Significance of the antimicrobial resistance gene, Nramp 1, in resistance to virulent Mycobacterium tuberculosis infection

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and eliminate the infection at the sites of acute infection.

localize

References Echtenacher, B., Falk, W., Mlnnel, D.N. & Krammer, P.H. (1990). Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. .I. Immunol., 145, 3762-3766. Echtenacher, B., Falk, W., M%nnel, D.N. & Krammer, P.H. (1993), Survival from cecal ligation and puncture and the formation of fibrous adhesions in the peritoneal cavity depend on endogenous tumor necrosis factor, in “Host defense dysfunction in trauma, shock and sepsis” (E. Faist, J.L. Meakins & F.W. Schildberg) (pp. 755758). Springer Verlag, Heidelberg. Echtenacher, B., Hiiltner, L. & Marmel, D.N. (1996a). Cellular and molecular mechanisms of TNF protection in septic peritonitis. J. Inflammation (in press). Echtenacher, B., Mlnnel, D.B. & Hultner, L. (1996b), Critical protective role of mast cells in a model of acute septic peritonitis. Nature (Lond.), 381, 75-77. Egger, D., Geuenich, S., Denzlinger, E., Mailhammer, R., Ehrenreich, H., Dormer, P. & Htiltner, L. (1995), II-4 renders mast cells functionally responsive to endothelin-1. J. Immunol., 154, 1830-1837. Galli, S.J., Gordon, J.R. & Wershil, B.K. (1991), Cytokine production by mast cells and basophils. Curr. Opin. Immunol., 3, 865-872. Galli, S.J. & Wershil, B.K. (1996), The two faces of the mast cell. Nature (Lond.), 381, 21-22.

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Gordon, J.R. & Galli, S.J. (1990), Mast cells as a source of both preformed and immunologically inducible TNFalpha/cache&in. Nature (Lond.), 346, 274-276. Malaviya, R., Ikeda, T., Ross, E. & Abraham, S.N. (1996), Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature (Lond.), 381, 77-80. Marmel, D.N. & Echtenacher, B. (1993), The role of tumor necrosis factor (TNF) in the host defense mechanisms against infection, in “Mediators of sepsis from pathophysiology to therapeutic approaches” (J.-D. Baumgartner, T. Calandra & J. Carlet) (pp. 63.69), Flammarion, Paris. Ramos, B.F., Qureshi, R., Olsen, K.M. & Jakschik, B.A. (1990). The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice. J. Immunol., 145, 1868-1873. Ramos, B.F., Zhang, Y. & Jakschik, B.A. (1992), Mast cells contribute to fibrin deposition in reverse passive Arthus reaction in mouse skin. Eur. J. Immunol., 22, 2381-2385. Wershil, B.K., Mekori, Y.A., Murakami, T. & Galli, S.J. (1987), 125I-fibrin deposition in IgE-dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells. J. Immunol., 139, 26052614. Zhang, Y., Ramos, B.F. & Jakschik, B.A. (1992), Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis. Science, 258, 1957-1959.

Significance of the antimicrobial resistance gene, Nrampl, in resistance to virulent Mycobacterium tuberculosis infection R.J. North (*) and E. Medina The Trudeau Institute, P.O. Box 59, Saranac Lake N.Y. 12983 (USA)

Introduction It has been established for some time (Bradley et al., 1979 ; Plant and Glynn, 1979 ; Skamene et al., 1982) that the superior resistance of some strains of mice over others to infection with certain microbial

Received December (*) Corresponding

including Salmonella typhimurium, Leishmania donovani, and the attenuated vaccine strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), is under the control of an autosomal gene located at a locus on chromosome 1, referred to as the Ity/LsWBcg locus. Mapping the gene to this pathogens,

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locus was achieved by backcrossing experiments with DBA/2 mice homozygous for the dominant resistant allele of the gene and BALB/c mice homozygous for the recessive susceptible allele. These experiments led to the generation of congenitally resistant BALB/c mice that are genetically the same as conventional BALB/c mice except for carrying the Zry/Z.W&g locus of DBA/2 mice (see Mock et al., 1994). The genetic and physical intervals defining the ItynsWBcg locus were recently used to clone several candidate transcriptional units of the gene (Vidal et al., 1993). Because one of these proved to be expressed exclusively by macrophages, it was considered the most likely candidate and was given the name, Nrumpl, for natural resistance-associated macrophage protein (Malo et al., 1994). It was also determined that susceptibility to infection was associated with a substitution of adenine for guanine at base position 169 of the gene, resulting in glycine to aspartic acid replacement in one of the predicted transmembrane domains of the protein. Subsequent elegant experimentation with mice in which this gene had been functionally deleted by homologous recombination showed quite clearly (Vidal et al., 1995) that the resistant allele of Nrampl was responsible for superior natural (preimmunity) resistance to L. donovani, S. typhimurium and BCG. It has been assumed that resistance to virulent M. tuberculosis is under the control of this same gene. Because our laboratory is engaged in analysing immunity to tuberculosis in mice, we considered it important to determine the role of Nrumpl in this immunity. Evidence against a role for Nrampl to M. tuberculosis

