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Human leukocyte antigens in tuberculosis and leprosy
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Human leukocyte antigens in tuberculosis and leprosy Christian G. Meyer, Jürgen May and Klaus Stark
M
ycobacterium leprae Human mycobacterial infections are NRAMP1, has also been idenand Mycobacterium characterized by a spectrum of clinical and tified and is currently being tuberculosis are the immunological manifestations. Specific analysed for its possible ascausative agents of leprosy and human leukocyte antigen (HLA) factors sociation with susceptibility/ tuberculosis, respectively. Most are associated with the subtypes of leprosy resistance to tuberculosis and individuals exposed to these that develop and the course of tuberculosis leprosy2,3. pathogens mount efficient imafter infection. The identification of For many years, major histomune responses, but individprotective mycobacterial antigens compatibility complex (MHC) uals who do develop disease presented by a broad variety of HLA genes have also been considered exhibit a variety of clinical molecules will have important implications as candidate genes involved in manifestations. for the design of vaccines. conferring susceptibility or proThe manifestations of leptection in animal and human C.G. Meyer*, J. May and K. Stark are in the rosy span a spectrum of clinical/ mycobacterial disease (Box 1). Institute for Tropical Medicine, histological characteristics, In mice infected with M. bovis Spandauer Damm 130, 14050 Berlin, Germany. ranging from the polar paucibacille Calmette–Guérin (BCG), *tel: +49 30 30116 810, fax: +49 30 30116 888, bacillary form, which correMHC genes profoundly affect e-mail: [email protected] sponds to tuberculoid leprosy the antibody repertoire against (TT), to the multibacillary form, which corresponds mycobacterial antigens4. Furthermore, MHC-dependent to lepromatous leprosy (LL). Intermediate forms are antimycobacterial protection to M. bovis BCG has classified as borderline tuberculoid (BT), midborder- been elegantly demonstrated in knockout mice with line (BB) and borderline lepromatous (BL) leprosy. In defined MHC deficiencies: MHC class II-deficient general, disease is characterized by skin and nerve in- Aβ–/– mice succumbed to M. bovis BCG infection, vasion by bacteria, with resulting lesions. Most clinical whereas controls were capable of inhibiting bacterial symptoms of leprosy reflect immune reactions against growth. After infection, bacterial infiltrates of the livantigenic constituents of M. leprae. In TT, a few cir- ers of β2-microglobulin-deficient mice were more difcumscribed skin and nerve lesions are found (Fig. 1a), fuse than those of controls, pointing to an important but marked cellular immune responses limit bacterial role of MHC class I molecules and cytotoxic T cells5. However, the situation is more complex. A model spread. In contrast, LL is a severe systemic disease and can involve multiple organs, including limbs, eyes, nose, derived from studies of twins in Gambia suggests that pharynx and testes (Figs 1b,c). Specific immunosup- the cumulative effect of human non-MHC genes expression in LL allows uncontrolled dissemination of ceeds the contribution of MHC class II genes in immune responses to both purified protein derivative (PPD) of the bacteria. In tuberculosis, bacterial growth and spread are usu- M. tuberculosis and to malarial antigens6. ally suppressed, but impaired control can result in severe disease. The most common clinical manifestation Human leukocyte antigens and associations with is pulmonary tuberculosis (PTB). Dissemination can mycobacterial disease also lead to extrapulmonary tuberculosis and the event- A variety of approaches can be used to study the association of human leukocyte antigen (HLA) factors, or ual involvement of other organs. The observations that particular ethnic groups and other genetic markers, with disease. In family studies, distinct families are more susceptible to leprosy and non-random segregation of HLA factors in diseased tuberculosis than others and that monozygotic twins children, compared with their healthy siblings, indidevelop comparable manifestations of mycobacterial cates HLA association with susceptibility to disease. disease support the view that genetic elements contrib- Segregation patterns are analysed by the lod score staute to determining the course of these infections. There tistic (the log odds ratio between the observed and the is now substantial evidence that host genetic factors in- expected distribution of the genetic markers in chilfluence not only initial susceptibility to mycobacterial dren). In case-controlled population studies, the distriinfections but also the spectrum of clinical and immuno- bution of HLA markers is compared between unrelated logical responses. The Bcg/Lsh/Ity gene of mice con- patients with unambiguous diagnoses and healthy controls susceptibility (Bcgs) and innate resistance (Bcgr) to trols, selected according to age, ethnic group and other Mycobacterium bovis, Mycobacterium lepraemurium demographic factors. The advantages of family studand other mycobacterial species1, with Bcgr being domi- ies are that they exclude biased data resulting from nant over Bcgs. The corresponding human homologue, population heterogeneity and that they may reveal an Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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PII: S0966-842X(98)01240-2
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association between the genetic marker and disease that is not observable at the population level. HLA associations with mycobacterial disease have been extensively studied in M. leprae and, to a lesser degree, in M. tuberculosis infection. Some of the associations have been consistently found in different ethnic groups, but others have not. More recent studies suggest that disease per se is controlled by non-HLA, rather than HLA, genes, which is similar to the observations made with the Bcg gene of mice7. One or more recessive non-HLA genes controlling susceptibility to leprosy per se, and non-lepromatous leprosy in particular, have been described7. However, there is strong evidence that HLA factors are associated with the type and severity of disease in mycobacterial infections (Table 1). Tuberculosis Few family studies have reported HLA associations with PTB. Siblings with the haplotype HLA-A2-B5 have more severe manifestations of PTB than those carrying only one of these alleles8. In multicase families in India, HLA-DR2 is significantly more common in children with PTB than in healthy siblings9. One recent study has shown a strong association of HLA-DR2 with advanced and, in particular, drug-resistant PTB (Ref. 10). Although the HLA-DR2 association has been consistently reported in several population studies (Table 1), another recent study from northern India found no relationship11. Nevertheless, HLA-DR3 has been found at significantly lower frequencies in PTB patients than in controls12. The early findings on HLA-DR and HLA-DQ associations with tuberculosis have been refined using DNAbased HLA-typing. DRB1*1501 and DQB1*0502 have been reported to be more common in PTB patients than in controls13. In this study, two haplotypes were observed at higher frequencies in patients with advanced PTB (Table 1). Other researchers have found increased frequencies of both DRB1*1501 and DRB1*1502 in PTB patients10. One very recent study reports an association between HLA-DQB1*0503 and the development of clinical PTB in Cambodian patients14. This is compatible with the previous observation that the frequent occurrence of HLA-DQ5, haplotypically linked to HLA-DR15(DR2), correlates with fulminant multibacillary tuberculosis13. In contrast, other studies suggest that HLA class II allele distributions do not differ in terms of the observed severity of tuberculosis (for example, Ref. 15).
Fig. 1. (a) Tuberculoid leprosy, showing limited spread of bacteria and circumscribed skin lesions. (b) and (c) Lepromatous leprosy, with multiple confluent skin lesions and unrestricted spread of bacteria.
The findings on HLA-DR and HLA-DQ associations with leprosy have also been further resolved by DNAbased HLA-typing. DRB1*1501, DRB1*1502 and DQB1*0601 occur more frequently in all forms of leprosy than in controls, but the associations are stronger with LL than with TT (Ref. 24). In the same study, DQA1*0103 was associated with LL, and DQA1*0102
Leprosy An HLA-A11 association with erythema nodosum leprosum has been described16; however, data on HLA class I associations with leprosy are inconsistent. Nevertheless, convincing results have been obtained from studies on HLA class II associations with leprosy (Table 1). HLA-DR2 is found significantly more frequently in LL and TT patients compared with healthy controls. TT is also associated with the occurrence of HLA-DR317–20, which is found at lower frequencies in LL patients18,20. Furthermore, HLA-DQ1 has often been found to be associated with LL and immunosuppression21–23.
