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(Journal of Leukocyte Biology. 2001;69:138-148.)
© 2001 by Society for Leukocyte Biology

Impaired IL-15 production associated with susceptibility of murine AIDS to mycobacterial infection

Masayuki Umemura*, Kenji Hirose{dagger}, Worawidh Wajjwalku{ddagger}, Hitoshi Nishimura*, Tetsuya Matsuguchi*, Yoshitaka Gotoh§, Masahide Takahashi||, Masahiko Makino# and Yasunobu Yoshikai*

* Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine
{dagger} Department of Bacteriology, National Institute of Infectious Diseases, Tokyo
{ddagger} Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Nakhonpathom, Thailand
§ Department of Veterinary Microbiology, Faculty of Agriculture, Miyazaki University, Japan
|| Center of Excellence, Department of Pathology II, Nagoya University School of Medicine, Japan
# Division of Human Retrovirus, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, Japan

Correspondence: Yasunobu Yoshikai, M.D., Ph.D., Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan. E-mail: yyoshika{at}tsuru.med.nagoya-u.ac.jp


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ABSTRACT
 
LP-BM5 murine leukemia virus (MuLV) injection causes murine AIDS (MAIDS), a disease characterized by many functional abnormalities of immunocompetent cells. We show that MAIDS mice are susceptible to Mycobacterium bovis Bacille Calmette-Guérin (BCG) infection as assessed by survival rate and bacterial counts. The peritoneal exudate macrophages from MAIDS mice produced a significant level of interleukin (IL)-12 soon after inoculation with BCG, whereas IL-15 and tumor necrosis factor (TNF) production were severely impaired in BCG-infected MAIDS mice. The appearance of natural killer (NK) and CD4+ T helper type 1 (Th1) cells specific for mycobacterial antigen were depressed in MAIDS mice after BCG infection. Thus, it appeared that impaired production of IL-15, besides other inflammatory cytokines, in MAIDS mice may be involved in the poor responses of the NK and Th1 cells, resulting in an increased susceptibility to BCG.

Key Words: interleukin-15 • macrophages • interferon-{gamma} • T helper type1 cells • infectious immunity • Mycobacterium bovis BCG


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INTRODUCTION
 
Infection with LP-BM5 murine leukemia virus (MuLV), including replication-competent and replication-defective viruses, causes a fatal immunodeficiency syndrome in mice, known as murine AIDS (MAIDS) [1 2 3 4 5 6 ]. MAIDS is characterized by activation and proliferation of T and B cells, impaired T and B cell functions, an aberrant regulation of cytokine production, hypergammaglobulinemia, decreased natural killer (NK) cell functions, the development of B cell lymphoma, and susceptibility to opportunistic infections [5 6 7 8 ]. There are several lines of evidence that infection with LP-BM5 MuLV, causative of MAIDS, renders mice susceptible to infections with microbes, including Cryptosporidium parvum [8 ], Candida albicans [9 ], Toxoplasma gondii [10 ], Cryptococcus neoformans [11 ], and Leishmania major [12 ]. On the other hand, we have reported that MAIDS mice were relatively resistant to infection with a facultative intracellular bacterium, Listeria monocytogenes, generating Th0-like CD4+ T cells producing both interferon (IFN)-{gamma} and interleukin (IL)-10 [13 ]. Furthermore, the in vivo response of {alpha}ß T cells after injection with bacterial superantigens was normal in MAIDS mice, although the in vitro response of the T cells to superantigen was severely impaired [14 ]. Thus, in vivo immune responses may be somewhat preserved in mice with advanced MAIDS, and the susceptibility of MAIDS mice may differ among infections with various microbes.

Innate immunity mediated by neutrophils, NK, and {gamma}{delta} T cells serves to protect against L. monocytogenes [15 , 16 ]. On the other hand, protection against infection with Mycobacteria, obligatory intracellular bacteria, depends mainly on Th1 cell responses mediated by CD4+ and CD8+{alpha}ß T cells [15 16 17 18 19 ], although several studies suggest that NK and {gamma}{delta} T cells are also involved in protection against mycobacterial infection [20 ]. Mycobacterium is representative of a number of opportunistic pathogens that cause systemic disease in compromised hosts with impaired T cell-mediated host defenses [21 ]. Mycobacterial infection is often disseminated in patients with immunodeficiency diseases, resulting in a reduced life expectancy. Therefore, it is interesting to determine the susceptibility and the immune responses in MAIDS mice after mycobacterial infection.

