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.     

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.     

Quantitative effect of H-2-linked gene(s) on antibody response to human gamma-globulin

H-2-linked gene(s) have been found to play a role in the quantitative regulation of response to human gamma-globulin (HGG) in mice selected for high or low antibody responsiveness to sheep erythrocytes. Unexpectedly, in a random genetically heterogeneous population of F2 interline hybrids, the gene(s) linked to the H-2 phenotype of H mice has a "low" effect, and the gene(s) linked to the H-2 phenotype of L mice a "high" effect on the magnitude of antibody response to HGG. In H and L mice, the non-specific polygenic control of antibody responsiveness is able to compensate/counteract the inverse effect of HGG-specific H-2-linked gene(s) since the usual interline difference is observed.     

Influence of Route of Mycobacterium lepraemurium Injection on Susceptibility to Mouse Leprosy and on Lymphoblastic Transformation

Groups of female C57BL/6 and C3H/St mice were inoculated intraperitoneally (i.p.) with 10⁹, 10⁷, and 10⁵ bacilli and into the right hind footpad with 10⁷ and 10⁵ bacilli of Mycobacterium lepraemurium. The incidence of death from leprosy and the mean survival time of leprous mice were recorded. In addition, the blastogenic responses to the T-cell mitogens phytohemagglutinin and concanavalin A and to the B-cell mitogens lipopolysaccharide and dextran sulfate were evaluated at various times during the course of infection in the spleen and peripheral lymph nodes of mice infected with 10⁷ bacilli. When M. lepraemurium was administered i.p., the two strains of mice succumbed to the disease at about the same time, except for the C57BL/6 mice infected with 10⁹ bacilli, which died earlier than the C3H/St mice. Moreover, in both strains of mice, a progressive depression of blastogenesis, first to the T-cell mitogens and then to the B-cell mitogens in the spleen, and to the T-cell mitogens in the peripheral lymph nodes, occurred during the course of the infection, whereas the response to the B-cell mitogens in the nodes increased slowly during the advanced stage of the disease. When 10⁷ and 10⁵ bacilli were injected into the footpad, the C3H/St mice succumbed to the disease at 298 and 344 days, respectively, and the modifications of blastogenesis were similar to those observed in i.p.-infected C3H/St mice. In contrast, the C57BL/6 mice appeared resistant to footpad inoculation of M. lepraemurium, since they lived until the end of the observation period (466 days postinfection) and the depression of blastogenesis was not detectable until 1 year after the infection. Thus, it is concluded that for the C57BL/6 mice (but not for the C3H/St mice), the route of administration of M. lepraemurium can markedly influence the susceptibility or resistance to leprosy.     

Resistance to Mycobacterium lepraemurium is correlated with the capacity to generate macrophage activating factor(s) in response to mycobacterial antigens in vitro.

The kinetics of cell-mediated immunity developed during the course of Mycobacterium lepraemurium infection were determined in resistant (C57BL) and susceptible (BALB/c) mice. Control of M. lepraemurium growth following footpad infection was T-cell dependent in C57BL mice as shown by the finding that T-cell deprived mice had enhanced bacterial counts in the footpad. In contrast, T-cell deprivation did not significantly alter the course of infection in BALB/c mice. However a T-cell dependent inflammatory response, resulting in an increase in size of the infected footpad, occurred in both strains, although it developed slightly later in BALB/c mice. Cells isolated from the lymph nodes, draining the infected foot-pads, were assayed for their proliferative responses to heat-killed M. lepraemurium (HK-MLM) antigens. Although lymph node cells from both mouse strains proliferated to HK-MLM early in the infection (1-2 weeks) both C57BL and BALB/c mice developed diminished in vitro proliferative reactivity within 4-6 weeks post-infection. Supernatants derived from cultures of lymph-node cells that had been stimulated with concanavalin A (Con A) or HK-MLM antigens, were assayed for the presence of macrophage-activating factor (MAF) activity using a tumour cytostasis assay and interferon (IFN) activity using a viral growth inhibition assay. Significantly higher levels of MAF and IFN were found in culture supernatants deprived from HK-MLM stimulated lymph-node cells from infected C57BL mice than from BALB/c mice during the first 8 weeks of infection. However, cells from infected mice of both strains produced similar amounts of both MAF and IFN in response to Con A.     