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Box 1. Human leukocyte antigen function and diversity Human leukocyte antigens (HLAs) provide a framework for T-cell recognition of antigenic peptide fragments. Different HLA variants present different peptides, and the HLA system is one of the mediators that, by virtue of its polymorphism, guarantees immunological specificity. The immense variability of HLAs is ancient and pre-dates human speciation. Genetic diversity would not persist without good reason and there is evidence that human MHC genes are still subject to variation. Balancing selection certainly operates, preventing gene loss. The concept of infection-driven selection promoting HLA polymorphism is attractive and is consistent with the primary function of HLA molecules: the presentation of peptide antigens in the fight against microorganisms. HLA heterozygosity increases the functional repertoire. Fluctuating patterns of lethal epidemics, emerging diseases, drug resistance and other factors contribute to selection and have probably modified HLA diversity during different historical episodes and in different geographical regions. Although this statement is speculative, it is supported by observations of epidemics that arose when Mycobacterium tuberculosis, measle viruses and other pathogens were introduced in previously unprimed native populations54,55.
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Table 1. Associations between HLA class II factors and mycobacterial disease Study size (n) HLA class II alleles
Direction of association
Tuberculoid leprosy (TT) DR2 DR2 DR3 DR2 DR2; DQw1 DR1; DR2; DRw9; DQw1; DR4; DRw53; DQw3 DRB1*1501; DRB1*1502; DQB1*0601; DQB1*0503 DRB1*1502; DQB1*0601; DRB1*1501–DRB5*0101–DQA1*0102–DQB1*0502 DR3 DR3 DR3 DR2 DQw1
Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Negative Positive Positive Positive Positive Positive
Lepromatous leprosy (LL) DR2; DQw1; DRw9; DRw53 DR1; DR2; DRw9; DQw1; DR4; DRw53; DQw3 DR2 DR2; DRw8; DQw1 DRB1*1501; DRB1*1502; DQA1*0103; DQB1*0601 DRB*1501; DRB5*0101; DQB1*0602 DR3 DR3 DR5 DR2 DQw1
Positive Negative Positive Negative Positive Positive Positive Positive Negative Negative Positive Positive Positive
Pulmonary tuberculosis (PTB) DR2 DR2; DRw6 DR2; DQw1; DQw3 DR2 DRB1*1501; DQB1*0502; DRB1*1501–DRB5*0101–DQA1*0103–DQB1*0601; DRB1*1501–DRB5*0101–DQA1*0102–DQB1*0502 DR2; DRB1*1501; DRB1*1502 DR2; DRw53 DR3 DQB1
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Ref.
Indian Egyptian Venezuelan Japanese Thai Korean
56 57 17 21 58 22
28
47
Indian
24
39
46
Indian
13
29 30 32 259 149
92 100 212 813 423
Surinamese Mexican Venezuelan Pooled data Pooled data
18 19 20 59
295
110
Japanese
21
152
155
Korean
23
50 68 65 62 23 35 35 474 417
50 237 47 114 92 212 32 703 472
Turkish Indian Indian Japanese Surinamese Venezuelan Thai Pooled data Pooled data
60 23 24 25 18 20 58 59
Indian Indian
9 61
Indonesian
62
25 multicase families 90 60
Positive Positive Negative Positive
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Controls
8 multicase families 15 multicase families 28 multicase families 74 110 32 32 152 155
Positive Positive Negative Positive Negative Positive Positive
was associated selectively with borderline leprosy. The frequency of the haplotype DRB1*1501–DRB5*0101– DQA1*0102–DQB1*0502 is lower in TT patients13. In Japanese leprosy patients, DRB1*1501, DRB1*1502, DRB5*0101, DRB5*0102, DQA1*0102, DQB1*0602 occur at an increased frequency in all patients, but DRB1*1501, DRB5*0101 and DQB1*0602 are only more frequent in LL patients25. Arginine, at positions 13 or 70/71 of the DRβ molecule, is present in large subsets of DRβ alleles and is significantly increased in TT patients26. Recently, particular polymorphic TAP (transporter associated with
Patients
101
64
204 20
404 46
Indian Indian
63 13
153 38 51 48
289 291 54 39
Indian Tuvinian Mongol Mexican Cambodian
10 64 12 14
antigen processing) elements have also been shown to correlate with the occurrence of tuberculosis and leprosy: TAP2-A/F correlates positively with PTB, and TAP2-B correlates with TT (Ref. 27). Limitations It is important to consider the possible limitations of the studies described above. In the earlier studies, the HLA patterns were determined by conventional techniques that might have resulted in a lower resolution of HLA types. The development of DNA-based HLA-typing methods has improved the validity of HLA-typing.
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In some studies, valid HLA associations might not have been detected because of small sample sizes and a lack of statistical power. The high number of HLA factors requires multiple statistical testing and increases the possibility that an apparent disease association may be found by chance. Some studies correct for multiple testing (Bonferroni correction), but this conservative approach may underestimate true associations. A substantial body of evidence now exists that specific HLA types are associated with mycobacterial disease. Associations of DR2 and DQ1 with LL, DR2 with PTB, and DR3 with TT have been described in different ethnic groups. However, other associations have not been confirmed. In addition to the methodological limitations, this may result from the enormous HLA polymorphism and the variable distributions of HLA alleles in different ethnic populations. Associations of nonclassical HLA class I genes with tuberculosis and leprosy have not yet been studied. Immunology HLA-restricted mycobacterial epitopes Little is known about the molecular nature of HLArestricted mycobacterial antigens eliciting either protective or impaired immune responses. A variety of HLA-restricted mycobacterial antigens, including heat-shock proteins (hsps) and secreted fibronectin-binding proteins, are presented to T cells by macrophages and other cells (Fig. 2). Fragments of M. leprae hsp70, hsp65 and hsp18 can induce vigorous CD4+ T-cell-proliferative responses that are restricted by multiple DR molecules during TT and LL (Ref. 28). BCG vaccinees and tuberculosis patients with various DR specificities also respond to M. leprae hsp70. Many T-cell determinants, rather than a few immunodominant epitopes, exist throughout the molecule, and different individuals and populations respond to different determinants29. Although M. leprae hsp70 is similar to its human homologue, human hsp70 does not stimulate M. leprae hsp70-reactive T cells. The response to hsp70 is obviously directed at specific mycobacterial determinants30. Particular epitopes of fibronectin-binding proteins and mycobacterial hsp peptides, such as hsp65 peptide p2-12 (p3-13), bind selectively to HLA-DR3 (Refs 31, 32). Dominance of the TCRBV5 family of TCR genes, with exclusive usage of the TCRBV55S1 segment, is observed in a TT-derived DR3–hsp65 p3-13-restricted Tcell line33. In LL patients, only a few hsp fragments can trigger T cells. Hsp65 induces the release of tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1) but does not activate human mononuclear phagocytes34, indicating that this protein contributes both to host defence and tissue damage in the inflammatory lesions. Mycobacterial lipid antigens (protease-resistant mycobacterial lipid antigens and mycobacterial lipoarabinomannan) also stimulate T-cell responses in the context of HLA-independent (including DMA/B) CD1band CD1c-restriction35–37 (Fig. 2). A common antigenic moiety, presented by CD1b to CD4-/CD8-α/β+ T cells, appears to be shared by many mycobacterial species37.
