2002). Two distinct pools of ManLAMhave been described, namely parietal and cellular, whichdiffer based on their phosphatidylinositol anchor and theextent of mannose capping (Gilleron

et al

., 2000), and intheir immunological properties (Nigou

et al

., 1997).Although there is significant heterogeneity between LAMmolecules with respect to glycosylation and acylation(Nigou

et al

., 2003), differences in biological activitybetween the major classes of LAM have been attributedprimarily to the heavy mannose capping of ManLAM or todifferences in the phosphatidylinositol anchor (Barnes

et al

., 1992; Chatterjee

et al

., 1992b).The sulpholipids of

M. tuberculosis

are five structurallyrelated sulphatides. Sulpholipid-I (SL-I), the principal sul-

phatide of the family, has been identified as a 2,3,6,6

¢

-tetraacyl-

a

,

a¢

-

D

-trehalose 2

¢

-sulphate (Goren

et al

.,1976a). The sulpholipids are mycobacterial cell envelopemolecules with pleiotrophic biological activities, includingantitumour activity (Yarkoni

et al

., 1979). The extent ofsulpholipid expression by various

M. tuberculosis

strainshas been correlated with strain virulence in the guinea pigmodel (Goren, 1982). Recent work has identified essentialgenes for SL-I biosynthesis, including

pks2

(Sirakova

et al

., 2001) and

mmpL8

(Converse

et al

., 2003), thusproviding the opportunity to study the virulence of thesegene mutants in animal models. Interestingly, althoughneither mutant can produce SL-I,

pks2

mutants demon-strate no reduction in replication, persistence or pathoge-nicity in mice or guinea pigs (Rousseau

et al

., 2003b),whereas

mmpL8

mutants are attenuated for growth in themouse model of TB (Converse

et al

., 2003), suggestingthat SL-I

per se

is not required for bacillary growth

in vivo

.The difference in growth phenotypes between these

Fig. 1.

Schematic representation of the cell envelope of

M. tuberculosis

(Besra and Chatterjee, 1994; Brennan and Nikaido, 1995; Iseman, 2000). The components include the (A) plasma membrane, (B) peptidoglycans, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasma membrane- and cell envelope-associated proteins, (F) mycolic acids and (G) glycolipid surface molecules associated with the mycolic acids.

F

Immune-mediated effects of

M. tuberculosis

lipids

107

© 2003 Blackwell Publishing Ltd,

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, 105–116

mutants may be explained if the

mmpL8

gene productacts as a transporter for other surface molecules in addi-tion to SL-I that mediate host–pathogen interactions, or ifthe accumulation of a precursor molecule in the sulpho-lipid biosynthesis pathway in

mmpL8

mutants interfereswith

in vivo

growth-related pathways.Mycolic acids are high-molecular-weight

a

-alkyl,

b

-hydroxy fatty acids found in the genera

Mycobacterium

,

Nocardia

,

Corynebacterium

and

Rhodococcus

(Brennanand Nikaido, 1995). Covalently attached to arabinogalac-tan, mycolic acids, together with other lipids forming theouter leaflet, constitute a very hydrophobic barrier(Draper, 1998), responsible for resistance to certain drugs(Nikaido, 2001). Mycolic acid-containing glycolipids and,in particular, trehalose-6,6

¢

-dimycolate (TDM), which isthe most prominent and best-studied representative of thisclass of molecules, exert a number of immunomodifyingeffects. TDM, also known as cord factor, was first identifiedin virulent

M. tuberculosis

, which led to speculation con-cerning its role in virulence and in the process of serpen-tine cording, a phenomenon observed in virulentmycobacteria (Bloch, 1950). However, its role in cordingwas later challenged when TDM was also found in othernon-cording mycobacterial species (Goren, 1972). TDMhas a number of biological activities including high toxicityto mice (Behling

et al

., 1993), potent adjuvant activity bystimulating cell-mediated immunity with co-administrationof protein antigen (Ribi

et al

., 1982), induction of non-specific resistance to infectious agents (Parant

et al

.,1977) and antitumour (Nolibe

et al

., 1986) and proangio-genic activities (Saita

et al

., 2000; Sakaguchi

et al

., 2000).The 19 kDa lipoprotein is a member of a family of

prokaryotic lipoproteins that have been found extensivelyin both Gram-positive and Gram-negative bacteria, includ-ing