in resistance

To investigate the importance of Nrampl in resistance to tuberculosis we examined the ability of BALB/mice (homozygous for Nrampl asp*69), DBA/2 mice (homozygous for NrampIs’Y’69) and congenic C.D2-N20 mice (homozygous for NramplslY1@) to resist infection initiated by a standard lo5 i.v. inoculum of the H37Rv strain of M. tuberculosis. Resistance was measured in terms of the ability to resist bacterial growth in major organs and the length of time the mice survived infection. It was found (Medina and North, 1996) that Nrampl was not involved in resistance to infection with H37Rv. On the contrary, BALB/c mice were more, rather than less, resistant to infection with the virulent pathogen, and C.D2-N20 congenics were resistant like BALB/c. Moreover, the F, progeny of BALB/c and DBA/2 parents were resistant, like BALB/c mice, showing that resistance to infection with the virulent organism is a dominant trait. The superior resistance of BALB/c over DBA/2 mice to infection was manifest as a superior ability

IN IMMUNOLOGY to restrict the growth of H37Rv in their lungs before, and especially after, the time of onset of expression of acquired immunity. Consequently, BALB/c mice contained 2 logs less bacilli in their lungs at day 100 of infection than DBA/2 mice, and this was associated with much slower development of lung pathology. In the liver and spleen, in contrast, both strains of mice were equally capable of controlling infection and of stabilizing pathology. Consequently, the pathology in these organs in both strains was seen as numerous, small, compact granulomas containing very few acid-fast bacilli. Therefore, in keeping with the results of a previous study (Dunn and North, 1995), it is apparent that tuberculosis in the two strains of mice examined in this study is a progressive disease only in the lungs, as is the case in humans, and that death is caused by pulmonary insufficiency. The results of this histopathology study argue against the notion that mice carrying the resistance allele of Nrumpl are more susceptible to M. tuberculosis infection because this allele causes them to generate a more intense inflammatory response against the pathogen at sites of infection, thereby causing more destructive pathology. Similar results were obtained with mice infected via the respiratory route (Medina and North, 1996).

Resistance to virulent M. tuberculosis segregates independently of Nrampl in F, generation mice Further evidence that Nrampl does not determine resistance to infection with virulent M. tuberculosis came from experiments with F, mice that were examined individually for their Nrampl genotype and their resistance to infection with H37Rv (Medina et al., 1996). This involved producing F, progeny from F, mice of BALB/c and DBA/2 parents, typing the mice according to whether they were homozygous for one or another Nrumpl allele, or heterozygous, and determining the length of time that each mouse survived infection caused by the standard i.v. inoculum of H37Rv. The Nrumpl genotype of individual mice was determined by employing a hot-start PCR procedure on genomic DNA using primers specific for the resistance or susceptibility allele of the gene. The results of this study showed that of the 100 F2 mice typed, 22 were homozygous for NrampAaP’69, 26 were homozygous for NrampsLY’69 and 52 were heterozygous, in keeping with Mendel’s first law. It was next revealed that the survival times of these mice following inoculation of H37Rv was not determined by their Nrumpl genotype, in that they died randomly over a 200&y period, regardless of whether they were homozygous for one or another allele or heterozygous. As a positive control for the foregoing experiment, DBA/2, BALB/c and CD2Fl mice were tested for their ability to resist a challenge infection with

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typhimurium, as measured by survival. The results in figure 1 show, as expected from the literature (Plant and Glynn, 1979; Vidal et al., 19954 that mice homozygous for the resistant allele of Nrampl, as well as heterozygous mice, were much more resistant to salmonellosis than mice homozygous for the susceptibility allele of the gene. Results with F, mice (to be published) are in agreement with these findings.

