The Ity/Lsh/Bcg gene significantly affects mouse resistance to Mycobacterium lepraemurium.

Mouse resistance to infection with Mycobacterium lepraemurium was measured by counting the total number of intact acid-fast bacilli in the spleen 8 weeks after i.v. injection of a standard inoculation. The effect of Ityr on resistance to M. lepraemurium was confirmed and the results extended to two Ityr strains of mice, A and C57L, not previously tested. Resistance to M. lepraemurium was also examined in the F1, backcross and F2 generations of BALB/c X CBA crosses, and in the congenic strain B10.LLshr that is Ityr. In all experiments the results were consistent with the view that resistance to M. lepraemurium is significantly affected by a gene close to or identical to the Ity/Lsh/Bcg gene on mouse chromosome 1. Sex had a marked effect on resistance to M. lepraemurium, so that the males of some genetically resistant strains were almost as susceptible as some genetically susceptible females.     

Influence of the Ity/Lsh/Bcg gene on the development of suppressor cell precursors in the early phase of the infection of mice with Mycobacterium lepraemurium.

In vitro inducible suppressor cell precursors were detected in the spleen of BALB/c but not in DBA/2 mice infected intraperitoneally with 10(8) Mycobacterium lepraemurium bacilli, thus suggesting that their development is genetically controlled. Two pairs of mouse strains congenic at the Ity/Lsh/Bcg locus (BALB/c-C.D2 and B10.A-B10.A.Bcgr) were used to investigate whether this phenomenon is influenced by this gene known to control the relative susceptibility of mice to Myco. lepraemurium infection. This seems likely, as the detection of culture-induced suppressor activity was delayed for 5-6 weeks in C.D2 and B10.A.Bcgr mice infected intravenously with 10(4) Myco. lepraemurium bacilli. However, despite the retardation in the detection of suppressor cell precursors, the level of in vitro induced suppressor activity at onset in spleen cell suspensions of mice carrying the resistant allele was higher than in cell cultures derived from susceptible mice. As the resistant allele has a different effect when found on BALB/c or DBA/2 background, other genetic factors are apparently involved in the development of suppressor cell precursors. We finally observed that, in spleen cell cultures from intravenously infected Ity/Lsh/Bcg congenic mice on the BALB/c background, adherent and non-adherent cells were required in the inductive phase of suppressor cell development, whereas in vitro induced suppressor activity was found exclusively in the adherent cell fraction. Given these properties, we thus conclude that suppressor cell precursors detected in the spleen of these intravenously infected mice are similar to those previously observed in C3H mice infected intraperitoneally with a thousand times more bacilli.     

Antibacterial resistance in mice infected with Mycobacterium lepraemurium.

The differences in susceptibility among C57Bl/6, DBA/2 mice and their F1 hybrids to infections with M. lepraemurium were shown to depend upon the route of infection and size of the inoculum. A method was developed to measure the ability of lymphocytes obtained from M. lepraemurium-infected donors to effect adoptive immunization of syngeneic naive mice against infection with M. tuberculosis. This required sublethal irradiation of recipient mice prior to cell transfer and bacterial challenge. Using this method, it was found that mice infected subcutaneously generated antituberculous immune mechanisms concordantly with the development of delayed-hypersensitivity to antigens of M. lepraemurium. In contrast, intravenously infected mice demonstrated only a transient from of delayed hypersensitivity and little or no antimycobacterial immunity in that progression of infection was associated with a rapid decay of both these functions. Moreover, during the terminal stage, M. lepraemurium-infected mice lost the ability to control the growth of a sublethal intravenous inoculum of the antigenically unrelated bacterium. Listeria monocytogenes.     

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

H-2 linkage control of resistance to subcutaneous infection with Mycobacterium lepraemurium.

The H-2 linkage of the gene or genes controlling resistance to subcutaneous infection with 10(7) Mycobacterium lepraemurium organisms was investigated by using H-2 congenic strains on BALB and B10 backgrounds. Resistance was assessed by counting the organisms present at the infection site in the footpad and in the draining (right popliteal) lymph node 20 weeks after infection. When mice of BALB and B10 backgrounds with the same H-2 haplotype were compared, the BALB mice were always more susceptible. However, BALB/K (H-2k) mice were more susceptible than BALB/B (H-2b) mice, and BALB/B mice were more susceptible than BALB/c (H-2d) mice. There was no detectable difference in the resistance of B10.D2/n (H-2d) mice and B10 (H-2b) mice, but B10.BR (H-2k) mice were more susceptible than mice of the other two B10 strains. BALB/K was the only strain in which a high proportion of mice showed significant dissemination of organisms to the liver and spleen.     

Suppression of immunity to Mycobacterium lepraemurium infection.