Treponema pallidum

,

Mycoplasma

species and

Borre-lia burgdorferi

(Brightbill

et al

., 1999). In members of the

M. tuberculosis

complex, this lipoprotein is anchored inthe cell wall by its lipid moiety (Young and Garbe, 1991).Although its function remains largely unknown, the 19 kDalipoprotein has been implicated in a range of immunolog-ical responses. The immunological activity of the lipopro-tein has been attributed to the NH

2

-terminal triacylatedlipopeptide region, as removal of the fatty acyl moietiesabolishes the immunoregulatory functions of the parentmolecule (Brightbill

et al

., 1999).

Innate immune responses

Phagocytic cell chemotaxis


cell envelope lipids havebeen shown to have effects on neutrophil and monocytemigration. AraLAM, but not ManLAM, induces the chemo-taxis of monocyte-derived macrophages and peripheral

blood monocytes (Bernardo

et al

., 1998). Other studieshave shown that, compared with ManLAM, AraLAM is amore potent inducer of the chemoattractant cytokines JE(murine homologue of MCP-1), KC (murine homologue ofGRO

a

), MCP-3, pentraxin-3 and interleukin (IL)-8 (Roach

et al

., 1993; Zhang

et al

., 1995; Vouret-Craviari

et al

.,1997a,b). In a mouse model, intratracheal administrationof AraLAM or BCG LAM induced significant neutrophilrecruitment into the lungs, but intratracheally administeredManLAM failed to induce neutrophil migration (Strohmeierand Fenton, 1999). Therefore, mannose capping of LAMmay represent a strategy by virulent

M. tuberculosis

tocircumvent the activation of a pulmonary granulocyticresponse, which may be important in the early immuneresponse to mycobacterial infection (Appelberg

et al

.,1995; Saunders and Cheers, 1996) and the enhancementof mycobactericidal activity in the lungs (Fulton

et al

.,2002).

Like AraLAM, TDM has chemotactic activity for manyimmune cells, including neutrophils, monocytes and mac-rophages (Ofek and Bekierkunst, 1976; Lima

et al

., 2001).Whereas intraperitoneal inoculation of delipidated

M.tuberculosis

in mice results in intense recruitment of poly-morphonuclear cells into the peritoneal cavity, intraperito-neal injection of intact

M. tuberculosis

or of purified TDMresults in greater recruitment of mononuclear cells (Lima

et al

., 2001).

Phagocyte function


cell envelope lipids appear toinduce neutrophil activation. Both

M. bovis

BCG ManLAMand

M. smegmatis

phosphatidylinositol-capped LAM(PILAM) were able to activate the protein tyrosine kinaseHck in human neutrophils (Astarie-Dequeker et al., 2000).Interestingly, complete deacylation of ManLAM abolishedits effect on Hck activity, demonstrating that acylation ofLAM, but not mannosylation, is critical for Hck activation(Astarie-Dequeker et al., 2000). Like LAM, SL-I also acti-vates neutrophils in vitro. At low concentrations, SL-I canprime neutrophils to several stimulants, whereas at higherconcentrations, SL-I directly stimulates neutrophils to gen-erate superoxide anion (Zhang et al., 1991).

With respect to macrophage function, the effects of M.tuberculosis cell envelope lipids appear to be more varied.Mannose capping of LAM appears to be critical in direct-ing macrophage responses. AraLAM from avirulent myco-bacteria appears to be more effective than ManLAM ineliciting proinflammatory cytokine responses from mac-rophages (for reviews, see Chatterjee and Khoo, 1998;Strohmeier and Fenton, 1999). AraLAM is a potentinducer of tumour necrosis factor (TNF)a, whereas Man-LAM from the virulent M. tuberculosis Erdman straininduces little or no TNFa in macrophages (Emoto et al.,

108 P. C. Karakousis, W. R. Bishai and S. E. Dorman

© 2003 Blackwell Publishing Ltd, Cellular Microbiology, 6, 105–116

1999). The transcription factor NF-kB, which helps to reg-ulate TNFa expression in lipopolysaccharide (LPS)-stimulated murine macrophages, was more potentlyinduced in bone marrow-derived macrophages andmurine macrophages by AraLAM compared with ManLAM(Brown and Taffet, 1995).

