IL-12 and IL-18 synergistically induce the bactericidal activity of murine peritoneal cells against M. leprae.

We examined the effect of IL-12 and IL-18 on bactericidal activities of mouse peritoneal cell (PC) against Mycobacterium leprae (M. leprae). We demonstrated that IL-12 and IL-18 synergistically induced the NO-dependent bactericidal activity of PC by stimulating Natural Killer (NK) cells and T-cells through IFN-gamma production. IL-12 and IL-18 induced host cell death through NK-cells and T-cells. Therefore. IL-12 and IL-18 play an important role on direct killing of intracellular M. leprae and on indirect killing of them through inducing host cell death.

[Bacteria killing by macrophages via NF-IL6 gene dependent mechanism: the susceptibility to Mycobacterium leprae in NF-IL6 knockout mice].

Transcription factor, NF-IL6 recognizes the same nucleotide sequences as C/EBP, and it is predominantly expressed in macrophages. Tanaka et al. reported that NF-IL6 knockout mice are highly susceptible to Listeria monocytogenes and Salmonella typhimurium due to impairment of bacteria killing by activated macrophages. We have tried to see the susceptibility for Mycobacterium leprae infection with intraperitoneal(i.p.) or both hind foot pad (BHF) in the NF-IL6 knockout mice with wild control mice. Although we examined the cytokine genes expression and induction of such as IL-1 alpha, IL-6, IL-12, IL-18/IGIF, NO2. and TNF alpha in the peritoneal macrophages on 1 month after inoculation, also IL-2 and IL-10 by splenocytes on 1 and 8 months after infection. Following the inoculation of M. leprae with i.p. or BHF, the mice were sacrificed from 1 to 12 months after inoculation in order to confirm the multiplication and the dissemination of the infection. Many leprosy bacilli was found in the peritoneal macrophages of NF-IL6 knockout mice on 1 month after inoculation while that of the wild control mice was showing disappear. In the case of the intraperitoneal infection, NF-IL6 knockout mice shows predominantly multiplication of M. leprae on the abdemino-organs such as omentum and also scrotum with male. Although NF-IL6 knockout mice with BHF inoculation did not show any swelling at the site of inoculated foot, however the foot pad on 12 month after inoculation was processed for Fite-Faraco's stain and microscopy shows many leprosy bacilli in the intermuscular layer or around the blood vessels/sciatic nerve in the subcutaneous tissue and then the multiplication extended to the toes. Besides the induction of cytokines such as IL-1 alpha, TNF alpha and IL-12 production were observed stronger in culture supernatant of peritoneal macrophages of NF-IL6 knockout mice than that of the wild control mice. IL-2 production was also observed strong in culture supernatant in splenocytes of NF-IL6 knockout mice while that of IL-10 production never induced at anytime. This is doubtless the results of impairment of bacteria killing by macrophages via NF-IL6 gene dependent mechanism not to antigen specific immune system.

Leprosy in hypertensive nude rats (SHR/NCrj-rnu).

Since more than a decade ago, we have attempted to develop spontaneously hypertensive rats carrying the nude gene that permits high multiplication of Mycobacterium leprae. A congenic strain carrying nude (rnu) and hypertensive genes was produced using SHR/NCrj females and F344/NJcl-rnu males. Cross-intercross was carried out 12 times to establish the hypertensive nude rat congenic strain. As a result of the genetic monitoring test with NE12F2 generation rats, the genetic profile of the SHR/NCrj-rnu rats was the same as that of the SHR/NCrj rats except for the rnu gene. We have successfully developed a hypertensive congenic nude rat strain (SHR.F344Hfh11; SHR/NCrj-rnu). An increase in the blood pressure in nude rats was found to begin at a slightly delayed age when compared with their hairy litter mates. Both female and male rats showed the highest blood pressure at approximately 20 weeks of age--166 +/- 1.4 and 197 +/- 11 mm Hg in nude rats and 175 +/- 11 and 193 +/- 3.2 mm Hg in their hairy litter mates in female and male rats, respectively. In the present study, comparisons were made on the susceptibility to M. leprae in hypertensive SHR/NCrj-rnu and normotensive F344/NJcl-rnu rats. We have reconfirmed that hypertensive SHR/NCrj-rnu rats of the NE12F3 generation were highly susceptible to M. leprae. In the SHR/NCrj-rnu rats of both sexes excellent massive swelling due to multiplication of M. leprae was observed and, also, nodular lesions were produced in uninoculated fore feet and lips while those sites in the F344/NJcl-rnu rats showed only a slight swelling of the inoculated feet with mild nodular lesions. Although mild lymphocyte proliferation was seen only in the M. leprae-inoculated site with numerous bacilli and partial necrosis in the SHR/NCrj-rnu rats, at noninoculated sites, multiplication of M. leprae was only observed in the cells of the mononuclear phagocyte system. However, in F344/NJcl-rnu rats, lymphocyte proliferation with a few neutrophils was seen at the site of inoculated hind foot pads and everywhere at the site of multiplication of M. leprae. There was a wide difference in the susceptibility to M. leprae between the SHR/NCrj-rnu and the F344/NJcl-rnu rats.