IL-15 uses a heterotrimeric receptor composed of the ß- and {gamma}-chains of IL-2 receptor (R) and its own specific high-affinity binding {alpha}-chain (designated IL-15R{alpha}) [22 , 23 ]. Because IL-15 binds and signals through IL-2R subunits, this cytokine shares many biological activities with IL-2 [24 , 25 ]. IL-2 is exclusively produced by activated T cells, whereas IL-15 is produced by activated macrophages and epithelial cells [26 27 28 29 30 ]. Recently, Lodelce et al. showed that mice genetically lacking IL-15R{alpha} have no NK cells and a severely reduced number of memory type CD8+ T cells [31 ]. Thus, IL-15 is important for development and/or maintenance of NK cells and memory type CD8+ T cells [31 32 33 ]. There is evidence that IL-15 production is observed during infection with Salmonella [26 ], Mycobacterium tuberculosis [30 ], Toxoplasma gondii [34 ], human immunodeficienty virus (HIV) [35 ], or hepatitis C infection [36 ]. We have recently demonstrated that endogenous IL-15 is responsible for early protection by NK cells against infection with an avirulent strain of Salmonella choleraesuis in mice [37 ]. Thus, it appears that IL-15 has a potential role in host defense against mycobacterial infection.

In this study, we examined the host defense mechanisms to M. bovis BCG during the course of MAIDS. Our results revealed that the susceptibility of MAIDS mice to M. bovis BCG infection was significantly increased, coincident with impaired IL-15 production. The number of NK cells, early IFN-{gamma} production, and generation of purified protein derivative of tuberculin (PPD)-specific Th1 cells were severely depressed in MAIDS mice after M. bovis BCG infection. Impaired IL-15 production could at least partly underlie susceptibility in MAIDS mice to mycobacterial infection.


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MATERIALS AND METHODS
 
Animals
Female C57BL/6 mice, 4 weeks of age, were purchased from Charles River Japan (Hino, Japan). Mice were housed under standard conditions and offered food and water ad libitum.

Microorganisms
LP-BM5 viral stocks were obtained as cell-free supernatants of chronically infected SC-1 cells, clone G6, by cocultivation with uninfected SC-1 cells as described [3 , 4 ]. The titers of ecotropic and mink cell focus-inducing (MCF) MuLV contained in the pool were determined by XC plaque assay in SC-1 cells and by focus induction assay in mink lung cells or by the SC-1 UV-mink assay [38 , 39 ]. The obtained virus pool contained 104 XC plaque-forming units and 102 MCF-inducing units per milliliter. M. bovis BCG, strain Tokyo, was grown in Middlebrook 7H9 medium (Difco, Detroit, MI) supplemented with ADC enrichment (Difco) and Tween 80 (Difco) at 37°C. At the midlog phase the bacteria in the culture were stored in Middlebrook 7H9 medium supplemented with ADC enrichment and Tween 80 and 20% (vol/vol) glycerol at -80°C until used. Mice were inoculated intraperitoneally with 0.1 mL of LP-BM5 viral stock and were infected intraperitoneally 68 days later with 1 x 107 colony-forming units (CFU) of M. bovis BCG. Mice were analyzed individually and compared with age-matched, LP-BM5 MuLV-inoculated or M. bovis BCG-infected control mice.

Antibodies and reagents
Fluorescein isothiocyanate (FITC)-conjugated anti-CD3{varepsilon} monoclonal antibody (mAb) (145-2C11), phycoerythrin (PE)-conjugated anti-TCR{alpha}ß mAb (H57-597), PE-conjugated anti-NK1.1 mAb (PK136), biotin-conjugated anti-TCR{gamma}{delta} mAb (GL3), Cy-chrome-conjugated anti-CD4 mAb (L3T4), FITC-anti-IFN-{gamma} mAb (XMG1.2), FITC-anti-IL-4mAb (BVD4-1D11), FITC-conjugated rat IgG1 isotype control Ig, and FITC-conjugated rat IgG2b isotype control Ig were purchased from PharMingen (San Diego, CA). Streptavidin-RED613TM was purchased from Life Technologies (Gaithersburg, MD). 2.4G2 (anti-Fc{gamma}RII/III-specific mAb, rat IgG1, producing hybridoma) was obtained from American Type Culture Collection (Rockville, MD). Anti-murine IL-15 sera were produced in F344 rats by immunization with an emulsion containing approximately 0.2 mg Escherichia coli-expressed murine IL-15 protein in the form of an insoluble inclusion body preparation in CFA (Difco). A total of three booster injections were given, each at 2-week intervals after primary injection. Two weeks after the last immunization, collected blood from the heart. enzyme-linked immunosorbent assay (ELISA) for IL-15 in individual sera was performed in triplicate using purified anti-mouse IL-15 mAb (G277-3588, PharMingen), biotin-conjugated anti-mouse IL-15 mAb (G277-3960, PharMingen), and peroxidase-conjugated streptavidin (Genzyme Diagnostics, Cambridge, MA).