In addition to being a poor inducer of macrophage-activating cytokines, ManLAM may directly inhibitmacrophage responses, thus promoting the intracellularsurvival of M. tuberculosis. ManLAM attenuates TNFa andIL-12 expression in human mononuclear phagocytes andinduces monocyte expression of SHP-1, a tyrosine phos-phatase that may function to downregulate macrophageresponses (Knutson et al., 1998). Furthermore, ManLAMisolated from M. tuberculosis and M. bovis BCG failed toinduce TLR-dependent activation of macrophages (Meanset al., 1999). Although the role of TLR-mediated macroph-age activation in host defence against M. tuberculosis isnot fully known, there is evidence that IL-12-mediated Tcell activation is important (Nau et al., 2002). Failure ofManLAM to induce TLR-dependent macrophage IL-12production may therefore be an important strategy bywhich M. tuberculosis evades the host immune response.In addition to inhibiting interferon (IFN)g-mediated mac-rophage activation (Adams et al., 1993), ManLAM caninduce macrophage tumour growth factor (TGF)b produc-tion (Dahl et al., 1996), which may inhibit macrophage andT cell activation and lead to development of Th2-typeresponses and ineffective immunity against M. tuberculo-sis (Takeuchi et al., 1998).

Upon binding to macrophages via the mannose recep-tor, LAM is transported through the endosomal pathwayto lysosomes, where it may bind to CD1b receptors;CD1b–LAM complexes are then transported to the mac-rophage cell surface, where they can be recognized byantigen-specific T cells (Sieling et al., 1995; Prigozy et al.,1997). There is evidence that LAM may be released fromthis endocytic pathway as a virulence factor. LAM hasbeen observed in exocytic vesicles outside the M. tuber-culosis phagosome (Xu et al., 1994), and LAM and otherlipid products have been demonstrated to traffic through-out the host cell, even penetrating the endosomal/lysoso-mal network and Golgi apparatus (Beatty et al., 2000).Small vesicles containing LAM have been found in theculture medium after phagocytosis of mycobacteria bymacrophages, eventually appearing in neighbouring unin-fected cells (Beatty et al., 2000).

In contrast to their stimulatory effect on neutrophil acti-vation, sulpholipids have been implicated in enhancingresistance of M. tuberculosis to macrophage killing. Ini-tially thought to be involved in the inhibition of phago-some–lysosome fusion (Goren et al., 1976b), sulpholipidswere subsequently shown to promote intracellular survivalof the pathogen by inhibiting phagosome activation

(Goren et al., 1987). Furthermore, pretreatment with sul-phatide blocked priming of cultured human monocytes byLPS or IFNg, resulting in superoxide anion productionequivalent to that of unprimed monocytes after stimulationwith phorbol myristate acetate (Pabst et al., 1988). Thisinhibitory effect of SL-I on monocyte superoxide releasewas negated when SL-I was added after stimulation ofmonocytes by phorbol myristate acetate, suggesting thatSL-I interfered directly with monocyte priming and did notinhibit enzymes involved in superoxide production. How-ever, a subsequent study challenged these findings byshowing that sulphatide treatment of adherent monocytesprimed with IFNg resulted in augmented oxidative activityof these monocytes after stimulation in vitro (Zhang et al.,1988). The discrepancy in the results of these two studieswas attributed to differences in experimental methodology,including doses of SL-I, duration of in vitro culture ofmonocytes and the type of monocyte stimulus used(Zhang et al., 1988). Given these conflicting observations,however, the specific effects of SL-I on macrophage func-tion and their significance in TB pathogenesis requirefurther investigation.