Salmonella

Similar results are obtained with a smaller inoculum and different strain of hf. tuberculosis It has often been stressedby those who work in the field that the influence of Nrampl on BCG infection is best revealed by experiments in which susceptible and resistant mice are given a very small rather than a large inoculum of the organism. It might be argued, therefore, that the foregoing results showing an insignificant role for Nrampl in resistance to infection with M. tuberculosis would not apply to infection caused by smaller i.v. inocula. This proved not to be the case, however, as evidenced by the results of an experiment that examined the resistance of BALB/c and DBA/2 mice against infection caused by a loo-fold lower i.v. inoculum of H37Rv (103) than that normally given. As can be seen in figure 2, the superior anti-M. tuberculosis resistance of Nrampl”” BALB/c mice over Nrampl res DBA/2 mice was even more pro-

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nounced against a lower level of infection, as evidenced by 3 logs fewer bacilli in the lungs of the former mice 100 days postinoculation. This was found (to be published) to be associatedwith much more extensive pathology in the lungs of DBA/2 mice. The apparent superior ability of DBA/2 mice to handle infection in the spleen between days 10 and 50 was not associatedwith lesspathology in this organ. That the same conclusion applies to infection with a different strain of M. tuberculosis is shown in figure 3, where it can be seenthat BALB/c mice displayed more resistance than DBA/2 mice against infection with the virulent Erdman strain, as measured by survival time.

Nrampl reSmice can be more susceptible to infection with BCG and avirulent M. tuberculosis in the lung It needs to be brought to mind that the influence of Nrampl on resistance to BCG infection has been measured almost exclusively in the spleen against small numbers of bacteria over about the first 30 days of infection. In view of the importance of the lung in resistance and susceptibility to tuberculosis, as revealed by the forgoing discussionand published results (Dunn and North, 1995; North, 1995), it seemed important to detemine whether the results

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obtained with BCG would be similar to those obtained with virulent M. tuberculosis, if resistance were measured in the lungs, instead of the spleen, over a longer time period. The results of an experiment that followed the growth of BCG Montreal in the lungs and spleensof DBA/2 and BALB/c mice over 100 days of infection initiated by a lo5 i.v. inoculum is shown in figure 4. It can be seen, in agreement with the results of others, that NrumpreS DBA/2 and mice were more resistant than Nramp’“’ BALB/c mice in the spleen. This was the result of a superior ability of DBA/2 mice to restrict the growth of the organism during the first 30 or so days of infection. However, BALB/c mice proved more resistant to infection in the lungs, as evidenced by a superior ability to resolve infection in this organ after about day 40 of infection. Therefore, there need be no disagreementbetween results obtained with M. tuberculosis and those obtained with BCG, provided resistance is measured in the lungs after the time of onset of expression of specific immunity. An experi-

ment with the avirulent RlRv stain of M. tuberculosis (fig. 5) which does not kill mice even during very protracted infections (Dunn and North, 1996) showed that resistance to infection with this organism is superior in Nrampl SuTBALB/c and C.D2-N20 mice becauseof the superior ability of these strains to control bacterial growth in their lungs.

Discussion There is no doubt that Nrampl plays a key role in the resistanceof mice to infection with certain intracellular microbial pathogens. However, the experimental evidence discussedabove serves to show that Nrumpl does not play a significant role in resistance of mice to infection with virulent strains of M. tuberculosis. This evidence is supported by findings from another laboratory (Nikonenko et al., 1996). It shows that Nrampl resmice are less, rather than more, resistant to infection with A4. tuberculosis, as

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M. tuberculosis Erdmand

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DAYS Fig. 5. The superior resistance of BALB/c mice over DBA/2 and congenic C.D2-N20 mice to infection with virulent M. tuberculosis also applies to infection with the avirulent RlRv strain of the organism. Infection was initiated with ld bacteria i.v. Meansof 5 mice per group per time point.

measured by mycobacterial growth in major organs and by survival time. Moreover, the results presented throw doubt on the importance of NrampZ in resistance to BCG itself. They show that, although Nrampl reS mice proved more capable than NrampS”” mice of retarding the growth of BCG in the spleen, they were lesscapable of controlling BCG growth in the lungs, at least in the case of the Montreal strain of BCG. Therefore, there need be no disagreement between the results obtained here with M. tuberculosis and those obtained elsewhere with BCG. Furthermore, given that in mice, as in humans, tuberculosis is predominantly a disease of the lung, an understanding of why host defencesfail to control infection and infection-induced pathology in this, rather than in other organs, represents a major problem in tuberculosis research. Needlessto say, however, the studies discussedhere did not identify the genesresponsible for the superior resistance of some mouse strains over others to virulent A4. tuberculosis. This is the subject of ongoing research in this laboratory.