Bacterial counts
Bacterial counts in the peritoneal cavity, liver, and spleen on days 7, 14, and 28 after M. bovis BCG infection were determined as described [13 , 40 ]. Briefly, peritoneal exudates were obtained from the peritoneal cavity by lavage with 4 mL of Hanks’ balanced salt solution (HBSS) and were serially diluted with HBSS (Nissui, Tokyo, Japan). Serial dilutions of the exudate samples were plated on Middlebrook 7H10 medium (Difco) supplemented with OADC enrichment (Difco) and 20% (vol/vol) glycerol. For enumeration of viable counts in the liver, the liver was perfused with 20 mL of sterile HBSS to wash out bacteria in the blood vessels immediately after mice were bled. Bacterial counts in the liver were measured as described above. The numbers of colonies were determined after incubation for 3 weeks.

Cell preparation
Mice were injected intraperitoneally with 1 x 107 CFU of M. bovis BCG in a volume of 100 µL of phosphate-buffered saline (PBS). Peritoneal exudate cells (PEC) were obtained by lavage of the peritoneal cavity with HBSS on days 7, 14, and 28 after inoculation. PEC were prepared by centrifugation and resuspended in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 10 mM HEPES. Cells were plated and allowed to adhere for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Nonadherent cells were used as mononuclear cells (MNC), and adherent cells were washed several times with HBSS. Adherent cells were collected by scraping with a rubber policeman, washed, and counted. More than 95% of the cells retained by this procedure were macrophages.

Expression of cytokine genes
Total RNA was extracted from adherent PEC at specific times, basically according to the method of Chomczynski and Sacchi [41 ]. First-strand cDNA was synthesized from 2 µg of RNA using reverse transcriptase and 20 pmol of random primer in 20 µL of reaction buffer. Synthesized first-strand cDNA were amplified by polymerase chain reaction (PCR) using 40 pmol of each primer with 2.5 U of AmpliTaq Kit (Takara Shuzo, Kyoto, Japan) in a total volume of 50 µL of reaction buffer consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 0.2 mM dNTP. PCR cycles were run for 1 min at 94°C, 1 min at 54°C, and 30 s at 72°C. Before the first cycle, a denaturation step for 5 min at 94°C was included, and after 35 cycles, the extension was prolonged for 2 min at 72°C. The specific primers were as follows: IL-1{alpha} sense (5’, CTC TAG AGC ACC ATG CTA CAG AC, 3’), antisense (5’, TGG AAT CCA GGG GAA ACA CTG, 3’); IL-6 sense (5’, TGG AGT CAC AGA AGG AGT GGC TAA G, 3’), antisense (5’, TCT GAC CAC AGT GAG GAA TGT CCA C, 3’); IL-15 exon 7–8 sense (5’, GTG ATG TTC ACC CCA GTT GC, 3’), antisense (5’, TCA CAT TCT TTG CAT CCA GA, 3’); tumor necrosis factor (TNF)-{alpha} sense (5’, GGC AGG TCT ACT TTG GAG TCA TTG C, 3’), antisense (5’, ACA TTC GAG GCT CCA GTG AAT TCG G, 3’); ß-actin sense (5’, TGG AAT CCT GTG GCA TCC ATG AAA C, 3’), antisense (5’, TAA AAC GCA GCT CAG TAA CAG TCC G, 3’). The PCR product was subjected to electrophoresis on a 1.0% agarose gel and then was transferred to a Gene Screen Plus filter (DuPont NEN, Boston, MA). The internal oligonucleotide probes were labeled with [{gamma}-32P]ATP using a Megalabel 5’-labeling Kit (Takara Shuzo, Kyoto, Japan) according to the manufacturer’s instructions. The internal oligonucleotide probes were as follows: IL-1{alpha} (5’, AAT GAT GTA AGA ATA CCC AG, 3’), IL-6 (5’, TAG AAA TTC TTC AAG GAT T, 3’), IL-15 exon 7–8 (5’, GCA ATG AAC TGC TTT CTC CT, 3’), TNF-{alpha} (5’, CCA GGT CAC TGT CCC AGC AT, 3’), ß-actin (5’, TTC TGC ATC CTG TCA GCA AT, 3’). Pre-hybridization was performed by incubating the membrane in 1 M NaCl, 1.0% sodium dodecyl sulfate (SDS), and 10% dextran sulfate for 1 h. In hybridization, the filters were incubated in 1 M NaCl, 1.0% SDS, 10% dextran sulfate, and 100 mg/mL heat-denatured salmon sperm DNA with labeled probes for 18 h at 60°C, and then the filters were washed in 2 x SSC, 1.0% SDS for 15 min at 60°C. The radioactivity of each band of PCR product was analyzed with the Fujix BAS2000 Bio-Image analyzer (Fuji PhotoFilm, Tokyo, Japan).