TDM can influence innate immune responses in M.tuberculosis infection by expansion of the natural killer(NK) cell population (Tabata et al., 1996; Ryll et al.,2001a). Unlike ManLAM and SL-I, TDM has been shownto activate macrophages and induce non-specific resis-tance to intracellular pathogens. Pretreatment of mac-rophage cultures with TDM resulted in enhancedphagocytosis and killing of Trypanosoma cruzi (Kierszen-baum et al., 1984) and Salmonella typhi (Yarkoni andBekierkunst, 1976) by TDM-treated macrophages.Furthermore, TDM promotes the development of a Th1response through induction of IL-12 and IFNg (Oswaldet al., 1997) and by depletion of IL-4-producing NK cells(Emoto et al., 1999). However, despite the stimulatoryeffects of TDM on M. tuberculosis-infected macrophages,there is no evidence to suggest that these effects resultin enhanced killing of ingested bacilli by macrophages. Onthe contrary, while delipidation of M. tuberculosis signifi-cantly reduced survival of the pathogen in mouse bonemarrow-derived macrophages, bacterial survival in mac-rophages was restored upon reconstitution of the bacteriawith purified TDM (Indrigo et al., 2002). Indrigo et al.(2002) showed that delipidation resulted in marked reduc-tion in cytokine levels of IL-1b, TNF-a, IL-6, IL-12 and MIP-1a, which were restored upon reconstitution with purifiedTDM. TDM has been indirectly implicated in the inhibitionof phagosome–lysosome fusion because it inhibits fusionbetween phospholipid vesicles in vitro (Spargo et al.,1991). Taken together, these findings support a role forTDM in activation of macrophages and induction of a Th1-type response after M. tuberculosis infection, but also inenhancement of mycobacterial survival within infected

Immune-mediated effects of M. tuberculosis lipids 109


macrophages, perhaps as a result of early phagosomearrest.

Microbial lipoproteins have been shown to be key sig-nalling molecules of the innate immune system. The M.tuberculosis 19 kDa lipoprotein was identified as a majorinducer of IL-12 by human macrophages and causedincreased transcription of inducible nitric oxide synthaseand production of nitric oxide (Brightbill et al., 1999).These activation pathways are regulated through a humanToll-like receptor 2 (TLR-2)-dependent mechanism andrequire the fatty acyl moieties of the lipoprotein (Brightbillet al., 1999). On the other hand, infection of human mono-cyte-derived macrophages with recombinant M. smegma-tis expressing the 19 kDa lipoprotein resulted in markedlyreduced production of TNF-a and IL-12, which may leadto reduced T cell activation (Post et al., 2001). Consistentwith this, although the rapidly growing species M. vaccaeand M. smegmatis, which do not express the 19 kDalipoprotein, induce some protection in vaccinated miceagainst challenge with M. tuberculosis (Abou-Zeid et al.,1997; Skinner et al., 1997), this protective immunity wasabrogated when mice were vaccinated with a recombinantM. smegmatis strain expressing the 19 kDa lipoprotein(Post et al., 2001).

In addition to their effects on macrophage function, bac-terial lipoproteins have been shown to induce apoptosisof THP-1 monocytic cells through human TLR-2 (Aliprantiset al., 1999). Specifically, the 19 kDa lipoprotein has beenidentified as a key molecule mediating M. tuberculosis-induced apoptosis of monocytes and macrophages (Cia-ramella et al., 2000). Although some evidence indicatesthat macrophage apoptosis is associated with increasedmycobacterial survival (Santucci et al., 2000), other stud-ies have shown that induction of alveolar macrophageapoptosis, especially by avirulent mycobacterial species,results in reduced bacillary survival (Keane et al., 2000;Spira et al., 2003). The apparent discrepancy between themonocyte-activating and proapoptotic properties of the19 kDa lipoprotein has been attributed to the differentmultiplicity of infection (MOI) used in the experiments(Ciaramella et al., 2000). In addition, the dual signallingcapacity of TLR-2 has a precedent in tumour necrosisfactor receptor-1, which mediates parallel pathways toapoptosis and NF-kB activation (Ashkenazi and Dixit,1998).