Application of the Autoanalyser to H-2 Typing in the Mouse

Supported in part by the National Institutes of Health (Contract PH-43-65-992), the Dutch Organization for Applied Research (TNO), the J. A. Cohen Institute for Radiology and Radiation Protection, the Whitehall Foundation, and the Dutch Organization for Pure Research (FUNGO).
The results of testing erythrocytes from eight mouse strains with 20 haemagglutinating antisera and five lymphocytotoxic antisera, using a Technicon Haemagglutinating Autoanalyser, are presented. The suitability of the autoanalyser to regular H–2 typing is proved and the adaptability of this methodology to an investigation of the haemagglutinating capacity of lymphocytotoxic antisera is demonstrated. The implications of specific haemagglutination in the autoanalyser by antisera defining the H–2 'cytotoxic' antigens are discussed.     

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.     

H-2-linked control of in vitro gamma interferon production in response to a 32-kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Guérin.

A 32-kilodalton protein antigen (P32) was previously purified to homogeneity from culture filtrate of Mycobacterium bovis BCG (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Spleen cells from BCG-sensitized mice produce significant amounts of gamma interferon (IFN-gamma) in response to this P32 protein. The amount of secreted IFN-gamma is influenced by mouse genotype, with C57BL/6 (H-2b), C57BL/10 (H-2b), and 129/Sv (H-2b) mice producing about four times more than BALB/c (H-2d), CBF1 (H-2d/b), and DBA/2 (H-2d) mice do. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments indicates a probable H-2-linked control of this IFN-gamma induction, with H-2b cells producing high titers and H-2d cells producing low titers in response to the P32 antigen.     

Elicitation and detection of in vitro H-2-linked Ir gene regulated antibody responses to poly(LTyr, Glu)-poly(DLAla)--poly(LLys)

A microculture system is described in which secreted antibody responses to the synthetic polypeptide (T,G)-A--L were obtained in vitro. Responses were highly reproducible, antigen-dependent, antigen-specific, and under H-2-linked Ir gene control. Critical elements in the system include the schedule of in vivo antigen-priming, removal of the stimulating antigen after 3 days of culture, and a sensitive detection system (double-antibody ELISA). This system should be useful in the analysis of the mechanism of action of Ir genes as well as the mechanisms by which anti-idiotype antibodies modulate immune responses.     

H-2-linked genetic control of antibody response to soluble calf skin collagen in mice

An autosomal dominant immune response gene could be demonstrated in congenic resistant strains of mice which is linked to the H-2 locus and controls the antibody response to soluble calf collagen. High responsiveness was associated with the H-2 alleles, b and f, low responsiveness with the H-2 alleles, d, k, m and r. Studies with calf procollagen, which contains an additional carrier moiety, indicated that these genetic differences might be expressed at the level of T cells.     

Selective reversal of H-2-linked genetic unresponsiveness to lysozymes. II. Alteration in the T helper/T suppressor balance, owing to gene(s) linked to Ir-2, leads to responsiveness in BALB.B