Confocal immunofluorescence microscopy
Peritoneal macrophages were subjected to detection of intracellular IL-15 with a confocal immunofluorescence microscope. Macrophages of PEC from M. bovis BCG-infected control or MAIDS mice on days 0, 7, 14, and 28 were resuspended in RPMI 1640 and adhered on cover slips. Macrophages were fixed in cold acetone for 30 min, then washed three times with PBS. Coverslips were incubated with a solution of 1.0% BSA for 30 min to reduce background levels. The macrophages were reacted with anti-mouse IL-15 rat serum diluted 1:100 in PBS containing 0.1% BSA for 2 h at room temperature, washed in excess PBS, and then reacted with a 1:100 dilution of FITC-conjugated goat anti-rat immunoglobulin in blocking solution for 1 h at room temperature. Fluorescent images were viewed and recorded with the Bio-Rad MRC-series confocal imaging system (Bio-Rad Laboratories, Hercules, CA).

Plastic nonadherent cells of PEC were subjected to an Ag stimulation assay for cytokine production. Nylon wool-passed plastic nonadherent cells of PEC from M. bovis BCG-infected control or MAIDS mice on days 0, 7, 14, and 28 were resuspended in RPMI 1640 and added to 96-well plates at a concentration of 2 x 105 cells/well. Cells were cultured without any stimulation or with 5 µg/mL of PPD (Japan BCG Association, Tokyo, Japan) or with 100 µg/mL of anti-TCR{alpha}ß mAb in the presence of mitomycin-treated splenocytes (2 x 105) from naive mice for 48 h at 37°C. Supernatants were collected and stored at -80°C until the cytokine assay. In some experiments cells were pulsed with [3H]TdR for 6 h before harvesting, then [3H]TdR incorporation was determined by liquid scintillation counting.

Cytokine ELISA
Cytokine levels in the culture supernatant or serum were determined by ELISA. IL-1{alpha}, IL-6, IL-10, IL-12 (p40), and TNF-{alpha} in the culture supernatant and IL-4 and IFN-{gamma} in the serum and culture supernatant were measured with an ELISA commercial kit provided by Toyobo (Tokyo, Japan). Briefly, adherent PEC were recovered and suspended in complete medium at 1 x 107 cells/mL. One hundred microliters of the cell suspension was distributed in each well of the microplate and incubated at 37°C for 24 h. The supernatant was used for the assay. ELISA for IL-15 in individual sera was performed in triplicate using purified anti-mouse IL-15 mAb (capture mAb), biotin-conjugated anti-mouse IL-15 mAb (second mAb), and peroxidase-conjugated streptavidin (detection reagent).

Flow cytometric (FCM) analysis
Plastic nonadherent cells of PEC were pre-incubated with a culture supernatant from 2.4G2 to prevent nonspecific staining. After washing, cells were stained with various combinations of mAbs. Staining with biotin-conjugated mAb was followed by treatment with streptavidin-RED613TM. For three-color analysis of T cell subsets, single-cell suspensions were stained with FITC-conjugated CD3{varepsilon}, PE-conjugated TCR{alpha}ß, NK1.1 mAbs, or biotin-conjugated TCR{gamma}{delta} and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The live lymphocytes were carefully gated by forward and side scattering. The data were analyzed with CELLQuestTM software (Becton Dickinson).