After injection of 10(8) live Mycobacterium lepraemurium (MLM) into the left hind footpad of mice, there is development of local swelling attributable to a granuloma of the cell-mediated immunity type. Concomitant intravenous inoculation of live MLM delays and may even suppress footpad swelling, the effects being proportional to the intravenous dose of organisms. Concomitant footpad infection and intravenous inoculation of 10(9) dead MLM also delays footpad swelling, but over a period of months the feet become excessively swollen. The excessive swelling is due to local enhancement of infection as evidenced by an increase in the number of MLM per footpad. Attempts were made to prevent such immunosuppression by splenectomy or treatment with BCG. Splenectomy was entirely without effect, but 10(7) live BCG administered intravenously 2 to 4 weeks before dead MLM prevented enhancement of infection. The mediator of the immunosuppressive mechanism that results in enhanced infection remains to be elucidated, but it is unlikely to be antibody or immune complexes.     

Genetic regulation of macrophage activation: understanding the function of Nramp1 (=Ity/Lsh/Bcg)

The Nramp1 gene was originally described as Ity/Lsh/Bcg, a single gene controlling resistance and susceptibility of inbred mice to a range of intramacrophage pathogens. Functional studies demonstrated that Ity/Lsh/Bcg had multiple pleiotropic effects on macrophage activation pathways, broadening interest in the gene to include its candidacy as an autoimmune disease susceptibility gene. In 1993 the gene was positionally cloned and found to encode a polytopic integral membrane protein of unknown function. Subsequent studies have localized the protein to late endosomal and lysosomal compartments, and demonstrated that it functions as an iron transporter. Precisely how this function influences macrophage activation pathways is still under investigation, but is likely to include direct effects on pathogen survival in the endosomal/lysosomal compartment as well as influences on intracellular signalling pathways and in regulating mRNA stability. Several studies now provide evidence for a role for NRAMP1 in determining human susceptibility to autoimmune (rheumatoid arthritis. juvenile rheumatoid arthritis, diabetes, Crohn's disease) and infectious (tuberculosis, leprosy) diseases. Amongst these. data are accumulating to support the hypothesis that a functional Z-DNA forming repeat polymorphism in the promoter region of human NRAMP1 contributes directly to disease susceptibility. Four alleles have been observed, alleles 1 and 4 are rare (gene frequencies approximately equal to 0.001), alleles 2 and 3 occur at gene frequencies approximately 0.25 and approximately 0.75, respectively. In the absence of exogenous stimuli, alleles 1, 2 and 4 are poor promoters of gene expression in a luciferase reporter gene system; allele 3 drives high expression. Allele 3 shows allelic association with autoimmune disease susceptibility, allele 2 with infectious disease susceptibility. Hence, balancing selection is likely to be maintaining these two alleles in human populations. Although the association of NRAMP1 with autoimmune disease susceptibility may be related to any one of the multiple pleiotropic effects associated with macrophage activation, the function of NRAMP1 as an iron transporter now prompts more interesting speculation that regulation of iron transport may contribute directly to the disease phenotype in arthritic disease. Patients suffering from rheumatoid arthritis show increased deposition of iron in the synovial membrane, which may contribute to free radical generation and local inflammation. Further analysis of NRAMP1 function will continue to be of importance in understanding the molecular basis to autoimmune and infectious disease susceptibility.     

Macrophage activity in resistant and susceptible mouse strains infected with Mycobacterium lepraemurium.

The level of activation of peritoneal macrophages following subcutaneous inoculation of resistant (C57BL) and susceptible (BALB/c) mice was assessed by monitoring superoxide anion and hydrogen peroxide production and also tumour cell cytostasis. The level of systemic macrophage activation appeared to correlate with bacterial load, rather than resistance to infection. It was observed that the more susceptible (BALB/c) strain developed higher and more sustained levels of systemic macrophage activation, whereas the more resistant (C57BL) strain showed only low transient levels of macrophage activation. In contrast, in vivo challenge of subcutaneously infected C57BL mice, via the intra-peritoneal route, with heat-killed Mycobacterium lepraemurium and thioglycollate resulted in a high level of macrophage activation compared with similarly treated uninfected mice. Similar treatment of susceptible BALB/c mice, however, did not result in enhanced macrophage activation. It was also observed that high levels of macrophage activation occurred in T-cell deprived C57BL mice following infection with M. lepraemurium.     

Enhancement of resistance in Mycobacterium lepraemurium infected C3H mice by treatment with sonicated M. lepraemurium or splenectomy.