Acknowledgements This work was supported by NIH grants HL-51960, AI-37844 and AI-4007 1 and a grant from the Mathers Charitable Foundation.

References Bradley, D.J., Taylor, B.A., Blackwell, J. et al. (1979), Regulations of Leishmania populations within the

host: III. Mapping of the locus controlling susceptibility to visceral leishmaniasis in the mouse. Clin. Exp. Immunol., 37, 7-14. Dunn, P.L. & North, R.J. (1995), Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice. Infect. bnmun., 63, 3428-3437. Dunn, P.L. & North, R.J. (1966), Persistent infection with virulent but not avirulent Mycobucterium tuberculosis in the lungs of mice causes progressive pathology. J. Med. Microbial., 45, 103-I 09.

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Malo, D., Vogan, K., Vidal, S. et al. (1994), Haplotype mappingand sequenceanalysisof the mouseNramp genepredictssusceptibilityto infection with intracellular parasites.Genomics,23, 51-61. Medina E. & North, R.J. (1996), Evidence inconsistent with a role for the Beg genein resistanceto infection with virulent M. tuberculosis. .I. Exp. Med., 183, 1045-1051. Medina, E., Rogerson,B.J. & North, R.J. ( 1996). The Nrampl antimicrobial resistancegene segregates independentlyof resistanceto virulent M. tuberculosis. Immunology, 88, 479-481. Mock, B.A., Holiday, D.L., Cerretti, D.P. et al. (1994), Construction of a series of congenic mice with recombinantchromosome1 regionssurroundingthe genetic loci for resistanceto intracellular parasites (Ity, Lsh, Beg), DNA repair responses (Rep-l), and cytoskeletal protein villin (Vil). Infect. Immun., 62, 325328. Nikonenko,A.S. eral. (1996), Influenceof the mouseBeg, Tbc-I and xid genes on resistance and immune

Macrophages,

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responsesto tuberculosisinfection and efficacy of bacille Calmette-Guerin (BCG) vaccination. Clin. Exp. Immunol.,

104, 37-43.

North, R.J. (1995). Mycobacterium tuberculosis is strikingly morevirulent for mice when given via the respiratory than by the intravenousroute.J. infect. Dis., 173, 1550-1553. Plant, J. & Glynn, A.A. (1979), Locating salmonellaresistance gene on mouse chromosome 1. Clin. Exp. Immunol., 37, 1-6.

Skamene,E., Gros, P., Gorget, A. et al. (1982), Genetic regulation of resistanceto intracellular pathogens. Nature (Lond.), 297, 506-509. Vidal, S.M., Malo, D., Vogan, K. et al. (1993), Natural resistanceto infection with intracellular parasites: identification of a candidategenefor Beg. Cell, 73, 469-485. Vidal, S., Tremblay, M.L., Govoni, G. et al. (1995), The /ty/LWBcg locus:naturalresistance to infection with intracellular parasitesis obrogatedby disruption of the Nrampl gene.J. Exp. Med., 182,655-666.

NK cells and neutrophils in the cytokine loop of Listeriu resistance E.R. Unanue

Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110 (USA)

This paper represents a brief comment on our research on the cellular events in Listeria infection. Our present interest is cent& on the early stageof the infection where the T-independent or cellular innate systemsplays a major role in resistance.Our coverage of referencesis not comprehensive.Our recent reviews have included a detailed analysis of them (Rogers et al, 1995; Tripp and Unanue, 1995; Unanue, 1996). Initially, in studying how the antigen-presenting function of macrophages was regulated, we were impressedby the very rapid and dramatic activation of macrophages during Listeria infection in the mouse (Belier et al., 1980). By 3 days after infection, the phenotype of tissue macrophages rapidly

ReceivedDecember13, 1996.

changed. These changes were particularly evident in the peritoneal macrophages, where its expression of class II MHC molecules shifted from a baseline of about lo-20% to close to 100% of positive macrophages. These macrophages were excellent presenting cells. The increase in class II-bearing macrophages was mostly accounted for by fresh new cells derived from blood (Scher et al., 1982). These early studies eventually identified interferon-y (IFNy) as the key cytokine responsible for this parameter of macrophage activation (for example, Sztein et al., 1984, reviewed in Unanue 1984), and also pointed to the T cell as a major producer of IFNy. Indeed, in transfer experiments, T cells from mice primed to