Intracellular cytokine staining
Enriched T cells (2 x 106 cells/mL) were incubated with 25 ng/mL PMA and 1 µg/mL ionomycin for 6 h at 37°C and 5% CO2, with 10 µg/mL brefeldin A (Sigma) added for the last 2 h in 24-well flat-bottomed plates (Falcon, Becton Dickinson, Oxford, UK) in a volume of 1 mL RPMI containing 10% FCS. After 6 h of culture, the cells were harvested, washed once in HBSS containing 2.5% newborn horse serum and 0.1% NaN3 (staining buffer), and surface-stained in staining buffer with Cy-chrome-conjugated anti-CD4 mAb and PE-conjugated anti-NK1.1 mAb. After surface staining, cells were subjected to intracellular cytokine staining using the Fast Immune Cytokine System (Becton Dickinson) according to the manufacturer’s instructions. For intracellular cytokine staining we used FITC-anti-IFN-{gamma}, IL-4 mAbs, or FITC-conjugated rat IgG1 or IgG2b as isotype control. Samples were acquired in a FACSCalibur flow cytometer and analyzed by CELLQuestTM software.

Statistical analysis
The statistical significance of the data was determined by Student’s t test. The statistical significance of the survival rate was determined by the generalized Wilcoxon’s test. P < 0.05 was considered significant.


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RESULTS
 
Susceptibility of MAIDS mice to mycobacterial infection
We first examined the survival of MAIDS mice after co-infection with M. bovis BCG. Survival rates of 10 MAIDS mice, which had been injected intraperitoneally with LP-BM5 MuLV 68 days previously, after a subsequent intraperitoneal infection with 1 x 107 CFU of M. bovis BCG are shown in Figure 1 . We chose 68 days after LP-BM5 MuLV injection for infection with M. bovis BCG because virus-infected mice developed late-stage two to three diseases at this stage [3 , 14 ]. All MAIDS mice died by day 72 after M. bovis BCG infection, whereas more than 50% of uninfected MAIDS mice survived. Age-matched control C57BL/6 mice showed resistance to the challenge with the same dose of M. bovis BCG. Thus, mycobacterial infection significantly shortened the survival times of MAIDS mice (P < 0.005).



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  		Figure 1. The survival rate of MAIDS mice or control mice infected intraperitoneally with Mycobacterium bovis BCG. Mice injected intraperitoneally with LP-BM5 MuLV 68 days previously were challenged intraperitoneally with 1 x 107 CFU of M. bovis BCG (on day 0). The same numbers of sex- and age-matched control mice were simultaneously challenged with M. bovis BCG. Data were obtained from three separate experiments and a representative results was shown. Significantly different from the value for MAIDS mice without infection with mycobacteria (*P < 0.005 by the generalized Wilcoxon’s test).

The bacterial growth in various organs of MAIDS was determined after M. bovis BCG infection. The kinetics of bacterial growth were also examined in the peritoneal cavity and liver of MAIDS mice after an intraperitoneal infection with 1 x 107 CFU of M. bovis BCG. As shown in Figure 2A , the bacterial number in PEC was gradually decreased with time in control mice, whereas the clearance of the bacteria was delayed in MAIDS mice (P < 0.05). The bacterial number in liver of MAIDS mice was significantly higher on days 14 and 28 after M. bovis BCG infection than in control mice (Fig. 2B , P < 0.005). Thus, MAIDS mice showed a severely impaired host defense against mycobacterial infection.



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  		Figure 2. Bacterial growth in MAIDS or control mice after infection with M. bovis BCG. MAIDS mice and age-matched control mice were challenged intraperitoneally with 1 x 107 of M. bovis BCG. The numbers of bacteria recovered from the peritoneal cavity (A) or the liver (B) of infected mice on the indicated days were determined. Data were obtained from at least three separate experiments and were expressed as the mean ± SD of five mice from a representative experiment. Statistical analysis was performed with Student’s t test. Significantly different from the value for control mice infected with mycobacteria (*P < 0.05, **P < 0.005).

Cytokine production by peritoneal macrophages of MAIDS mice after infection with M. bovis BCG
To elucidate the causes of the impaired host defense against M. bovis BCG infection in MAIDS mice, we first examined the absolute numbers of whole PEC and peritoneal adherent cells after an intraperitoneal infection with 1 x 107 CFU of M. bovis BCG. The number of adherent cells was significantly fewer in MAIDS mice on days 7 and 14 after M. bovis BCG infection than in control mice (7.8 ± 3.2 x 105 cells/MAIDS mouse vs. 19.6 ± 5.8 x 105 cells/control mouse on day 7 and 2.6 ± 1.8 x 105 cells/MAIDS mouse vs. 8.6 ± 4.1 x 105 cells/control mouse on day 14, P < 0.05). Thus, the macrophage response to M. bovis BCG infection appeared to be impaired in MAIDS mice.