C3H mice were first infected in a hind footpad with 10(7) freshly harvested Mycobacterium lepraemurium bacilli. Four weeks later, when a granulomatous reaction was detected at the inoculation site, the animals were treated with two doses of a whole sonicated preparation of M. lepraemurium administered 2 weeks apart in the contralateral footpad. Such treatment was found to prolong the survival time of infected mice by 55-60 days. To study the involvement of the spleen in the immunomodulation of resistance to M. lepraemurium infection, splenectomy was performed in mice prior to infection via two different routes. Splenectomy significantly prolonged the mean survival time of mice infected in the footpad but did not affect survival of those infected intraperitoneally. Treatment of splenectomized footpad infected mice with sonicated bacilli abrogated almost completely the beneficial effect of splenectomy.     

Inbred mouse strains differ in resistance to lethal Coccidioides immitis infection.

Inbred strains of mice were infected intraperitoneally with Coccidioides immitis, and the mean lethal dose was determined after 28 days. DBA/2N mice had a mean lethal dose of greater than 10(5) arthroconidia, whereas BALB/cAnN, C57BL/6N, and C57L/J mice had a mean lethal dose of less than or equal to 10(3). Since both BALB/c and DBA/2 mice are the H-2d haplotype, resistance is not primarily determined by the major histocompatibility locus. Resistance was the dominant phenotype. The pattern of C. immitis-resistant strains does not correspond to the strain distribution of the lsh gene or to the pattern of resistance to Blastomyces dermatitidis or Cryptococcus neoformans. Both resistant and susceptible mice, however, could be successfully immunized with a killed spherule vaccine, and susceptible BALB/cAnN mice were protected from an otherwise lethal infection by prior immunization with an attenuated mutant of C. immitis. Despite the evidence that BALB/cAnN mice could respond to immunization, nonimmune mice did not control the later phase of intraabdominal infection as well as DBA/2N mice. Dissemination of C. immitis to the lung occurred frequently in BALB/cAnN but not in DBA/2N mice. This suggests that BALB/cAnN mice cannot mount an effective immune response to C. immitis during the course of infection.     

Role of the major histocompatibility complex in resistance and granuloma formation in response to Mycobacterium lepraemurium infection.

Resistance to a subcutaneous infection with a moderate dose of Mycobacterium lepraemurium was investigated in C57BL/6 mice and in three congenic strains with the BALB background (BALB/c, BALB/B, and BALB/K). Resistance after 10 weeks of infection was found not to be linked to the major histocompatibility complex. The ability to develop a delayed hypersensitivity response to an ultrasonicate of M. lepraemurium was associated with the background genes, and this ability had no influence on resistance to M. lepraemurium. Granuloma formation at the infection site in the early stages appeared to be linked to the H-2b haplotype. The types of cells involved in the granulomas were also investigated.     

Mitsuda-type lepromin reactions as a measure of host resistance in Mycobacterium lepraemurium infection.

The footpad reaction to autoclaved whole Mycobacterium lepraemurium organisms (MLM lepromin) in high-resistance (C57BL) and low-resistance (BALB/c) mice was studied. Infected C57BL mice gave a prolonged footpad response persisting for 4 weeks after skin testing with high and low doses of lepromin. This was accompanied by mononuclear cell infiltration. Uninfected C57BL mice gave no response. The majority of infected BALB/c mice gave no increase in footpad thickness. However, a high proportion of infected and control BALB/c mice tested with the high dose showed mononuclear cell infiltration which resembled that in C57BL mice. The low dose caused little infiltration in infected or control BALB/c mice. The course of infection in the two strains was different. Dissemination of organisms from the infected footpad was minimal in C57BL mice 5 months after infection. In BALB/c mice, dissemination to the draining lymph node and to some extent to the liver had occurred by 5 months. The draining lymph node of BALB/c mice showed histological evidence of local antibody formation, which uas not found in C57BL mice. On the basis of these findings, it was possible to fit murine leprosy in these two strains into a classification similar to that used for human leprosy.     

A lack of correlation between antigen-specific cellular reactions and resistance to Mycobacterium lepraemurium infection in mice.

Following infection subcutaneously in the footpad with 10(7) Mycobacterium lepraemurium organisms C57BL mice were able to limit multiplication of organisms at the infection site for the 6 months studied and to limit organism spread to the draining lymph node. Large numbers of organisms were present in the footpad and draining lymph node of BALB/c mice at 6 months. In spite of this difference in local immunity the changes in cellular reactivity to specific antigen as assessed by the delayed footpad response and the in vitro proliferative response of draining lymph node cells were similar in the two strains over the time studied.     

H-2-linked genes which modify resistance of C57BL/10 mice to subcutaneous infection with Mycobacterium lepraemurium.

Strains of C57BL/10 mice with recombinants within the H-2 complex were used to map the genes which control the mononuclear cell response at the infection site and modify resistance to subcutaneous infection with Mycobacterium lepraemurium. Strains with b in the K-E beta regions of the H-2 complex mounted a more rapid cellular response in the infected footpad and were more resistant than mice with d or k in the K-E beta regions. Significant differences between strains with k in the K-E beta regions appeared to be controlled by a gene in the D region.