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.

Protective responses against experimental Mycobacterium leprae infection in mice induced by recombinant Bacillus Calmette-Guérin over-producing three putative protective antigen candidates.

The components of Ag85 (Ag85A, Ag85B, and Ag85C) are putative protective antigen candidates against mycobacterial infection. A recombinant Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) over-producing Ag85A, Ag85B, and MPB51 (rBCG/BA51) was constructed. rBCG/BA51 could secrete these antigens at levels more than five times higher than parental BCG. Immunization of C57BL/6 and BALB/c mice with this rBCG reduced the multiplication of Mycobacterium leprae in the foot pads of both strains of mice. The inhibition by rBCG/BA51 was more evident than that by parental BCG.

Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by recombinant Bacillus Catmette-Guérin (BCG).

Immunization of mice with recombinant Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) which over-produces a putative protective antigen candidate, the A component of antigen 85 complex (Ag85A), reduced the multiplication of Mycobacterium leprae in the foot pads of mice. The inhibition by this rBCG (rBCG/85A) was more evident than that with parental BCG. Repeated rBCG/85A immunization significantly could reduce M. leplae multiplication in mice. This is first report of rBCG to control mycobacterial infection in animal model. Therefore, rBCG technique may be useful for the development of a more effective mycobacteria vaccine.

The antigen 85 complex vaccine against experimental Mycobacterium leprae infection in mice.

The proteins in culture filtrate derived from Bacillus Calmette-Guérin (BCG) were examined for protection against infection by Mycobacterium leprae. Immunization with the major secreted proteins, antigen 85 complex (Ag 85) A, B and C, induced effective protective immunity against multiplication of M. leprae in the foot pads of mice. The most effective protection was observed when mice were immunized with Ag 85A. A single immunization with Ag 85 could induce antigen-specific interferon gamma (IFNgamma) synthesis and more effective protection than live BCG vaccine. This study demonstrates that Ag 85 is an important immunoprotective molecule against leprosy infection.

[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.

Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG.

Immunization of mice with the ribosomal fraction from ruptured Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and the culture filtrate reduced remarkably the multiplication of Mycobacterium leprae in the foot pads of mice. This is the first reported case of the protective activity against M. leprae multiplication in mice of the BCG ribosomal fraction and culture filtrate. The inhibition was more evident with the culture filtrate than with the ribosomal fraction. When the ribosomal proteins separated from ribosomal RNA were injected into mice, only slight inhibition was observed. Ribosomal RNA alone did not inhibit at all, in contrast to the conclusion reported by Youmans and Youmans.

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.

Immune response to heat-shock protein correlates with induction of insulitis in I-E alpha d transgenic NOD mice.

To evaluate the correlation between heat-shock protein (HSP) and insulitis, we compared lymphocyte proliferative response to Mycobacterium leprae HSP65 of NOD mice with that of I-E alpha d transgenic NOD (I-E+NOD) mice, which show no insulitis. We found that splenocytes from 15-week-old NOD mice showed a more marked proliferative response to HSP than did those from age-matched I-E+NOD mice (P < 0.05). We then transferred splenocytes from 12-week-old NOD mice into I-E+NOD mice to induce insulitis in the recipients and examined antibody levels against HSP. By 6 weeks posttransfer, insulitis was successfully transferred to four out of five recipients of NOD splenocytes and antibody levels against HSP were significantly higher in the NOD splenocyte-transferred group than in controls, which showed no insulitis (P < 0.01). These results suggest that immune response to HSP correlates with insulitis in NOD mice. Our results support the assertion that HSP is a useful antigen for investigating the etiology of IDDM.

Antibody production to heat shock proteins with Mr 65 kD (HSP65) in cutaneous inflammation: a possible relation to focal infection.

In order to correlate the immunomodulatory roles of homologous heat shock proteins with Mr 65 kD (HSP65) to skin diseases, antibody level to recombinant-HSP65 of Mycobacterium leprae was quantified with enzyme-linked immunosorbent assay (ELISA) in the sera of patients. In psoriasis, an insignificant increase was observed in anti-HSP65 IgG (0.111 +/- 0.053, mean +/- SD in 0D492 nm, n = 22), compared with a normal group (0.080 +/- 0.032, n = 9). However, psoriasis of acute guttate-type (PGA), which is often induced after tonsillar infection, showed a significant increase (0.178 +/- 0.032 n = 4, p <0.001), but psoriasis vulgaris did not (PV) (0.101 +/- 0.053, n = 12), nor generalized psoriasis pustulosa (PP) (0.087 +/- 0.025, n = 6). Similarly, patients with palmoplantar pustulosis (PPP) with tonsillar or periodontal infection showed significantly high anti-H5P65 IgG (0.230 +/- 0.065, n = 7, p <0.0001), compared with only a mild increase in PPP without suspected infectious foci (0.139 +/- 0.066, n = 13, p <0.05). Possible staphylococcal infection in the oral cavity was suggested by an additional ELISA assay to staphylococcal antigen: anti-staphylokinase IgG showed a significant increase in PPP with infectious foci (0.110 +/- 0.028 n = 3, p <0.01) compared with the normal group (0.039 +/- 0.014), while PPP without them showed only a mild change (0.060 +/- 0.017, n = 6, p <0.05). We assume that immunoreaction to H5P65 may be involved in psoriatic skin inflammation associated with focal infection.