Adaptive immune responses

Antigen presentation

Dendritic cells are critical in host defence as they arecapable of activating naïve lymphocytes, leading to initia-tion of protective immune responses. ManLAM has beenshown to have several inhibitory effects on dendritic cell

function. Nigou et al. (2001) found that ManLAM from M.bovis BCG was able to inhibit IL-12 production by humandendritic cells stimulated with LPS. Loss of the mannosecaps or the phosphatidylinositol anchor abrogated thisinhibitory activity. Because mannan as well as a mono-clonal antibody specific for the mannose receptor eachinhibited LPS-induced IL-12 production by dendritic cells,the authors concluded that engagement of the mannosereceptor by ManLAM delivers an inhibitory signal to den-dritic cells that may contribute to the persistence of viru-lent mycobacteria within phagocytic cells (Nigou et al.,2001). Recently, DC-SIGN, a dendritic cell-specific C-typelectin receptor, was found to mediate capture and inter-nalization of M. bovis BCG by dendritic cells (Geijtenbeeket al., 2003). Binding of ManLAM to DC-SIGN suppressedmycobacteria- or LPS-induced maturation of dendriticcells and enhanced IL-10 production by dendritic cells inresponse to LPS (Geijtenbeek et al., 2003), further sup-porting a role for ManLAM in mycobacterial evasion ofhost immune responses. Interestingly, Nigou et al. (1997)found that only the parietal form of ManLAM from M. bovisBCG stimulated TNFa secretion from human dendriticcells, and parietal ManLAMs were more potent stimulatorsof IL-8 production from dendritic cells compared with cel-lular ManLAMs. In the same study, the immunologicalactivities of ManLAM were abrogated after deacylationand were not observed with mannosylated arabinoman-nan, which lacks the phosphatidyl-myo-inositol anchor,suggesting that this anchor may play an essential role inthe immunomodulatory properties of the ManLAMs(Nigou et al., 1997).

In contrast to the inhibitory effects of ManLAM on den-dritic cell function, the effects of the 19 kDa lipoprotein onthese cells appear to be more varied. M. tuberculosis19 kDa lipoprotein and other microbial lipopeptides havebeen shown to stimulate dendritic cell maturation via TLR-2, as measured by increased expression of CD83, classII MHC (MHC-II) molecules, CD80, CD86, CD54 andCD58, and decreased CD32 expression and endocyticactivity (Hertz et al., 2001). These lipopeptide-matureddendritic cells displayed enhanced T cell stimulatory activ-ity in mixed leucocyte reactions, as measured by T cellproliferation and IFNg secretion (Hertz et al., 2001). Onthe other hand, Noss et al. (2001) found that electroelu-tion- or immunoaffinity-purified 19 kDa lipoprotein of M.tuberculosis inhibits macrophage expression of MHC-IImolecules and processing of soluble antigens via a TLR-2-dependent mechanism. Consistent with these findings,it was demonstrated recently that, through activation ofTLR-2, the 19 kDa lipoprotein inhibits IFNg receptor sig-nalling in human macrophages by downregulating HLA-DR expression, resulting in decreased MHC-II antigenprocessing and recognition by MHC-II-restricted CD4+ Tcells (Gehring et al., 2003). The mechanism by which the



19 kDa lipoprotein inhibits IFNg-induced MHC-II expres-sion is independent of suppressor of cytokine signalling(SOCS)1 and activation of Stat1, but appears to involvereduced induction of class II transactivator (Pai et al.,2003). It appears that the 19 kDa lipoprotein may promoteactivation of microbicidal and innate immune functionsearly in infection whereas, during later stages of macroph-age infection, this molecule may somehow inhibit MHC-IIexpression and antigen processing via a TLR-2-dependent mechanism, thus preventing the presentationof M. tuberculosis antigens and decreasing recognitionby T cells. The latter mechanism could potentially allowintracellular M. tuberculosis actively to evade immunesurveillance and maintain chronic infection (Noss et al.,2001; Pai et al., 2003).