To examine the qualitative differences in cytokine production by macrophages infected with M. bovis BCG, we next compared the expression levels of several cytokine genes by semiquantitative reverse transcriptase-PCR, including IL-1{alpha}, IL-6, IL-15, and TNF-{alpha} in the adherent PEC of control or MAIDS mice on days 0, 7, 14, and 28 after infection with M. bovis BCG. Typical results from three independent experiments are shown in Figure 3 . The TNF-{alpha} mRNA increased in the adherent PEC of control mice on day 28 after infection with M. bovis BCG. Expression of IL-15 mRNA reached a peak on day 14 after M. bovis BCG infection and then decreased markedly in control mice. On the other hand, the expressions of TNF-{alpha} and IL-15 mRNA were severely impaired in the peritoneal adherent cells from infected MAIDS mice, but the expression levels of IL-1{alpha} and IL-6 mRNA were rather increased in those of MAIDS mice before infection and on day 28 after infection. We further compared cytokine production of IL-10, IL-12 (p40), and TNF-{alpha} in the culture supernatant of macrophages from M. bovis BCG-infected MAIDS mice with that from M. bovis BCG-infected control mice assessed by ELISA (Fig. 4 ). The level of IL-12 production by the peritoneal exudate macrophages was higher on day 14 but lower on day 28 after M. bovis BCG challenge in the MAIDS than control mice. IL-10 and TNF-{alpha} were produced at lower levels in the peritoneal macrophages of MAIDS mice than those in control mice after M. bovis BCG infection. On the other hand, the levels of IL-1{alpha} and IL-6 in the peritoneal macrophages of MAIDS mice were significantly higher than those in control mice on day 28 after infection with M. bovis BCG (P < 0.05; IL-1{alpha}, 271.5 ± 68.4 pg/mL vs. 77.5 ± 11.5 pg/mL; and IL-6, 2,700.0 ± 490.2 pg/mL vs. 1,528.6 ± 418.0 pg/mL).



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  		Figure 3. Expression of cytokine genes in the adherent PEC from MAIDS mice or control mice after an intraperitoneal challenge with M. bovis BCG. Total RNA extracted from adherent PEC pooled from five mice of each group was reverse transcribed and then amplified using primers specific for the cytokines. The signal intensity is presented as relative value to the maximal radioactivity of each cytokine. Data of a representative experiment were shown from three separate experiments.



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  		Figure 4. Kinetics of cytokine production by the peritoneal exudate macrophages in MAIDS mice or control mice after M. bovis BCG challenge. MAIDS mice and control mice were challenged intraperitoneally with 1 x 107 CFU of M. bovis BCG. The concentrations of different cytokines (IL-10, IL-12, and TNF-{alpha}) in culture supernatants of the adherent PEC were determined by ELISA. Data were obtained from at least three separate experiments and were expressed as the means ± SD of five mice of each group from a representative experiment. Statistical analysis was performed with Student’s t test. *Significant difference from the value for control mice infected with M. bovis BCG, P < 0.05.

IL-15 synthesis in the peritoneal macrophages of control mice or MAIDS mice after infection with M. bovis BCG
To compare IL-15 synthesis between MAIDS and control mice after M. bovis BCG infection, we first examined the intercellular distribution of IL-15 by indirect immunofluorescence staining. At various times after infection, macrophages infected with M. bovis BCG were fixed with cold acetone, washed with PBS to exclude nonspecific binding, and incubated with the anti-mouse IL-15 rat serum. A typical staining pattern is shown in Figure 5A . Specific fluorescence became detectable in the macrophages of control mice infected with M. bovis BCG, but not in the macrophages of MAIDS mice. We next examined IL-15 protein levels in the serum in MAIDS and control mice infected with M. bovis BCG. Because recombinant murine IL-15 was not available at present, IL-15 levels were expressed as OD (Fig. 5B) . As shown in Figure 5B 5a significant level of IL-15 was detected in control mice after M. bovis BCG infection, whereas it was only marginally increased in MAIDS mice after M. bovis BCG infection.