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In the early stages of mycobacterial infection, HLAunrestricted and HLA class I-restricted γ/δ T-cell proliferation (V-γ 9/V-δ 2) and lysis of infected target cells occur, which is compatible with a superantigenlike phenomenon38,39. Immunosuppression Immunosuppression in mycobacterial infection, particularly in LL, is not fully understood (reviewed in Ref. 40). The fact that macrophages from LL patients can present M. leprae antigen to lymphocytes of healthy compatible siblings argues against defective antigen presentation in immunosuppression. T cells derived from LL patients and cultured in the absence of mycobacteria can be induced to proliferate in response to M. leprae, challenging the concept that M. leprae-specific T cells might be selectively deleted in LL. One possible mechanism underlying suppression is lysis of responder and/or antigen-presenting cells by cytotoxic T lymphocytes (CTLs), a mechanism absent when the cell-mediated response is restored by IL-2 or when cells are precultured before addition of antigen. The existence of suppressor T cells is controversial41; however, antigen-specific CD8 cells in unresponsive LL patients appear to act as T suppressor cells. These cells are HLA-DQ restricted and predominantly produce IL-4 (type 2 CD8 cells)42 (Fig. 2). Suppression can be inhibited by anti-DQ antibodies43. Cytotoxic T cells Mycobacteria also induce HLA class I-restricted CD8+ cells, which leads to killing of infected macrophages44 (Fig. 2). Evidence for this comes from studies in which CD8+ T cells are depleted with monoclonal antibodies, and from experiments with MHC-inbred and β2-microglobulin knockout mice that do not express class I antigens. Following mycobacterial infection, these mice die rapidly. Live M. bovis BCG can efficiently activate CD8+ cells from BCG vaccinees, showing that this T-cell subset plays an essential cytolytic role in the immune response to mycobacterial infection45. In addition to phagolysosomic presentation, HLA class I presentation of mycobacterial antigens can also occur by either circumventing the cytoplasmic pathway or after translocation of antigens from the phagosome46. There are few data on the identity of mycobacterial antigens recognized by CD8+ cells or on the mechanisms of T-cell activation, and only some CTL epitopes have been characterized so far47. T-cell dichotomy Differences in immune responses to M. leprae are reflected by the cytokine production patterns observed at the two extremes of the leprosy spectrum48 (Fig. 2). In TT, a T-helper type 1 (Th1)-like cytokine pattern is observed; mycobacterial spread is restricted, and CD4+ cells and type 1 cytokines [e.g. IL-2 and interferon γ (IFN-γ)] are mainly found in skin lesions (Th1 response). In contrast, in LL, abundant CD8+ cells and the production of IL-4 and IL-10 represent a type 2 (Th2) response. These cytokines inhibit cellular immunity and downregulate Th1 responses. Th2-like cytokine profiles
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Activation Inhibition
HLA molecule Mycobacteria
Antibody
Cytotoxicity
CD4 α/β
?
B CD1b/c IL-10
IFN-γ
IL-4
DR
T γ/δ
DR ? HLA classI I HLA class
M/M HLA class II
DQ
Th2
Anti-DQ Ab
TNF- α
CD8
IFN- γ IL-2
Anti-CD8 Ab Anti-TCR Ab IFN-γ
IL-12 Th1
Protection
Multibacillary disease
CD8 Ts
NK
Paucibacillary disease
Fig. 2. A model for immunological mechanisms in human mycobacterial infection. Green pathways indicate activation, and red pathways represent either inhibition or cytotoxicity. Intracellular mycobacterial antigens (small coloured circles) may or may not be presented in the context of HLA class I or class II molecules (grey boxes), leading to either strong or weak T-cell responses. Bacterial lipid antigen may also be presented to CD4+ T cells in the context of CD1b/c molecules. Activating responses result in CD4+ (including CD4+ γ/δ)- and CD8+-dependent killing of infected macrophages. Activation of T suppressor (Ts) cells inhibits cellular immunity. Several antibodies have been shown to block CD8 +-specific effects. The role of humoral immunity is unclear, but is probably negligible. The dichotomy of T-cell responses provides a basis for understanding the effective or impaired control of mycobacterial infection. T-helper type 1 (Th1) responses and the release of the respective cytokines result in protection or recovery from mycobacterial infection, whereas uncontrolled dissemination of bacteria is associated with a Th2 response involving interleukin 4 (IL-4) and IL-10 release. Balanced Th1–Th2 activation would, presumably, confer adequate protection. Abbreviations: Ab, antibody; B, B cell; CD4, CD4+ T cell; CD8, CD8+ T cell; HLA, human leukocyte antigen; IFN-γ, interferon γ; M/M, macrophage/monocyte; NK, natural killer cell; TNF-α, tumour necrosis factor α.
are found in both CD4+ and CD8+ cells isolated from LL patients. Patients with tuberculous pleuritis and a favourable prognosis have high concentrations of IFN-γ, IL-2 and their respective mRNAs in their pleural fluids. This also indicates the central role of Th1 responses associated with protection and recovery49. It is intriguing that mycobacterial antigens, which are associated with particular DR or DQ molecules, can preferentially trigger different Th populations. Thus,
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these antigens favour particular patterns of cytokine production, which eventually lead to the spectrum of responses to mycobacterial infection. The association of HLA-DQ variants with immunosuppression in human leprosy and with advanced multibacillary tuberculosis supports the observation that suppression in vitro is restricted by HLA-DQ (Ref. 50) but not by HLA-DR. Moreover, suppression can be inhibited by anti-DQ antibodies. Similar HLA-DQ associations with
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immunosuppression have also been reported in infections with the helminth Onchocerca volvulus51,52. Antigen-induced suppression can also be inhibited by antibodies specific to polymorphic T cell receptor β chains of CD8+ T-cell clones43. The impairment of immunity in LL, namely the absence of IL-2 and IFN-γ, could result from defective Th1 responses, whereas enhanced B-cell-specific and polyclonal activation and generation of cytotoxic (suppressor) cells could represent an overactivation of Th2 cells. Balanced activation of both T-cell populations would lead to successful defence against mycobacteria. Conclusions Several antigenic peptides triggering different types of immune responses have been identified in human mycobacterial infections. Although the exact mechanisms leading to specific immunosuppression are not completely understood, immunosuppression and progress of disease appear to be controlled by the HLA system. In contrast, innate resistance to mycobacterial infection appears to be dependent on non-HLA genes. However, universal T-cell epitopes of mycobacteria have not yet been characterized, although they have recently been described for the Plasmodium falciparum circumsporozoite protein53. The identification of mycobacterial antigens that fit the molecular shape of a broad variety of HLA molecules and are capable of inducing protective Th1 immune responses may lead to the design of subunit vaccines for both tuberculosis and leprosy. A general strategy for the development of successful vaccines for leprosy requires the identification of the binding motifs of common HLA molecules and the characterization of their anchor positions. This can be achieved by amino acid sequencing of bound peptide fragments (either individual or pooled fragment sequencing), followed by analysis of binding properties of polyalanine analogue peptides. Preparations of cocktails of synthesized peptides would allow binding to different HLA molecules and help to provide vaccine cover for different human populations. In addition, the motif affinity to HLA molecules would need to be validated and accompanied by cellular binding assays and Phase I clinical trials of candidate vaccines to assess the immunogenicity of subunits in several susceptible populations. This approach is currently being planned using computer-based algorithms for the prediction of promiscuous T-cell epitopes. Acknowledgements We thank Rolf Horstmann for encouragement, and Frank Mockenhaupt for valuable comments on the manuscript. References 1 Gros, P., Skamene, E. and Forget, A. (1981) J. Immunol. 127, 2417–2421 2 Cellier, M. et al. (1994) J. Exp. Med. 180, 1741–1752 3 Liu, J. et al. (1995) Am. J. Hum. Genet. 56, 845–853 4 Huygen, K. et al. (1993) Infect. Immun. 61, 2687–2693 5 Ladel, C.H., Daugelat, S. and Kaufmann, S.H.E. (1995) Eur. J. Immunol. 25, 377–384 6 Jepson, A. et al. (1997) Infect. Immun. 65, 872–876
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Questions for future research • Can associations of human leukocyte antigen (HLA) elements, including the association of arginine at DRβ positions 13 or 70/71 and that of the TAP genes, be confirmed in larger studies and in other ethnic groups? • What are the specific non-HLA genes that confer susceptibility and resistance in human mycobacterial diseases? • How do these genes act and what are their products? • What is the precise role of CD8+ and γ/δ T cells in mycobacterial infection? • What are the essential HLA-restricted and unrestricted ‘protective’ antigens? • Which antigens should be used in subunit vaccines?