T cell chemotaxis and function

In addition to its effects on neutrophil and macrophagechemotaxis, LAM also induces T cell migration. Superna-tants from M. tuberculosis-infected human alveolar mac-rophages can induce T cell chemotaxis, but this responsecan be abrogated by the addition of anti-LAM antibodies(Berman et al., 1996). Interestingly, although purifiedManLAM from both virulent H37Rv and attenuated H37Racan induce T cell chemotaxis, only supernatants frommacrophages infected with the virulent strain retained thischemotactic response, suggesting that only virulentstrains possess a mechanism for exporting LAM (Bermanet al., 1996). Both AraLAM and M. tuberculosis Erdmanstrain ManLAM are chemotactic for CD4+ and CD8+

peripheral blood T cells in vitro, whereas M. bovis BCGManLAM lacks these chemotactic properties (Bermanet al., 1996).

Similar to its inhibitory effects on macrophage activa-tion, ManLAM can suppress T cell proliferation and acti-vation in vitro. Specifically, M. tuberculosis ManLAM wasfound to inhibit antigen-induced proliferation of humanCD4+ T cell clones specific for influenza virus and prolif-eration of peripheral human T cells after stimulation withcrude mycobacterial antigen extracts (Moreno et al.,1988). This inhibitory effect on T cell proliferation appearsto be related to the processing of antigen by antigen-presenting cells, as the phenomenon was observed onlywhen the T cells were stimulated with intact virus, and notwhen they were stimulated with synthetic peptide alonecontaining the relevant epitope (Moreno et al., 1988).Interestingly, although ManLAM from M. leprae stimulatesthe proliferation of a/b T cells, this effect was not seen inresponse to stimulation with M. tuberculosis ManLAM,suggesting that structural differences within the ManLAMfamily may account for differences in immunological activ-ities. Furthermore, M. tuberculosis-derived ManLAM hasbeen found to inhibit T cell activation in vitro, as evidenced

by decreased expression of mRNAs for several cytokines,including IL-2, IL-3, granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-2 receptor alpha, in Tcells co-stimulated with phytohaemagglutinin-P and phor-bol myristate acetate (Chujor et al., 1992).

Unlike ManLAM, TDM induces proliferation of T cellsfrom rat thymus and lymph nodes in vitro (Kierszenbaumand Walz, 1981). In addition, TDM has been found topotentiate non-specific resistance in mice to lethal influ-enza virus infection by inducing a striking proliferation ofg/d T cells that accumulated in granulomatous lungs (Hoqet al., 1997). Injection of TDM into mice results in a rapidexpansion of the NK T cell population, followed by subse-quent depletion of CD4+ NK1.1+ TCR a/b cells after 7 daysand an increased proportion of Th1 cells after 3 weeks(Ryll et al., 2001a).

Terminally differentiated CD8+ T cells reactive to M.tuberculosis 19 kDa lipoprotein were found to be elevatedin a small study of patients with active pulmonary TB, anddeclined in most patients with response to therapy (Hohnet al., 2003). An HLA-A-binding epitope composed of res-idues 88–97 of the 19 kDa lipoprotein was found to berecognized by circulating CD8+ T cells from both healthytuberculin skin test-positive individuals and patients withactive TB, but not by tuberculin skin test-negative subjects(Mohagheghpour et al., 1998). Moreover, dendritic cellspulsed with this peptide induced MHC-I-restricted lympho-cytes from the T cells of healthy unsensitized persons, andcytotoxic T cells specific for this peptide were shown tolyse autologous monocytes that had been acutely infectedwith the H37Ra strain of M. tuberculosis (Mohagheghpouret al., 1998). Recently, the 19 kDa lipoprotein was shownto inhibit alternative MHC-I processing of latex-conjugateantigen by IFNg-activated macrophages and subsequentpresentation to CD8+ T cells (Tobian et al., 2003).

The important role of CD8+ T cells in immunity againstM. tuberculosis was demonstrated in experiments withexperimentally generated mice lacking functional CD8+ Tcells. These mice had enhanced susceptibility to infec-tion with M. tuberculosis (Flynn et al., 1992). Subse-quent studies revealed that TAP knock-out mice alsohave enhanced susceptibility to tuberculosis (Beharet al., 1999; Sousa et al., 2000), corroborating other evi-dence that M. tuberculosis-containing phagosomes maybe semi-permeable to macromolecules (Teitelbaumet al., 1999). Another potential mechanism by whichmycobacterial antigens may be presented to CD8+ Tcells was recently revealed by Schaible et al. (2003),who demonstrated that apoptosis of M. tuberculosis-infected macrophages causes release of apoptotic vesi-cles carrying mycobacterial antigens, which are engulfedby antigen-presenting cells and can be presentedthrough MHC-I and CD1b to CD8+ T cells. Furthermore,when expressed as a recombinant protein in rapidly



growing mycobacteria, the 19 kDa lipoprotein was foundto be exported from the mycobacterial phagosomewithin the first hour after phagocytosis and was able todeliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism(Neyrolles et al., 2001).