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  		Figure 5. Confocal microscopic images of intracellular IL-15 protein in the adherent PEC of MAIDS mice or control mice after M. bovis BCG challenge. MAIDS mice and control mice were challenged intraperitoneally with 1 x 107 CFU of M. bovis BCG. The adherent PEC from control mice (a, c) and MAIDS mice (b, d) were recovered on day 14 of infection with M. bovis BCG for immunofluorescence staining (A). The concentrations of IL-15 in individual sera was determined by ELISA. ELISA for IL-15 was performed in triplicate (B). Data were expressed as the means ± SD of three mice of each group from a representative experiment. Statistical analysis was performed with Student’s t test. **Significant difference from the value for control mice infected with M. bovis BCG, P < 0.01.

Kinetics of NK cells and T cells in the peritoneal cavity of MAIDS mice after infection with M. bovis BCG
To monitor the kinetics of intraperitoneal lymphocyte populations after mycobacterial infection, FCM analysis for the expression of CD3{varepsilon}, TCR{alpha}ß, and NK1.1 was carried out on nonadherent PEC on days 0, 7, 14, and 28. A typical result is shown in Figure 6 , and the data are summarized in Table 1 . The absolute number of CD3- NK1.1+ cells in the peritoneal cavity of control mice and MAIDS mice before infection was 0.78 ± 0.39 x 105 cells/mouse and 2.88 ± 0.38 x 105 cells/mouse, respectively (Table 1) . The number of CD3- NK1.1+ cells in control mice was increased to about 9.3 x 105 cells/mouse on day 7 after M. bovis BCG infection, whereas it was severely reduced in the peritoneal cavity of infected MAIDS mice (Fig. 6A , Table 1 ). On the other hand, the proportion of CD3+ TCR{alpha}ß+ cells in the peritoneal cavity of MAIDS mice was larger than that in control mice after infection with M. bovis BCG (Fig. 6B) .



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  		Figure 6. FCM profile of the T cell subsets in the PEC from MAIDS mice or control mice infected with M. bovis BCG. MAIDS mice and age-matched control mice were infected with 1 x 107 CFU of M. bovis BCG. Nonadherent PEC were stained with various mAbs and then the cells were analyzed by flow cytometry. A typical result from five mice was shown in two-color profile.


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  		Table 1. Lymphocyte Subsets of the PEC from MAIDS Mice or Control Mice after M. bovis BCG Infection

Cytokine production in serum of MAIDS mice after M. bovis BCG infection
We next examined the serum level of IFN-{gamma} and IL-4 after M. bovis BCG infection. As shown in Figure 7 , the IFN-{gamma} level in serum of control mice was maximal on day 14, whereas the level was only marginally increased in serum of MAIDS mice after infection with M. bovis BCG. Serum IL-4 was not detected in either control or MAIDS mice at any stage of M. bovis BCG infection (data not shown).



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  		Figure 7. IFN-{gamma} levels in serum from MAIDS mice or control mice infected with M. bovis BCG. MAIDS mice and control mice were bled from the retroorbital plexus at the indicated times after infection with M. bovis BCG. The concentration of IFN-{gamma} in individual sera was determined by ELISA. Data were obtained from separate experiments and were expressed as the mean ± SD of five mice from a representative experiment. Statistical analysis was performed with Student’s t test. Significantly different from the value for control mice infected with M. bovis BCG (*P < 0.05).

Cytokine production by Ag-stimulated T cells in the peritoneal exudate cells of MAIDS mice after infection with M. bovis BCG
To investigate whether Th1 cells were generated in MAIDS mice during the course of M. bovis BCG infection, T cells were isolated from PEC of MAIDS mice on days 0, 14, and 28 post-intraperitoneal infection with M. bovis BCG and were cultured with or without PPD in the presence of antigen-presenting cell (APC), or on anti-TCR{alpha}ß mAb-coated dishes, and the culture supernatants were examined for IFN-{gamma} release by ELISA. The proliferative response of T cells to anti-TCR{alpha}ß mAb was severely impaired in MAIDS mice compared with control mice (data not shown). However, the T cells from MAIDS mice produced much the same level of IFN-{gamma} in response to TCR{alpha}ß cross-linking as those from control mice (Fig. 8A ). As shown in Figure 8A , T cells from control mice on days 14 and 28 after infection produced a significant level of IFN-{gamma} in response to PPD, suggesting that BCG-specific Th1 cells are generated in control mice after M. bovis BCG infection. On the other hand, IFN-{gamma} production was significantly depressed in the culture supernatants of T cells from MAIDS mice on days 14 and 28 after infection (P < 0.05). To determine population of the responder cells, we utilized cytokine FACS analysis for expression of CD4 and intracellular IFN-{gamma}. As shown in Figure 8B , most of the IFN-{gamma}-producing T cells were of CD4+ NK1.1- phenotype. The relative numbers of IFN-{gamma}-producing CD4+ T cells were small in MAIDS mice compared with those in control mice after M. bovis BCG infection. These results indicated that the generation of CD4+ Th1 cells was severely impaired in MAIDS mice infected with M. bovis BCG.