7 Abel, L. et al. (1989) Int. J. Lepr. Other Mycobact. Dis. 57, 465–471 8 Hafez, M. et al. (1992) Dis. Markers 10, 143–149 9 Singh, S.P. et al. (1983) J. Infect. Dis. 148, 676–681 10 Rajalingam, R. et al. (1996) J. Infect. Dis. 173, 669–676 11 Ghosal, A.E. et al. (1996) J. Indian Med. Assoc. 94, 328–330 12 Cox, R.A. et al. (1988) J. Infect. Dis. 158, 1302–1308 13 Mehra, N.K. et al. (1995) Int. J. Lepr. Other Mycobact. Dis. 63, 241–248 14 Goldfeld, A.E. et al. (1998) J. Am. Med. Assoc. 279, 226–228 15 Sanjeevi, C.B. et al. (1992) Tubercle 73, 280–284 16 Agrewala, J.N. et al. (1989) Tissue Antigens 33, 486–487 17 van Eden, W. et al. (1985) J. Infect. Dis. 151, 9–14 18 van Eden, W. et al. (1982) Hum. Immunol. 4, 343–350 19 Gorodezky, C. et al. (1987) Lepr. Rev. 58, 401–406 20 Ottenhoff, T.H.M. and de Vries, R.R.P. (1987) Int. J. Lepr. Other Mycobact. Dis. 55, 521–534 21 Izumi, S. et al. (1982) Vox Sang. 42, 243–247 22 Kim, S.J. et al. (1987) Tissue Antigens 29, 146–153 23 Rani, R., Zaheer, S.A. and Mukherjee, R. (1992) Tissue Antigens 40, 124–127 24 Rani, R. et al. (1993) Tissue Antigens 42, 133–137 25 Joko, S. et al. (1996) Nippon Rai Gakkai Zasshin 65, 121–127 26 Zerva, L. et al. (1996) J. Exp. Med. 183, 829–836 27 Rajalingam, R., Singal, D.P. and Mehra, N.K. (1997) Tissue Antigens 49, 168–172 28 Mustafa, A.S., Lundin, K.E.A. and Oftung, F. (1993) Infect. Immun. 61, 5294–5301 29 Oftung, F. et al. (1994) Infect. Immun. 62, 5411–5418 30 Adams, E. et al. (1994) Scand. J. Immunol. 39, 588–596 31 van Schooten, W.C. et al. (1989) Eur. J. Immunol. 19, 2075–2079 32 Geluk, A. et al. (1994) Eur. J. Immunol. 24, 3241–3244 33 Struyk, L. et al. (1995) Hum. Immunol. 44, 220–227 34 Peetermanns, W.E. et al. (1994) Scand. J. Immunol. 39, 613–617 35 Porcelli, S., Morita, C.T. and Brenner, M.B. (1992) Nature 360, 593–597 36 Sieling, P.A. et al. (1995) Science 269, 227–230 37 Beckman, E.M. et al. (1996) J. Immunol. 157, 2795–2803 38 Holoshitz, J. et al. (1993) Int. Immunol. 5, 1437–1443 39 Tsuyuguchi, I. et al. (1991) Infect. Immun. 59, 3053–3059 40 Ottenhoff, T.H.M. (1994) Int. J. Lepr. Other Mycobact. Dis. 62, 108–121 41 Bloom, B.R., Modlin, R. and Salgame, P. (1992) Annu. Rev. Immunol. 10, 453–488 42 Mutis, T. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9456–9460 43 Salgame, P., Convit, J. and Bloom, B.R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2598–2602 44 Flynn, J.L. et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12013–12017
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45 46 47 48 49 50 51 52 53 54 55 56
Turner, J. and Dockrell, H.M. (1996) Immunology 87, 339–342 Pfeifer, J.D. et al. (1993) Nature 361, 359–362 Vordermeier, H.M. (1995) Eur. Respir. J. 20, S657–S667 Modlin, R.L. (1994) J. Invest. Dermatol. 730, 828–832 Barnes, P.F. et al. (1993) Infect. Immun. 61, 3482–3489 Sasazuki, T. et al. (1989) Immunology 2, 21–24 Meyer, C.G. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7515–7519 Meyer, C.G. and Kremsner, P.G. (1996) Parasitol. Today 12, 179–186 Calvo-Calle, J.M. et al. (1997) J. Immunol. 159, 1362–1373 Calmette, A. (1931) Presse Med. 2, 17 Goodman, R.M. and Motulsky, A.G. (1979) Genetic Diseases among Ashkenazi Jews, Raven Press van Eden, W. et al. (1980) J. Infect. Dis. 141, 693–701
57 Dessoukey, M.W., el Shiemy, S. and Sallam, T. (1996) Int. J. Dermatol. 35, 257–264 58 Schauf, V. et al. (1985) Tissue Antigens 26, 243–247 59 Todd, J.R., West, B.C. and McDonald, J.C. (1990) Rev. Infect. Dis. 12, 63–74 60 Cem, M. et al. (1988) Int. J. Dermatol. 27, 246–247 61 Singh, S.P. et al. (1983) Tissue Antigens 21, 380–383 62 Bothamley, G.H. et al. (1989) J. Infect. Dis. 159, 549–555 63 Brahmajothi, V. et al. (1991) Tubercle 72, 123–132 64 Pospelov, L.E. et al. (1996) Tubercle Lung Dis. 77, 77–80 65 Bellamy, R. et al. (1998) New Engl. J. Med. 338, 640–644 Note added in proof In a very recent study it has been shown that NRAMP1 polymorphisms affect susceptibility to tuberculosis in West Africans65.
Antibacterial and anti-inflammatory agents that target endotoxin Timna J.O. Wyckoff, Christian R.H. Raetz and Jane E. Jackman
G
ram-negative bacteria Antibiotic-resistant bacterial infections are production of cytokines and incause about half of all a major clinical problem. Lipid A, the flammatory mediators, includserious human infecactive part of lipopolysaccharide ing tumor necrosis factor α tions, and complications of seendotoxins in Gram-negative bacteria, is (TNF-α) and interleukin 1β vere Gram-negative sepsis may an intriguing target for new antibacterial (IL-1β), damages the microaccount for as many as 100 000 and anti-inflammatory agents. Inhibition vasculature5–7. A full systemic deaths per year in the USA of lipid A biosynthesis kills most response to lipid A, which is the (Ref. 1). Over the past two dec- Gram-negative bacteria, increases bacterial active component of LPS endoades, many bacteria, including permeability to antibiotics and decreases toxins, leads to Gram-negative Gram-negative species, have endotoxin production. septic shock1,8, a syndrome characquired resistance to the diacterized by increased vascular verse antibiotics once thought T.J.O. Wyckoff, C.R.H. Raetz* and J.E. Jackman are permeability, severe hypotenin the Dept of Biochemistry, Duke University to control all infections2. Dursion, multiple organ failure and Medical Center, Durham, NC 27710, USA. death. ing the current revival of anti*tel: +1 919 684 5326, fax: +1 919 684 8885, Recent insights into the bibacterial research, many come-mail: [email protected] ology and chemistry of lipid A panies are continuing to explore new variations of existing antibiotics directed against (Ref. 7) offer new opportunities to address simultawell-studied targets, such as protein, nucleic acid and neously the issues of antibiotic resistance, outer memcell wall biosynthesis. However, bacteria have dem- brane impermeability and Gram-negative septic shock. onstrated their ability to evade earlier versions of such antibiotics2, and resistance to the next generation will Unique properties of lipid A The outer leaflet of the outer membrane of Gramsurely arise. One obstacle to developing new drugs against Gram- negative bacteria is composed mainly of LPS (Ref. 7). negative bacteria is their outer membrane (Fig. 1), which In Escherichia coli, the lipid A anchor of LPS (Fig. 1) acts as a very efficient permeability barrier3,4. Antibiotics is a hexaacylated disaccharide of glucosamine, substisuch as erythromycin, rifampicin and bacitracin, al- tuted with phosphate at the 1 and 4′ positions5,7 (Fig. 2). though active against their targets in Gram-negative bac- The minimal LPS structure required for growth of E. teria, often cannot penetrate the outer membrane3. coli contains five acyl chains on lipid A (Ref. 9) and Even when antibiotics are effective during severe Gram- two Kdo (3-deoxy-D-manno-octulosonic acid) residues negative infections, the lipid A moiety of lipopolysac- (Figs 2,3)7. The biosynthesis of Kdo2-lipid A in E. coli is charide (LPS) (Fig. 1), which may be shed from dying well characterized (Fig. 3)7. The seven enzymes needed bacteria, can cause excessive activation of macrophages to make the key precursor Kdo2-lipid IVA (Fig. 3) are and endothelial cells5–7. The resulting systemic over- encoded by single-copy genes that are required for the Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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Human leukocyte antigens in tuberculosis and leprosy Christian G. Meyer, Jürgen May and Klaus Stark
M