Granuloma formation

Effective adaptive immune responses to M. tuberculosisinfection involve T cell-dependent granuloma formation inan attempt to contain the bacilli, prevent their spread andallow for their eradication by cell-mediated immunity (Ryllet al., 2001b). The ability of cord factors to induce pulmo-nary granulomas in mice resembling those seen aftermycobacterial infection was recognized many years ago(Bekierkunst, 1968). More recently, TDM has been shownto elicit both foreign body-type (non-immune) andhypersensitivity-type (immune) lung granulomas in miceby acting as a non-specific irritant and T cell-dependentantigen respectively (Yamagami et al., 2001). In the samestudy, the authors demonstrated that CD4+ cells, alongwith the cytokines IL-1, IL-12, TNF-a and IFN-g, and thechemokine MIP-1a, were more abundant in lung granulo-mas of mice immunized with TDM compared with unim-munized mice after TDM challenge (Yamagami et al.,2001). These specific cytokines are thought to be keycomponents in the process of granuloma formation (Koba-yashi and Yoshida, 1996). Moreover, intravenous injectionof TDM in rabbits induces weight loss, prominent hepaticand pulmonary granulomas and thymic and splenic atro-phy resulting from apoptosis, whereas intravenous injec-tion of sulpholipid has none of these effects (Hamasakiet al., 2000). Similarly, intravenous injection of purifiedTDM, but not SL-I, from M. tuberculosis in BALB/c miceresults in thymic atrophy via apoptosis and pulmonarygranulomas by day 7 (Ozeki et al., 1997). These effectswere attributed to induction of TNFa as they were almostcompletely abrogated by administration of anti-TNFa anti-body (Ozeki et al., 1997).

In the last decade, considerable progress has beenmade in understanding the cell signalling events underly-ing the action of TDM. CD1 proteins were identified inhumans as non-polymorphic MHC class I-like moleculesresponsible for presenting lipid antigens to T cells and,specifically, human CD1b was identified as the receptorinvolved in mycolic acid presentation (Beckman et al.,1994). Partial unfolding of CD1 helices and exposure oflipid antigen-binding domains occurs at acidic pH, per-haps in the endosomal pathway (Ernst et al., 1998). Themolecular interactions of mycolic acid with the bindinggroove of CD1b (Niazi et al., 2001) and subsequent pre-sentation to the T cell receptor have been describedrecently (Melian et al., 2000).