Antibodies to 65Kd heat-shock protein were elevated in rheumatoid arthritis.

Antibodies to 65Kd heat-shock protein (hsp) of mycobacterium leprae were measured by enzyme-linked immunosorbent assay (ELISA) in the three immunoglobulin classes in paired sera and synovial fluids of patients with rheumatoid arthritis (RA). Titers of anti-hsp antibody were expressed by optical density (OD) values for sera or indexes (OD values divided by amounts of immunoglobulin in each class) for synovial fluids and for their paired sera. Indexes of anti-hsp antibody were higher in synovial fluids than those in sera at 15/18 for IgG, 17/18 for IgA and 16/18 for IgM class. These results suggest the participation of anti-hsp antibodies to synovitis in RA.

Antibody to 65-kD stress protein (HSP65) of Mycobacterium leprae in various inflammatory skin diseases. A preliminary report.

Cells from prokaryotes and eukaryotes exposed to environmental changes produce a series of highly homologous proteins called stress proteins, or heat shock proteins (HSPs). Recent investigators suggested that the reaction to the shared antigenic epitope between HSPs may link infections with induction of autoimmune processes. In the present study, antibody level to HSP with 65 kDa (HSP65) of Mycobacterium leprae was investigated with enzyme-linked immunosorbent assay (ELISA) in various skin diseases. Comparing to normal group (n = 9) including patients with nevus cell nevus showing 0.097 +/- 0.039 (mean +/- SD) in anti-HSP65 IgG level (OD492), 20 patients with palmoplantar pustulosis (PPP) and 22 with psoriasis demonstrated elevated (0.170 +/- 0.079, 0.111 +/- 0.053, respectively) level. Among them patients judged as focal infection-related PPP or psoriasis showed significantly higher level of anti-HSP65 (p < 0.01) than those without focal infection. Anaphylactoid purpura (0.125 +/- 0.085, n = 5), Behcet disease (0.178 +/- 0.045), atopic dermatitis (0.218 +/- 0.096, n = 13), urticaria (0.185 +/- 0.079, n = 30), and herpes zoster (0.193 +/- 0.092, n = 13) showed likewise elevated anti-HSP65 antibody. Similar tendency was found in anti-HSP65 IgM level but not in anti-HSP65 IgA. Western blotting confirmed specific immunoreaction bands to HSP65 in blood samples with high titer. Immunomodulation by stress proteins of bacterial or host cells is assumed in pathophysiology of inflammatory skin disorders, especially in relation to focal bacterial infection as observed in cases with PPP and psoriasis.

Detection of heat shock protein in patients with insulin-dependent diabetes mellitus.

We have investigated whether antibodies to heat shock protein (hsp) 65 are present in sera from patients with insulin-dependent diabetes mellitus by using Mycobacterium leprae hsp65. Fifty-two sera from patients with IDDM, 36 from patients with unclassified insulin-treated diabetes mellitus and 41 from normal healthy controls were examined by ELISA assay. Seventeen (32.7%) out of 52 IDDM sera and 10 (27.8%) out of unclassified insulin-treated diabetic sera were positive for anti-Mycobacterium (anti-M. leprae) hsp65 antibodies while none of the healthy control sera were positive. Based on western blot analysis, 12 of the 17 IDDM sera and 1 of 2 sera from the unclassified insulin-treated diabetics were positive for anti-M.leprae hsp65 antibodies while all normal control sera were negative. These results support the idea that hsp65 may play a role in the pathogenesis of IDDM. Future studies are necessary to elucidate the role of hsp65 in the pathogenesis of IDDM.

Overproduction, affinity purification and characterization of 65-kDa protein of Mycobacterium leprae in Escherichia coli.

The 65-kDa protein of Mycobacterium leprae was produced in an Escherichia coli strain carrying a plasmid harboring the recloned gene coding for the protein. The protein was purified through affinity chromatography prepared with the IgG fraction of a monoclonal antibody which was prepared against the 65-kDa protein. The purified 65-kDa protein also reacted immunologically with the monoclonal antibody IIIE9, which recognizes the epitope for M. leprae, prepared by Buchanan, et al. BALB/c mice were inoculated with M. leprae and 4 months later were skin tested with the purified 65-kDa protein. Gross changes were observed at the skin-test site. The role of the protein in protective immunity against M. leprae foot pad infection in mice was also studied.