ycobacterium leprae Human mycobacterial infections are NRAMP1, has also been idenand Mycobacterium characterized by a spectrum of clinical and tified and is currently being tuberculosis are the immunological manifestations. Specific analysed for its possible ascausative agents of leprosy and human leukocyte antigen (HLA) factors sociation with susceptibility/ tuberculosis, respectively. Most are associated with the subtypes of leprosy resistance to tuberculosis and individuals exposed to these that develop and the course of tuberculosis leprosy2,3. pathogens mount efficient imafter infection. The identification of For many years, major histomune responses, but individprotective mycobacterial antigens compatibility complex (MHC) uals who do develop disease presented by a broad variety of HLA genes have also been considered exhibit a variety of clinical molecules will have important implications as candidate genes involved in manifestations. for the design of vaccines. conferring susceptibility or proThe manifestations of leptection in animal and human C.G. Meyer*, J. May and K. Stark are in the rosy span a spectrum of clinical/ mycobacterial disease (Box 1). Institute for Tropical Medicine, histological characteristics, In mice infected with M. bovis Spandauer Damm 130, 14050 Berlin, Germany. ranging from the polar paucibacille Calmette–Guérin (BCG), *tel: +49 30 30116 810, fax: +49 30 30116 888, bacillary form, which correMHC genes profoundly affect e-mail: [email protected] sponds to tuberculoid leprosy the antibody repertoire against (TT), to the multibacillary form, which corresponds mycobacterial antigens4. Furthermore, MHC-dependent to lepromatous leprosy (LL). Intermediate forms are antimycobacterial protection to M. bovis BCG has classified as borderline tuberculoid (BT), midborder- been elegantly demonstrated in knockout mice with line (BB) and borderline lepromatous (BL) leprosy. In defined MHC deficiencies: MHC class II-deficient general, disease is characterized by skin and nerve in- Aβ–/– mice succumbed to M. bovis BCG infection, vasion by bacteria, with resulting lesions. Most clinical whereas controls were capable of inhibiting bacterial symptoms of leprosy reflect immune reactions against growth. After infection, bacterial infiltrates of the livantigenic constituents of M. leprae. In TT, a few cir- ers of β2-microglobulin-deficient mice were more difcumscribed skin and nerve lesions are found (Fig. 1a), fuse than those of controls, pointing to an important but marked cellular immune responses limit bacterial role of MHC class I molecules and cytotoxic T cells5. However, the situation is more complex. A model spread. In contrast, LL is a severe systemic disease and can involve multiple organs, including limbs, eyes, nose, derived from studies of twins in Gambia suggests that pharynx and testes (Figs 1b,c). Specific immunosup- the cumulative effect of human non-MHC genes expression in LL allows uncontrolled dissemination of ceeds the contribution of MHC class II genes in immune responses to both purified protein derivative (PPD) of the bacteria. In tuberculosis, bacterial growth and spread are usu- M. tuberculosis and to malarial antigens6. ally suppressed, but impaired control can result in severe disease. The most common clinical manifestation Human leukocyte antigens and associations with is pulmonary tuberculosis (PTB). Dissemination can mycobacterial disease also lead to extrapulmonary tuberculosis and the event- A variety of approaches can be used to study the association of human leukocyte antigen (HLA) factors, or ual involvement of other organs. The observations that particular ethnic groups and other genetic markers, with disease. In family studies, distinct families are more susceptible to leprosy and non-random segregation of HLA factors in diseased tuberculosis than others and that monozygotic twins children, compared with their healthy siblings, indidevelop comparable manifestations of mycobacterial cates HLA association with susceptibility to disease. disease support the view that genetic elements contrib- Segregation patterns are analysed by the lod score staute to determining the course of these infections. There tistic (the log odds ratio between the observed and the is now substantial evidence that host genetic factors in- expected distribution of the genetic markers in chilfluence not only initial susceptibility to mycobacterial dren). In case-controlled population studies, the distriinfections but also the spectrum of clinical and immuno- bution of HLA markers is compared between unrelated logical responses. The Bcg/Lsh/Ity gene of mice con- patients with unambiguous diagnoses and healthy controls susceptibility (Bcgs) and innate resistance (Bcgr) to trols, selected according to age, ethnic group and other Mycobacterium bovis, Mycobacterium lepraemurium demographic factors. The advantages of family studand other mycobacterial species1, with Bcgr being domi- ies are that they exclude biased data resulting from nant over Bcgs. The corresponding human homologue, population heterogeneity and that they may reveal an Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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association between the genetic marker and disease that is not observable at the population level. HLA associations with mycobacterial disease have been extensively studied in M. leprae and, to a lesser degree, in M. tuberculosis infection. Some of the associations have been consistently found in different ethnic groups, but others have not. More recent studies suggest that disease per se is controlled by non-HLA, rather than HLA, genes, which is similar to the observations made with the Bcg gene of mice7. One or more recessive non-HLA genes controlling susceptibility to leprosy per se, and non-lepromatous leprosy in particular, have been described7. However, there is strong evidence that HLA factors are associated with the type and severity of disease in mycobacterial infections (Table 1). Tuberculosis Few family studies have reported HLA associations with PTB. Siblings with the haplotype HLA-A2-B5 have more severe manifestations of PTB than those carrying only one of these alleles8. In multicase families in India, HLA-DR2 is significantly more common in children with PTB than in healthy siblings9. One recent study has shown a strong association of HLA-DR2 with advanced and, in particular, drug-resistant PTB (Ref. 10). Although the HLA-DR2 association has been consistently reported in several population studies (Table 1), another recent study from northern India found no relationship11. Nevertheless, HLA-DR3 has been found at significantly lower frequencies in PTB patients than in controls12. The early findings on HLA-DR and HLA-DQ associations with tuberculosis have been refined using DNAbased HLA-typing. DRB1*1501 and DQB1*0502 have been reported to be more common in PTB patients than in controls13. In this study, two haplotypes were observed at higher frequencies in patients with advanced PTB (Table 1). Other researchers have found increased frequencies of both DRB1*1501 and DRB1*1502 in PTB patients10. One very recent study reports an association between HLA-DQB1*0503 and the development of clinical PTB in Cambodian patients14. This is compatible with the previous observation that the frequent occurrence of HLA-DQ5, haplotypically linked to HLA-DR15(DR2), correlates with fulminant multibacillary tuberculosis13. In contrast, other studies suggest that HLA class II allele distributions do not differ in terms of the observed severity of tuberculosis (for example, Ref. 15).
Fig. 1. (a) Tuberculoid leprosy, showing limited spread of bacteria and circumscribed skin lesions. (b) and (c) Lepromatous leprosy, with multiple confluent skin lesions and unrestricted spread of bacteria.
The findings on HLA-DR and HLA-DQ associations with leprosy have also been further resolved by DNAbased HLA-typing. DRB1*1501, DRB1*1502 and DQB1*0601 occur more frequently in all forms of leprosy than in controls, but the associations are stronger with LL than with TT (Ref. 24). In the same study, DQA1*0103 was associated with LL, and DQA1*0102
Leprosy An HLA-A11 association with erythema nodosum leprosum has been described16; however, data on HLA class I associations with leprosy are inconsistent. Nevertheless, convincing results have been obtained from studies on HLA class II associations with leprosy (Table 1). HLA-DR2 is found significantly more frequently in LL and TT patients compared with healthy controls. TT is also associated with the occurrence of HLA-DR317–20, which is found at lower frequencies in LL patients18,20. Furthermore, HLA-DQ1 has often been found to be associated with LL and immunosuppression21–23.