Humoral immune responses

Humoral immune responses to LAM, SL-I, TDM and the19 kDa lipoprotein have been described in animals afterexperimental administration of these antigens and inhumans during the natural course of M. tuberculosis infec-tion. Several immunomodulatory effects for these antibod-ies have been described. Anti-LAM IgG was reported tomediate classical complement activation, which may beimportant for phagocytosis of bacilli by mononuclearphagocytes (Hetland et al., 1998). In addition, anti-LAMIgM enhanced serum clearance and altered organ depo-sition of intravenously administered LAM in mice(Glatman-Freedman et al., 2000). The ability of TDM toinduce humoral responses was recognized many yearsago (Bekierkunst et al., 1971). The injection of TDMbefore antigen administration in the same footpads ofmice resulted in higher antibody production to the injectedantigen compared with that resulting from antigen injec-tion into the contralateral footpads (Bekierkunst et al.,1971). Furthermore, treatment with TDM increases themagnitude of the IgM antibody response of mice to typeIII pneumococcal polysaccharide (Baker et al., 1989).Recent studies have shown that injection of TDM into miceor exposure of macrophages in vitro to TDM results in theinduction of a variety of cytokines, including IL-4, IL-6 andIL-10 (Perez et al., 2000; Lima et al., 2001), which maycreate a cytokine milieu conducive to antibody production.In addition to inducing antibodies against unrelated anti-gens, TDM can induce antibody production against its ownmolecular components, including its carbohydrate moiety(Kato, 1972) and mycolic acid components (Fujiwaraet al., 1999a,b). Bacterial lipoproteins are potent poly-clonal activators of B lymphocytes (Bessler et al., 1985).When given as an adjuvant in mice, a synthetic lipoproteinderivative was found significantly to enhance and accel-erate the humoral immune response to tetanus toxoid,resulting in a shift from a Th1-type to a Th2-type immuneresponse (Mittenbuhler et al., 2003). Whereas the pre-dominant anti-LAM antibody isotype during human infec-tion is IgG2 (Da Costa et al., 1993), immunization of micewith various 20-mer peptides contained within the 19 kDalipoprotein elicited strong IgG1 isotype responses, indicat-ing that these peptides could possibly stimulate lympho-cytes of a Th2 phenotype (Ashbridge et al., 1992).Although these lipid antigens induce antibody responses,the precise role of humoral immunity to these antigenswithin the overall host immune response to M. tuberculo-sis infection requires further investigation.

Antibodies to mycobacterial lipid antigens have beenstudied as potential diagnostic tools in human TB. In gen-eral, diagnostic tests based on antibody detection havesuboptimal sensitivity and specificity, and none is recom-mended for use in TB control programmes. A more



detailed discussion of antibody-based diagnostic tests forTB is outside the scope of this review.

Conclusions

Mycobacterium tuberculosis is a highly successful patho-gen, in large part because of its intracellular nature, hav-ing evolved mechanisms to invade and survive within thehost. Latent tuberculosis infection probably represents abalance between pathogen virulence factors and the hostimmune response, leading to long-term containment, butnot eradication, of persistent bacilli (Manabe and Bishai,2000). When host immune mechanisms wane, such aswith intercurrent infection with human immunodeficiencyvirus, the balance shifts in favour of the pathogen, result-ing in bacillary replication and active tuberculosis disease.The cell envelope of M. tuberculosis has several structuralcomponents that probably play a role in directing thesehost–pathogen interactions.

This review summarizes the immunomodulatoryproperties of the cell envelope-associated lipid-containingmolecules lipoarabinomannan, sulpholipids, mycolic acid-containing glycolipids and the 19 kDa lipoprotein. Thepleiotrophic and often seemingly antagonistic immunolog-ical effects of these molecules make it difficult to discernthe precise mechanism by which each contributes to theimmunopathogenesis of tuberculosis. Occasionally, dis-crepancies have been reported in the literature regardingthe immunological effects of individual molecules. Onepotential explanation for some of these conflicting findingsmay be that they are a function of the specific experimen-tal conditions used to study them. On the other hand, theymay reflect a complex strategy by which M. tuberculosisis able to survive within the host for prolonged periods oftime. The net immunological effect produced by these cellenvelope-associated lipids may be to recruit and activatephagocytes in order for the pathogen to gain entry intothese cells, only to subvert the microbial killing mecha-nisms of these cells and promote intracellular survival ofthe pathogen.

The advent of molecular genetic tools to manipulatemycobacteria and the availability of the M. tuberculosisgenome (Cole et al., 1998) have now provided the oppor-tunity to generate mutations experimentally in the genesrequired for biosynthesis of these cell envelope-associ-ated lipid-containing molecules and to test directly the roleof each molecule in the immunopathogenesis of M. tuber-culosis. If these mutated strains show an attenuated phe-notype in vivo, they may serve as potential vaccinecandidates. Finally, elucidation of the biosynthetic path-ways involved in the assembly of these molecules mayprovide molecular targets for new drugs to combat thishighly successful pathogen.

Acknowledgements

This work was supported by a postdoctoral research award fromthe Potts Memorial Foundation, the National Institutes of Healthgrants AI 54491, AI 43846, AI 36973 and AI 37856, and aresearch grant from the American Lung Association of Maryland.

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Mycobacterium tuberculosis cell envelope lipids and the host immune response

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