Immune responsiveness to lysozyme in H-2b mice is under the control of H-2-linked Ir genes, with T suppressor (Ts) cells playing a dominant role in strains such as C57BL/6 (B6), C57BL/10 and A.BY. However, non-H-2-genes were found to be capable of specific reversal of the effect of the H-2-linked genes in responsiveness to chicken lysozyme (HEL), but not to human lysozyme (HUL). Therefore, studies were performed to identify any lesion in the suppressor circuit in BALB.B. It was known that HUL-induced suppressor cells could cross-suppress the anti-HEL response in B10.Q mice, which are responsive to HEL but nonresponsive to HUL. Similarly, BALB.B Ts cells were able to suppress the anti-HEL response, using as T helper (Th) source a T cell line (BB-1), derived from HEL-primed BALB.B periaortic and inguinal lymph node cells. A protocol designed to examine the in vivo suppression by the use of HUL-induced suppressor cells also demonstrated a significant suppression of the anti-HEL response. Since the suppressive circuitry seemed intact in the BALB.B, the possibility was examined that a step in T-B cell collaboration was more efficient in this strain than in the B6 nonresponder. With a B6-derived HEL-specific T cell line, BO1H, the B cell and antigen-presenting (B/APC) populations from B6 required addition of concanavalin A supernatant for anti-HEL antibody formation, whereas BALB.B B/APC were capable of responding to HEL in culture without the addition of concanavalin A supernatant. In agreement with this finding, when B/APC cell populations from BALB.B and B6 were compared for their extent of anti-HEL responsiveness, as measured with BB-1 Th cells, BALB.B B/APC populations responded significantly higher than B6 populations when the responses were activated by picogram/nanogram amounts of HEL. The response level of (BALB.B X B6)F1 B/APC measured in the same assay resembled that of B6. However, when HEL was used at the microgram level, both B6 and BALB.B strains responded equivalently. The above data are consistent with the expression of the reversing non-H-2 Ir gene(s) resulting from the balance of antigen presentation to Th and Ts cells in the H-2b mouse. In the B6, processing and handling of antigen may be inefficient in activating response-enhancing Th, and more effective in triggering Ts cells, while the reverse may be true for the BALB.B.     

Local reactivity, local resistance and systemic dissemination in Mycobacterium lepraemurium (MLM) infection.

Local reactivity measured as swelling of the infected footpad, local resistance to bacterial multiplication, and capacity to limit systemic dissemination were studied in C57BL, C3H/Bom, C3H/HeJ, and A/Sn mice inoculated with Mycobacterium lepraemurium. C57BL mice developed a strong local reaction with a sudden onset, and effectively limited local multiplication as well as systemic dissemination of bacteria to the liver and spleen as determined 19 weeks after the inoculation. C3H/Bom mice showed no local reaction, had high numbers of bacteria locally, and had extensive systemic dissemination of the infection. C3H/HeJ mice, on the other hand, developed a small local reaction and had less systemic dissemination of bacteria than C3H/Bom mice. In C57BL mice and in the two C3H substrains local reactivity, local resistance to infection, and resistance to systemic spread of the infection paralleled each other. In contrast, A/Sn mice showed a small local reaction, had the most extensive bacterial multiplication at the site of inoculation of the four mouse strains tested, and at the same time were the mice that most effectively restricted systemic dissemination of the infection. Thus, the mechanisms restricting local bacterial multiplication may be different from the mechanisms limiting bacterial dissemination. Neither bacterial growth locally at the site of subcutaneous inoculation in the footpad, nor systemic dissemination of the infection, followed a mouse strain pattern consistent with the Ity/Lsh/Bcg gene model. In experimental mycobacterial infection both local bacterial growth at the site of inoculation and systemic dissemination should be determined.     

Influence of Lsh, H-2, and an H-11-linked gene on visceralization and metastasis associated with Leishmania mexicana infection in mice

Visceral infection and metastatic lesion development following intravenous or subcutaneous inoculation of Leishmania mexicana amastigotes were examined in different B10 congenic mouse strains carrying alternative alleles at Lsh, H-2, or H-11. The results show that, despite a failure to observe any differences in rates of expansion of primary lesions in mice inoculated subcutaneously, each of these genes could be shown to exert some influence during visceralization and metastatic spread of L. mexicana infection. Of particular interest were (i) the continuous advantage observed throughout 160 to 200 days of infection in Lshr versus Lshs mice, (ii) the association between structural gene polymorphism at H-2 and profound visceral and metastatic spread of the parasite producing disease phenotypes akin to diffuse cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis in humans, and (iii) similar effects observed in mice differing at H-11, the functional basis for which involves modified expression of major histocompatibility complex class II molecules. The results are discussed in relation to the human disease and the possibility that homologs for each of these genes regulate leishmanial infections in humans.     

Influence of H-2 genes on growth of Mycobacterium tuberculosis in the lungs of chronically infected mice.