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Box 1. Human leukocyte antigen function and diversity Human leukocyte antigens (HLAs) provide a framework for T-cell recognition of antigenic peptide fragments. Different HLA variants present different peptides, and the HLA system is one of the mediators that, by virtue of its polymorphism, guarantees immunological specificity. The immense variability of HLAs is ancient and pre-dates human speciation. Genetic diversity would not persist without good reason and there is evidence that human MHC genes are still subject to variation. Balancing selection certainly operates, preventing gene loss. The concept of infection-driven selection promoting HLA polymorphism is attractive and is consistent with the primary function of HLA molecules: the presentation of peptide antigens in the fight against microorganisms. HLA heterozygosity increases the functional repertoire. Fluctuating patterns of lethal epidemics, emerging diseases, drug resistance and other factors contribute to selection and have probably modified HLA diversity during different historical episodes and in different geographical regions. Although this statement is speculative, it is supported by observations of epidemics that arose when Mycobacterium tuberculosis, measle viruses and other pathogens were introduced in previously unprimed native populations54,55.
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Table 1. Associations between HLA class II factors and mycobacterial disease Study size (n) HLA class II alleles
Direction of association
Tuberculoid leprosy (TT) DR2 DR2 DR3 DR2 DR2; DQw1 DR1; DR2; DRw9; DQw1; DR4; DRw53; DQw3 DRB1*1501; DRB1*1502; DQB1*0601; DQB1*0503 DRB1*1502; DQB1*0601; DRB1*1501–DRB5*0101–DQA1*0102–DQB1*0502 DR3 DR3 DR3 DR2 DQw1
Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Negative Positive Positive Positive Positive Positive
Lepromatous leprosy (LL) DR2; DQw1; DRw9; DRw53 DR1; DR2; DRw9; DQw1; DR4; DRw53; DQw3 DR2 DR2; DRw8; DQw1 DRB1*1501; DRB1*1502; DQA1*0103; DQB1*0601 DRB*1501; DRB5*0101; DQB1*0602 DR3 DR3 DR5 DR2 DQw1
Positive Negative Positive Negative Positive Positive Positive Positive Negative Negative Positive Positive Positive
Pulmonary tuberculosis (PTB) DR2 DR2; DRw6 DR2; DQw1; DQw3 DR2 DRB1*1501; DQB1*0502; DRB1*1501–DRB5*0101–DQA1*0103–DQB1*0601; DRB1*1501–DRB5*0101–DQA1*0102–DQB1*0502 DR2; DRB1*1501; DRB1*1502 DR2; DRw53 DR3 DQB1
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Ref.
Indian Egyptian Venezuelan Japanese Thai Korean
56 57 17 21 58 22
28
47
Indian
24
39
46
Indian
13
29 30 32 259 149
92 100 212 813 423
Surinamese Mexican Venezuelan Pooled data Pooled data
18 19 20 59
295
110
Japanese
21
152
155
Korean
23
50 68 65 62 23 35 35 474 417
50 237 47 114 92 212 32 703 472
Turkish Indian Indian Japanese Surinamese Venezuelan Thai Pooled data Pooled data
60 23 24 25 18 20 58 59
Indian Indian
9 61
Indonesian
62
25 multicase families 90 60
Positive Positive Negative Positive
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8 multicase families 15 multicase families 28 multicase families 74 110 32 32 152 155
Positive Positive Negative Positive Negative Positive Positive
was associated selectively with borderline leprosy. The frequency of the haplotype DRB1*1501–DRB5*0101– DQA1*0102–DQB1*0502 is lower in TT patients13. In Japanese leprosy patients, DRB1*1501, DRB1*1502, DRB5*0101, DRB5*0102, DQA1*0102, DQB1*0602 occur at an increased frequency in all patients, but DRB1*1501, DRB5*0101 and DQB1*0602 are only more frequent in LL patients25. Arginine, at positions 13 or 70/71 of the DRβ molecule, is present in large subsets of DRβ alleles and is significantly increased in TT patients26. Recently, particular polymorphic TAP (transporter associated with
Patients
101
64
204 20
404 46
Indian Indian
63 13
153 38 51 48
289 291 54 39
Indian Tuvinian Mongol Mexican Cambodian
10 64 12 14
antigen processing) elements have also been shown to correlate with the occurrence of tuberculosis and leprosy: TAP2-A/F correlates positively with PTB, and TAP2-B correlates with TT (Ref. 27). Limitations It is important to consider the possible limitations of the studies described above. In the earlier studies, the HLA patterns were determined by conventional techniques that might have resulted in a lower resolution of HLA types. The development of DNA-based HLA-typing methods has improved the validity of HLA-typing.
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In some studies, valid HLA associations might not have been detected because of small sample sizes and a lack of statistical power. The high number of HLA factors requires multiple statistical testing and increases the possibility that an apparent disease association may be found by chance. Some studies correct for multiple testing (Bonferroni correction), but this conservative approach may underestimate true associations. A substantial body of evidence now exists that specific HLA types are associated with mycobacterial disease. Associations of DR2 and DQ1 with LL, DR2 with PTB, and DR3 with TT have been described in different ethnic groups. However, other associations have not been confirmed. In addition to the methodological limitations, this may result from the enormous HLA polymorphism and the variable distributions of HLA alleles in different ethnic populations. Associations of nonclassical HLA class I genes with tuberculosis and leprosy have not yet been studied. Immunology HLA-restricted mycobacterial epitopes Little is known about the molecular nature of HLArestricted mycobacterial antigens eliciting either protective or impaired immune responses. A variety of HLA-restricted mycobacterial antigens, including heat-shock proteins (hsps) and secreted fibronectin-binding proteins, are presented to T cells by macrophages and other cells (Fig. 2). Fragments of M. leprae hsp70, hsp65 and hsp18 can induce vigorous CD4+ T-cell-proliferative responses that are restricted by multiple DR molecules during TT and LL (Ref. 28). BCG vaccinees and tuberculosis patients with various DR specificities also respond to M. leprae hsp70. Many T-cell determinants, rather than a few immunodominant epitopes, exist throughout the molecule, and different individuals and populations respond to different determinants29. Although M. leprae hsp70 is similar to its human homologue, human hsp70 does not stimulate M. leprae hsp70-reactive T cells. The response to hsp70 is obviously directed at specific mycobacterial determinants30. Particular epitopes of fibronectin-binding proteins and mycobacterial hsp peptides, such as hsp65 peptide p2-12 (p3-13), bind selectively to HLA-DR3 (Refs 31, 32). Dominance of the TCRBV5 family of TCR genes, with exclusive usage of the TCRBV55S1 segment, is observed in a TT-derived DR3–hsp65 p3-13-restricted Tcell line33. In LL patients, only a few hsp fragments can trigger T cells. Hsp65 induces the release of tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1) but does not activate human mononuclear phagocytes34, indicating that this protein contributes both to host defence and tissue damage in the inflammatory lesions. Mycobacterial lipid antigens (protease-resistant mycobacterial lipid antigens and mycobacterial lipoarabinomannan) also stimulate T-cell responses in the context of HLA-independent (including DMA/B) CD1band CD1c-restriction35–37 (Fig. 2). A common antigenic moiety, presented by CD1b to CD4-/CD8-α/β+ T cells, appears to be shared by many mycobacterial species37.