Susceptibility to Mycobacterium leprae of ALY (alymphoplasia) mice and IFN-gamma induction in the culture supernatant of spleen cells.

The aly/aly (alymphoplasia) mice from a mutation of a colony of the C57BL/6J mouse strain, which has a systemic absence of lymph nodes and Peyer's patches, are deficient in both T- and B-cell-mediated immune functions. We have undertaken a comparison of susceptibility to Mycobacterium leprae of ALY (aly/aly, aly/+) mice with C57BL/6J mice. The aly/aly mouse was found to have an excellent high susceptibility to M. leprae with no distinction between female and male. The aly/+ mouse also was more susceptible to M. leprae at an earlier stage than the C57BL/6J mouse. Therefore, we examined and compared the cytokine gene expression and gamma interferon (IFN-gamma) induction in the splenocytes of ALY mice. The expression of interleukin 4 (IL-4), IL-10 and IL-12 mRNA was weakly stimulated with ML-lysate in inoculated aly/aly mice but IL-2, IL-6, IGIF/IL-18 and IFN-gamma mRNA were not observed. None of the cytokine genes used appeared, except the mRNA for IL-1-alpha, when uninfected cultured spleen cells were stimulated with ML-lysate. Also, IFN-gamma production was not induced. However, the appearance of these cytokine genes was observed when stimulated with concanavalin A (ConA), and IFN-gamma production was also induced in the culture supernatant by aly/+ and even aly/aly mice stimulated with ConA. To examine the reason why IFN-gamma cannot be produced by splenocytes of ALY mice inoculated with M. leprae, we detected cytokine gene expression and IFN-gamma induction in the presence of recombinant murine IL-12 or IGIF/IL-18. IL-2 mRNA expression was detected in all of the mice tested in the presence of IL-12 but not in aly/aly mice under IGIF/IL-18, and iNOS mRNA expression was not observed in aly/aly mice under IL-12 or IGIF/IL-18. IL-4 and IL-10 mRNA were detected by aly/aly mice only by exposure to IGIF/IL-18. In culture, the supernatant with ML antigens of the aly/aly mice did not produce IFN-gamma in spite of the presence of IL-12 and IGIF/IL-18, while IFN-gamma was weakly induced in aly/+ mice stimulated with ML-lysate and in the presence of IGIF/IL-18. Nevertheless, IFN-gamma production was observed in splenocytes of the aly/aly mice stimulated with ConA and also with IGIF/IL-18 plus anti-CD3 antibody. Our results suggest that ALY mice might be showing a high susceptibility to M. leprae because of deficient priming for activation of T cells with the leprosy bacilli infection. Moreover, it is possible that the phagocytic activities of the macrophages of ALY mice are also impaired.

Cytokine gene expression in the foot pad and spleen of BALB/cAJcl mice infected with M. leprae.

The cytokine mRNAs expressed in the foot pads and spleens of BALB/cAJcl mice infected with Mycobacterium leprae were studied by the reverse transcriptase-polymerase chain reaction (RT-PCR) method using cytokine-specific primers for interleukin-1 alpha (IL-1 alpha), -2, -4, -6, -10, -12-(p40), gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and TNF-beta, and then for CD4 and CD8 markers. The pattern of cytokine gene expression in the foot pad which supports M. leprae growth was different from the expression in the spleen which does not permit M. leprae multiplication in mice. Before BALB/cAjcl mice were infected with M. leprae, IL-1 alpha and TNF-beta mRNAs were expressed physiologically in the foot pad while all of the cytokine genes examined were expressed in the spleen. In the foot pads of mice inoculated with M. leprae, in addition to the physiological appearance of IL-1 alpha and TNF-beta mRNAs, these signals were intensified. TNF-alpha expression was induced by the infection. On the other hand, in the spleens of mice inoculated with M. leprae, CD4 mRNA expression disappeared on day 1 of the infection, which was accompanied by the reduced expression of IL-2, -4, -6, and -12 mRNAs. The recovery of CD4 mRNA expression at a latter stage was accompanied by a corresponding increase of the cytokine mRNA expression. It was suspected that these results might permit restricted growth of M. leprae in the foot pads of normal mice. Furthermore, our study suggests that tissue-specific, local, immunologic characteristics are important in M. leprae growth.

[BCG vaccination to Mycobacterium leprae infection in mice].