Mice infected by intraperitoneal injection of Mycobacterium tuberculosis were studied over a 23-week period. They showed progressive infection in the lung (with increasing microbial count and granuloma size) whereas viable bacillary counts remained largely stationary in the spleen and in the liver. The influence of H-2 genes on the progression of the lung infection was studied in four congenic strains of animals with B10 and three congenic strains of animals with BALB backgrounds. H-2k mice had significantly higher bacterial counts in the lung than H-2b mice on both B10 and BALB backgrounds, BALB. K (H-2k) mice were also more susceptible than BALB/c (H-2d) mice. Results with recombinant strains showed that bacillary counts and granulomatous infiltration were lower in the B10 (KbAbE-Db) compared with B10.A(3R) (KbAbEbDd) strain and in B10.A(4R) (KkAkE-Db) compared with B10.BR (KkAkEkDk) mice. This resistance to the late expansion of tuberculous infection in the lungs may be associated with the lack of an expressed I-E molecular or with the expression of the Db molecule.     

Adrenal Function in the Diabetic Mutant Mouse (Gene Symbol dbm)

Naeser , P. Adrenal function in the diabetic mutant mouse (gene symboI dbm). Acta physioi. scand. 1976. 98. 395–399. The adrenal function in diabetes mutant mice with misty coat colour (dbm) was investigated by measurements of serum corticosteroids, adrenal weights and adrenal corticosteroid content. Furthermore, the adrenal corticosteroid content was studied in obese-hyperglycemic mice (ob). In the dbm-mice the serum corticosteroid levels were elevated at the age of 5 and 12 months although the adrenal weight only was significantly elevated at the age of 5 months. The adrenal corticosteroid content was significantly lower in the 12 months old dbm-mice. In the ob-mice the adrenal corticosteroid content was elevated at the age of 5 weeks, 5 and 12 months. It is concluded that in both the dbm-mouse and the ob-mouse there is an increased functional activity of the adrenal cortex which may reflect a pituitary hypersection of ACTH, perhaps as a manifestation of a common hypothalamic disorder.     

Genetic regulation of delayed-type hypersensitivity responses to poly (Tyr,Glu)-poly(DLAla)--poly(Lys): expression of the genetic defect in the induction and manifestation phases in H-2s and H-2f mice.

The genetic defect of H-2s and H-2s non-responder mouse strains in both the induction and manifestation phases of delayed-type hypersensitivity (DTH) responses to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L] was analysed. Utilizing an in vitro system to activate DTH effector T cells, we observed that non-adherent T cells of (H-2f X H-2b) F1 or (H-2s X H-2b)F1 responder mice, could not be activated on antigen bearing adherent cells of H-2f or H-2s haplotypes. On the other hand, these T cells were effectively sensitized on adherent cells derived from either F1 or parental (H-2b) responder mice. These results indicate that in these mouse strains the genetic defect, in the induction phase of DTH, is expressed at the level of the antigen presenting cell. In subsequent experiments, we were able to "correct' the non-responsiveness of H-2s recipients by transfer of educated and irradiated (H-2s X H-2b)F1 T cells together with normal F1 adherent cells. Normal non-adherent and nylon wool enriched T cells failed to restore these responses. Similarly, antigen-pulsed F1 irradiated peritoneal exudate cells could stimulate DTH responses in SJL recipients of (SJL X C57BL/6)F1 (T,G)-A--L educated cells. The genetic defect of H-2s mice in the manifestation phase of the DTH reaction is thus also expressed on the antigen presenting cell.     

H-2-linked control of the Yaa gene-induced acceleration of lupus-like autoimmune disease in BXSB mice.