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In the early stages of mycobacterial infection, HLAunrestricted and HLA class I-restricted γ/δ T-cell proliferation (V-γ 9/V-δ 2) and lysis of infected target cells occur, which is compatible with a superantigenlike phenomenon38,39. Immunosuppression Immunosuppression in mycobacterial infection, particularly in LL, is not fully understood (reviewed in Ref. 40). The fact that macrophages from LL patients can present M. leprae antigen to lymphocytes of healthy compatible siblings argues against defective antigen presentation in immunosuppression. T cells derived from LL patients and cultured in the absence of mycobacteria can be induced to proliferate in response to M. leprae, challenging the concept that M. leprae-specific T cells might be selectively deleted in LL. One possible mechanism underlying suppression is lysis of responder and/or antigen-presenting cells by cytotoxic T lymphocytes (CTLs), a mechanism absent when the cell-mediated response is restored by IL-2 or when cells are precultured before addition of antigen. The existence of suppressor T cells is controversial41; however, antigen-specific CD8 cells in unresponsive LL patients appear to act as T suppressor cells. These cells are HLA-DQ restricted and predominantly produce IL-4 (type 2 CD8 cells)42 (Fig. 2). Suppression can be inhibited by anti-DQ antibodies43. Cytotoxic T cells Mycobacteria also induce HLA class I-restricted CD8+ cells, which leads to killing of infected macrophages44 (Fig. 2). Evidence for this comes from studies in which CD8+ T cells are depleted with monoclonal antibodies, and from experiments with MHC-inbred and β2-microglobulin knockout mice that do not express class I antigens. Following mycobacterial infection, these mice die rapidly. Live M. bovis BCG can efficiently activate CD8+ cells from BCG vaccinees, showing that this T-cell subset plays an essential cytolytic role in the immune response to mycobacterial infection45. In addition to phagolysosomic presentation, HLA class I presentation of mycobacterial antigens can also occur by either circumventing the cytoplasmic pathway or after translocation of antigens from the phagosome46. There are few data on the identity of mycobacterial antigens recognized by CD8+ cells or on the mechanisms of T-cell activation, and only some CTL epitopes have been characterized so far47. T-cell dichotomy Differences in immune responses to M. leprae are reflected by the cytokine production patterns observed at the two extremes of the leprosy spectrum48 (Fig. 2). In TT, a T-helper type 1 (Th1)-like cytokine pattern is observed; mycobacterial spread is restricted, and CD4+ cells and type 1 cytokines [e.g. IL-2 and interferon γ (IFN-γ)] are mainly found in skin lesions (Th1 response). In contrast, in LL, abundant CD8+ cells and the production of IL-4 and IL-10 represent a type 2 (Th2) response. These cytokines inhibit cellular immunity and downregulate Th1 responses. Th2-like cytokine profiles
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Activation Inhibition
HLA molecule Mycobacteria
Antibody
Cytotoxicity
CD4 α/β
?
B CD1b/c IL-10
IFN-γ
IL-4
DR
T γ/δ
DR ? HLA classI I HLA class
M/M HLA class II
DQ
Th2
Anti-DQ Ab
TNF- α
CD8
IFN- γ IL-2
Anti-CD8 Ab Anti-TCR Ab IFN-γ
IL-12 Th1
Protection
Multibacillary disease
CD8 Ts
NK
Paucibacillary disease
Fig. 2. A model for immunological mechanisms in human mycobacterial infection. Green pathways indicate activation, and red pathways represent either inhibition or cytotoxicity. Intracellular mycobacterial antigens (small coloured circles) may or may not be presented in the context of HLA class I or class II molecules (grey boxes), leading to either strong or weak T-cell responses. Bacterial lipid antigen may also be presented to CD4+ T cells in the context of CD1b/c molecules. Activating responses result in CD4+ (including CD4+ γ/δ)- and CD8+-dependent killing of infected macrophages. Activation of T suppressor (Ts) cells inhibits cellular immunity. Several antibodies have been shown to block CD8 +-specific effects. The role of humoral immunity is unclear, but is probably negligible. The dichotomy of T-cell responses provides a basis for understanding the effective or impaired control of mycobacterial infection. T-helper type 1 (Th1) responses and the release of the respective cytokines result in protection or recovery from mycobacterial infection, whereas uncontrolled dissemination of bacteria is associated with a Th2 response involving interleukin 4 (IL-4) and IL-10 release. Balanced Th1–Th2 activation would, presumably, confer adequate protection. Abbreviations: Ab, antibody; B, B cell; CD4, CD4+ T cell; CD8, CD8+ T cell; HLA, human leukocyte antigen; IFN-γ, interferon γ; M/M, macrophage/monocyte; NK, natural killer cell; TNF-α, tumour necrosis factor α.
are found in both CD4+ and CD8+ cells isolated from LL patients. Patients with tuberculous pleuritis and a favourable prognosis have high concentrations of IFN-γ, IL-2 and their respective mRNAs in their pleural fluids. This also indicates the central role of Th1 responses associated with protection and recovery49. It is intriguing that mycobacterial antigens, which are associated with particular DR or DQ molecules, can preferentially trigger different Th populations. Thus,
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these antigens favour particular patterns of cytokine production, which eventually lead to the spectrum of responses to mycobacterial infection. The association of HLA-DQ variants with immunosuppression in human leprosy and with advanced multibacillary tuberculosis supports the observation that suppression in vitro is restricted by HLA-DQ (Ref. 50) but not by HLA-DR. Moreover, suppression can be inhibited by anti-DQ antibodies. Similar HLA-DQ associations with
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immunosuppression have also been reported in infections with the helminth Onchocerca volvulus51,52. Antigen-induced suppression can also be inhibited by antibodies specific to polymorphic T cell receptor β chains of CD8+ T-cell clones43. The impairment of immunity in LL, namely the absence of IL-2 and IFN-γ, could result from defective Th1 responses, whereas enhanced B-cell-specific and polyclonal activation and generation of cytotoxic (suppressor) cells could represent an overactivation of Th2 cells. Balanced activation of both T-cell populations would lead to successful defence against mycobacteria. Conclusions Several antigenic peptides triggering different types of immune responses have been identified in human mycobacterial infections. Although the exact mechanisms leading to specific immunosuppression are not completely understood, immunosuppression and progress of disease appear to be controlled by the HLA system. In contrast, innate resistance to mycobacterial infection appears to be dependent on non-HLA genes. However, universal T-cell epitopes of mycobacteria have not yet been characterized, although they have recently been described for the Plasmodium falciparum circumsporozoite protein53. The identification of mycobacterial antigens that fit the molecular shape of a broad variety of HLA molecules and are capable of inducing protective Th1 immune responses may lead to the design of subunit vaccines for both tuberculosis and leprosy. A general strategy for the development of successful vaccines for leprosy requires the identification of the binding motifs of common HLA molecules and the characterization of their anchor positions. This can be achieved by amino acid sequencing of bound peptide fragments (either individual or pooled fragment sequencing), followed by analysis of binding properties of polyalanine analogue peptides. Preparations of cocktails of synthesized peptides would allow binding to different HLA molecules and help to provide vaccine cover for different human populations. In addition, the motif affinity to HLA molecules would need to be validated and accompanied by cellular binding assays and Phase I clinical trials of candidate vaccines to assess the immunogenicity of subunits in several susceptible populations. This approach is currently being planned using computer-based algorithms for the prediction of promiscuous T-cell epitopes. Acknowledgements We thank Rolf Horstmann for encouragement, and Frank Mockenhaupt for valuable comments on the manuscript. References 1 Gros, P., Skamene, E. and Forget, A. (1981) J. Immunol. 127, 2417–2421 2 Cellier, M. et al. (1994) J. Exp. Med. 180, 1741–1752 3 Liu, J. et al. (1995) Am. J. Hum. Genet. 56, 845–853 4 Huygen, K. et al. (1993) Infect. Immun. 61, 2687–2693 5 Ladel, C.H., Daugelat, S. and Kaufmann, S.H.E. (1995) Eur. J. Immunol. 25, 377–384 6 Jepson, A. et al. (1997) Infect. Immun. 65, 872–876
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Questions for future research • Can associations of human leukocyte antigen (HLA) elements, including the association of arginine at DRβ positions 13 or 70/71 and that of the TAP genes, be confirmed in larger studies and in other ethnic groups? • What are the specific non-HLA genes that confer susceptibility and resistance in human mycobacterial diseases? • How do these genes act and what are their products? • What is the precise role of CD8+ and γ/δ T cells in mycobacterial infection? • What are the essential HLA-restricted and unrestricted ‘protective’ antigens? • Which antigens should be used in subunit vaccines?