BCG vaccine (Tokyo strain) was given in BALB/cA mice intradermally 1 or 3 months before Mycobacterium leprae (M. leprae) challenge as modified Shepard's method. The vaccine dosage was 10(7-8) or 10(6). The BCG gave good protection in both dosages and both challenges against M. leprae infection. Lymphocytes proliferations of BCG-vaccinated splenocyte cultures in response to M. leprae lysate or BCG components (hsp65, 38 kD, 30 kD or 12 kD protein) were tested, and potent proliferative responses were seen in the cultures with M. leprae lysate and hsp65. Furthermore, gamma-IFN productions were positive in the cultures with M. leprae lysate or hsp65, but negative with other antigens. The production of gamma-IFN with hsp65 was never inhibited with polymyxin B, but inhibited with IL-10. These results show that BCG (Tokyo strain) is a useful vaccine for M. leprae infection in mice, and one of the components of BCG, hsp65, may be a effective antigen component for protection of M. leprae infection inducing Th1 type cytokine.

Down regulation of Ia expression in macrophages following incubation with mycobacteria.

Macrophages are known to release cytokines in response to various kinds of stimulators. In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M. lepraemurium, M. intracellulare or M. gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities. It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media. The amount of release was dose dependent on mycobacteria employed. In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria. Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro. More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia. These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria. Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages.

IL-12 regulates T helper type 1 cytokine responses in human infectious disease.

We investigated the role of IL-12 in regulating T cell and cytokine responses in human infectious disease by using the spectrum of leprosy as a model. Tuberculoid patients mount strong T cell responses to Mycobacterium leprae, with production of the type 1 cytokines IL-2 and IFN-gamma in lesions; whereas lepromatous patients manifest weak T cell responses to M. leprae, with production of the type 2 cytokines IL-4 and IL-10 in lesions. We found expression of IL-12 p40 mRNA, as measured by PCR amplification, and IL-12 p70, as measured by immunohistochemistry, to be 10-fold greater in tuberculoid lesions than in lepromatous lesions. The ability of M. leprae to stimulate release of IL-12 from monocytes was inhibited by rIL-4 and rIL-10. M. leprae-induced T cell proliferation in tuberculoid patients was blocked by the addition of neutralizing Abs to IL-12. Furthermore, rIL-12 stimulated proliferation of CD4+ type 1 T cell clones from tuberculoid lesions, but not CD8+ type 2 T cell clones from lepromatous lesions; however, both responded to rIL-2, rIL-12 augmented M. leprae-specific T cell proliferation in lepromatous patients, thereby causing the selective expansion of CD4+ T cells and increasing T cell IFN-gamma production. These data indicate that IL-12 is an important mediator in the generation of the type 1 cytokine response in human infectious disease.

Susceptibility of severe combined immunodeficient (SCID) mice to Mycobacterium leprae: multiplication of the bacillus and dissemination of the infection at early stage.

Inoculation of M. leprae were made into the both hind feet at a dose of 4.8 x 10(6) bacilli per foot in order to determine the susceptibility to M. leprae of SCID mice which is severely deficient in both T- and B cell immunity. SCID mice was found to have an extremely high susceptibility to M. leprae, and the progress of infection observed in the SCID mice were shown a rapid systemic spread of infection at the all over the tissues as well as the growth of the leprosy bacilli at the site of inoculation. Therefore, SCID mice can be used as a suitable multibacillary model for the study of leprosy.

The experimental inoculation with Mycobacterium leprae in autoimmune mice: results of MRL/lpr mice inoculated into the right hind foot (continued).

Using MRL/lpr, NOD and Crj:CD-1 (ICR) mice, inoculations of M. leprae were made into the right hind foot at a dose of 5.8 x 10(6) bacilli per foot in order to study the influence of the immunobiological characteristics of their mice on the growth of M. leprae. To summarize of the results, MRL/lpr mice showed the high susceptibility to M. leprae, while that of NOD mice were poor. In conclusion, the immunobiological characteristics of MRL/lpr, autoimmune mice bearing lpr gene had effect on the multiplication of M. leprae, this mouse is a suitable multibacillary model for the study of leprosy.

The nude mouse as an experimental lepromatous leprosy model: the enhancing effect of thymus cells in infected / ?

In sumary, it may well be conculuded that the nude mouse with thymus cell grafting as a severe lepromatous leprosy model was developed in C57BL/6-nu/nu as well as CF#1-nu/nu, comparing with untreated nude mice. In addtion, comparison of sex in treated group, susceptibility to M. leprae in the some males were higher than in foot swelling of the females, also, difference of individuality occured in both sex at a latter stage after infection, therefore, further investigation is under way to look for preferable nude mouse and hairless-athymic mouse which they are established by ourselves