The accelerated development of lupus-like autoimmune disease in male BXSB mice (H-2b, I-E-) is associated to the presence of a mutant gene, designated Yaa, located on their Y chromosome. To investigate whether the H-2b haplotype and/or the lack of expression of I-E molecules play any role in the Yaa-linked acceleration of autoimmune disease, an I-E+ BXSB.H-2d congenic strain was created by backcross procedures. We compared the development of autoimmune disease in the novel BXSB.H-2d (I-E+) strain to that of BXSB.H-2b (I-E-) and BXSB.H-2b/d (I-E+) heterozygous mice. Male BXSB.H-2d (I-E+) mice exhibited only a limited production of autoantibodies and a lower incidence of glomerulonephritis with a markedly prolonged survival rate, which were essentially identical to those of female BXSB mice of both-H-2b and H-2d haplotypes. However, BXSB.H-2b/d (I-E+) heterozygous males developed an accelerated disease comparable to that of conventional BXSB.H-2b (I-E-) male mice. These results indicate that the expression of I-E molecules and consequent clonal deletion or anergy of I-E reactive T cells does not appear to be responsible for the prevention of accelerated autoimmune disease in BXSB.H-2d (I-E+) male mice. The finding that the Yaa gene-induced acceleration of lupus-like autoimmune disease is modulated by gene(s) within or closely linked to the H-2 complex underlines the crucial role of the major histocompatibility complex and the polygenetic nature of autoimmune disease in BXSB mice.     

Mouse major histocompatibility complex (H-2) and fetal lung development: implications for human pulmonary maturation

Using C57/10Sn (B10, H-2b) and B10.A/SgSn (B10.A,H-2a) congenic mice, we measured 1) the level of endogenous pulmonary corticosterone during mouse development; 2) the degree of lung morphological maturation on gestation day 17, with or without corticosteroid treatment; and 3) the maternal influence on normal lung development and fetal response to corticosteroids. The results of our study indicate that there was a progressive increase in the level of endogenous hormone with time in fetal B10 (H-2b) and B10.A (H-2a) mice; throughout mid- to late gestation, the detectable amount of hormone was almost identical in lungs of both strains. Evaluating the degree of lung maturation by morphometry, B10.A mouse lungs were found to be less mature than B10 mouse lungs. Following corticosteroid treatment on day 12 of gestation, H-2a lungs were equal to or more mature than H-2b lungs. We also compared heterozygous mouse lungs from reciprocal crosses (B10.B10.A, b/a and B10.A.B10, a/b). Mice with a maternally derived H-2a haplotype had less mature lungs than those with a maternally derived H-2b haplotype, suggesting a maternal effect. When exogenous hormone was administered, all heterozygous mouse lungs increased in maturity regardless of the origin of the H-2a haplotype. The treated a/b or b/a lungs were more mature than homozygous b/b and less mature than homozygous a/a lungs. We conclude that progressive lung maturation is associated with a gene(s) at or near the H-2 complex, as is the ability to respond to corticosteroids.     

Specificity of H-2-linked Ir gene control in mice: recognition of defined sequence analogs of (T, G)-A--L.

In order to study the specificity of H-linked Ir gene control in more detail the polypeptide poly(LTyr, LGlu>poly(DLAla)–poly(LLys) ((T, G)-A-L) was modified by replacing the (T, G) copolymers by various defined tyrosine-containing oligopeptides. It was found that the antibody response to all of the analogs of (T, G)-A–L tested was influenced to some extent by H-2-linked Ir gene(s), the response pattern being concordant with that of (T, G)-A–L. Some of the antigens carrying structurally and serologically distinct oligopeptides were as efficient with respect to high/low responder discrimination as (T, G)-A–L, others including the core polypeptide A-L itself were weakly immunogenic and gave only a small high/low responder split. Certain experimental data indicate that in addition to the tyrosine peptides the poly-DL-alanine side chains may play an important role for the recognition of these polypeptides. The possibility that the common response pattern could be due to an Ir gene specific mainly for the DL-alanine peptides is discussed. Specificity of genetic control would then be relatively independent of the serological specificity of the tyrosine peptides. On the other hand, recognition of the analogs and of (T, G)-A–L could be controlled by separate but closely linked Ir genes specific for each of the terminal peptide determinants which probably include the adjacent alanine residues. Genetic factors outside the H-2 complex have in addition to the H-linked Ir genes been found to exert a strong influence on the antibody response to some of the analogs of (T, G)-A–L.