7 Abel, L. et al. (1989) Int. J. Lepr. Other Mycobact. Dis. 57, 465–471 8 Hafez, M. et al. (1992) Dis. Markers 10, 143–149 9 Singh, S.P. et al. (1983) J. Infect. Dis. 148, 676–681 10 Rajalingam, R. et al. (1996) J. Infect. Dis. 173, 669–676 11 Ghosal, A.E. et al. (1996) J. Indian Med. Assoc. 94, 328–330 12 Cox, R.A. et al. (1988) J. Infect. Dis. 158, 1302–1308 13 Mehra, N.K. et al. (1995) Int. J. Lepr. Other Mycobact. Dis. 63, 241–248 14 Goldfeld, A.E. et al. (1998) J. Am. Med. Assoc. 279, 226–228 15 Sanjeevi, C.B. et al. (1992) Tubercle 73, 280–284 16 Agrewala, J.N. et al. (1989) Tissue Antigens 33, 486–487 17 van Eden, W. et al. (1985) J. Infect. Dis. 151, 9–14 18 van Eden, W. et al. (1982) Hum. Immunol. 4, 343–350 19 Gorodezky, C. et al. (1987) Lepr. Rev. 58, 401–406 20 Ottenhoff, T.H.M. and de Vries, R.R.P. (1987) Int. J. Lepr. Other Mycobact. Dis. 55, 521–534 21 Izumi, S. et al. (1982) Vox Sang. 42, 243–247 22 Kim, S.J. et al. (1987) Tissue Antigens 29, 146–153 23 Rani, R., Zaheer, S.A. and Mukherjee, R. (1992) Tissue Antigens 40, 124–127 24 Rani, R. et al. (1993) Tissue Antigens 42, 133–137 25 Joko, S. et al. (1996) Nippon Rai Gakkai Zasshin 65, 121–127 26 Zerva, L. et al. (1996) J. Exp. Med. 183, 829–836 27 Rajalingam, R., Singal, D.P. and Mehra, N.K. (1997) Tissue Antigens 49, 168–172 28 Mustafa, A.S., Lundin, K.E.A. and Oftung, F. (1993) Infect. Immun. 61, 5294–5301 29 Oftung, F. et al. (1994) Infect. Immun. 62, 5411–5418 30 Adams, E. et al. (1994) Scand. J. Immunol. 39, 588–596 31 van Schooten, W.C. et al. (1989) Eur. J. Immunol. 19, 2075–2079 32 Geluk, A. et al. (1994) Eur. J. Immunol. 24, 3241–3244 33 Struyk, L. et al. (1995) Hum. Immunol. 44, 220–227 34 Peetermanns, W.E. et al. (1994) Scand. J. Immunol. 39, 613–617 35 Porcelli, S., Morita, C.T. and Brenner, M.B. (1992) Nature 360, 593–597 36 Sieling, P.A. et al. (1995) Science 269, 227–230 37 Beckman, E.M. et al. (1996) J. Immunol. 157, 2795–2803 38 Holoshitz, J. et al. (1993) Int. Immunol. 5, 1437–1443 39 Tsuyuguchi, I. et al. (1991) Infect. Immun. 59, 3053–3059 40 Ottenhoff, T.H.M. (1994) Int. J. Lepr. Other Mycobact. Dis. 62, 108–121 41 Bloom, B.R., Modlin, R. and Salgame, P. (1992) Annu. Rev. Immunol. 10, 453–488 42 Mutis, T. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9456–9460 43 Salgame, P., Convit, J. and Bloom, B.R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2598–2602 44 Flynn, J.L. et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12013–12017
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45 46 47 48 49 50 51 52 53 54 55 56
Turner, J. and Dockrell, H.M. (1996) Immunology 87, 339–342 Pfeifer, J.D. et al. (1993) Nature 361, 359–362 Vordermeier, H.M. (1995) Eur. Respir. J. 20, S657–S667 Modlin, R.L. (1994) J. Invest. Dermatol. 730, 828–832 Barnes, P.F. et al. (1993) Infect. Immun. 61, 3482–3489 Sasazuki, T. et al. (1989) Immunology 2, 21–24 Meyer, C.G. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7515–7519 Meyer, C.G. and Kremsner, P.G. (1996) Parasitol. Today 12, 179–186 Calvo-Calle, J.M. et al. (1997) J. Immunol. 159, 1362–1373 Calmette, A. (1931) Presse Med. 2, 17 Goodman, R.M. and Motulsky, A.G. (1979) Genetic Diseases among Ashkenazi Jews, Raven Press van Eden, W. et al. (1980) J. Infect. Dis. 141, 693–701
57 Dessoukey, M.W., el Shiemy, S. and Sallam, T. (1996) Int. J. Dermatol. 35, 257–264 58 Schauf, V. et al. (1985) Tissue Antigens 26, 243–247 59 Todd, J.R., West, B.C. and McDonald, J.C. (1990) Rev. Infect. Dis. 12, 63–74 60 Cem, M. et al. (1988) Int. J. Dermatol. 27, 246–247 61 Singh, S.P. et al. (1983) Tissue Antigens 21, 380–383 62 Bothamley, G.H. et al. (1989) J. Infect. Dis. 159, 549–555 63 Brahmajothi, V. et al. (1991) Tubercle 72, 123–132 64 Pospelov, L.E. et al. (1996) Tubercle Lung Dis. 77, 77–80 65 Bellamy, R. et al. (1998) New Engl. J. Med. 338, 640–644 Note added in proof In a very recent study it has been shown that NRAMP1 polymorphisms affect susceptibility to tuberculosis in West Africans65.
Antibacterial and anti-inflammatory agents that target endotoxin Timna J.O. Wyckoff, Christian R.H. Raetz and Jane E. Jackman
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ram-negative bacteria Antibiotic-resistant bacterial infections are production of cytokines and incause about half of all a major clinical problem. Lipid A, the flammatory mediators, includserious human infecactive part of lipopolysaccharide ing tumor necrosis factor α tions, and complications of seendotoxins in Gram-negative bacteria, is (TNF-α) and interleukin 1β vere Gram-negative sepsis may an intriguing target for new antibacterial (IL-1β), damages the microaccount for as many as 100 000 and anti-inflammatory agents. Inhibition vasculature5–7. A full systemic deaths per year in the USA of lipid A biosynthesis kills most response to lipid A, which is the (Ref. 1). Over the past two dec- Gram-negative bacteria, increases bacterial active component of LPS endoades, many bacteria, including permeability to antibiotics and decreases toxins, leads to Gram-negative Gram-negative species, have endotoxin production. septic shock1,8, a syndrome characquired resistance to the diacterized by increased vascular verse antibiotics once thought T.J.O. Wyckoff, C.R.H. Raetz* and J.E. Jackman are permeability, severe hypotenin the Dept of Biochemistry, Duke University to control all infections2. Dursion, multiple organ failure and Medical Center, Durham, NC 27710, USA. death. ing the current revival of anti*tel: +1 919 684 5326, fax: +1 919 684 8885, Recent insights into the bibacterial research, many come-mail: [email protected] ology and chemistry of lipid A panies are continuing to explore new variations of existing antibiotics directed against (Ref. 7) offer new opportunities to address simultawell-studied targets, such as protein, nucleic acid and neously the issues of antibiotic resistance, outer memcell wall biosynthesis. However, bacteria have dem- brane impermeability and Gram-negative septic shock. onstrated their ability to evade earlier versions of such antibiotics2, and resistance to the next generation will Unique properties of lipid A The outer leaflet of the outer membrane of Gramsurely arise. One obstacle to developing new drugs against Gram- negative bacteria is composed mainly of LPS (Ref. 7). negative bacteria is their outer membrane (Fig. 1), which In Escherichia coli, the lipid A anchor of LPS (Fig. 1) acts as a very efficient permeability barrier3,4. Antibiotics is a hexaacylated disaccharide of glucosamine, substisuch as erythromycin, rifampicin and bacitracin, al- tuted with phosphate at the 1 and 4′ positions5,7 (Fig. 2). though active against their targets in Gram-negative bac- The minimal LPS structure required for growth of E. teria, often cannot penetrate the outer membrane3. coli contains five acyl chains on lipid A (Ref. 9) and Even when antibiotics are effective during severe Gram- two Kdo (3-deoxy-D-manno-octulosonic acid) residues negative infections, the lipid A moiety of lipopolysac- (Figs 2,3)7. The biosynthesis of Kdo2-lipid A in E. coli is charide (LPS) (Fig. 1), which may be shed from dying well characterized (Fig. 3)7. The seven enzymes needed bacteria, can cause excessive activation of macrophages to make the key precursor Kdo2-lipid IVA (Fig. 3) are and endothelial cells5–7. The resulting systemic over- encoded by single-copy genes that are required for the Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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