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  		Figure 8. IFN-{gamma} release and intracellular expression of IFN-{gamma} in the peritoneal exudate T cells from MAIDS mice or control mice after M. bovis BCG infection. MAIDS mice and control mice were challenged intraperitoneally with 1 x 107 CFU of M. bovis BCG. The enriched peritoneal exudate T cells (2 x 105 cells) were cultured with PPD in the presence of mitomycin C-treated spleen cells (1 x 105 cells) from naive mice or an immobilized anti-TCR{alpha}ß mAb for 48 h at 37°C. The concentrations of IFN-{gamma} in the culture supernatants were determined by ELISA (A). Nonadherent PEC were pooled from five mice of each group on day 0, 14, or 28 after infection and cultured with 25 ng/mL PMA and 1 µg/mL ionomycin for 6 h at 37°C, with 10 µg/mL brefeldin A added for the last 2 h. After culture, the cells were surface-stained with Cy-chrome-conjugated anti-CD4 mAb, and then subjected to intercellular cytokine staining with FITC-conjugated anti-IFN-{gamma} mAb (B). Data were obtained from three separate experiments and a representative result is shown. Statistical analysis was performed with Student’s t test. Significantly different from the value for control mice infected with M. bovis BCG (*P < 0.05; NS, not significant).

We further examined IL-4 or IL-10 production by T cells from MAIDS mice infected with M. bovis BCG. T cells from neither control nor MAIDS mice produced IL-4 or IL-10 in response to PPD, suggesting that PPD-specific Th2 or regulatory T (Tr1) cells [42 ] are not generated in MAIDS mice in place of Th1 cells (data not shown).


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DISCUSSION
 
We show that mice with advanced MAIDS are susceptible to mycobacterial infection. IL-15 production by macrophages was severely impaired in MAIDS mice soon after an intraperitoneal infection with M. bovis BCG and concurrently, the serum IFN-{gamma} level and the number of NK cells in the peritoneal cavity were depressed in these mice. The generation of CD4+ Th1 cells was also impaired in MAIDS mice after M. bovis BCG infection. Impaired IL-15 production may be linked to the depressed activation of NK cells and Th1 cells during mycobacterial infection and at least partly underlie the increased susceptibility of MAIDS mice to mycobacterial infection.

Protection against infection by intracellular bacteria such as Mycobacteria mainly depends on cellular immunity mediated by {alpha}ß T cells including CD4+ and CD8+ T cells [15 , 18 , 19 ]. It is reported that C57BL/6 mice infected with LP-BM5 MuLV 4 weeks previously became susceptible to Leishmania amazonesis and showed diminished levels of IFN-{gamma} and high levels of IL-4 and IL-10 [43 ]. On the other hand, Lacroix et al. reported that LP-BM5 MuLV infection did not result in a significant increase in fungal burdens in the lungs or brains after co-infection with Cryptococcus neoformans [11 ]. We have previously reported that MAIDS mice display relative resistance to infection with Listeria monocytogenes [13 ]. Protection against Leishmania infection depends exclusively on CD4+ Th1 cells response [17 ], whereas innate immunity mediated by neutrophils, NK and {gamma}{delta} T cells serves at least partly to protect against L. monocytogenes [15 , 16 ]. Thus, different effector mechanisms used to clear pathogens may explain the difference in susceptibility of MAIDS mice to L. monocytogenes and Mycobacteria. This model of murine retroviral infection would seem suitable for studying opportunistic infection by obligatory intracellular bacteria in immunocompromised animals. Grassi et al. reported that the co-infection of C57BL/6 mice with LP-BM5 MuLV and Mycobacterium avium (MO-1 strain) did not induce an exacerbation of M. avium infection [44 ]. On the contrary, Orme et al. reported that LP-BM5 MuLV infection exacerbated M. avium infection (2-151 strain) [45 ]. We also found that MAIDS mice were susceptible to M. avium (Mino strain) infection [46, and data not shown]. This discrepancy may be due to the different effector mechanism whereby each strain of M. avium is cleared.

IL-15 is a novel cytokine that uses ß and common {gamma} ({gamma}c) of IL-2R for signal transduction, and shares many of the biological activities of IL-2 [22 , 23 , 47 , 48 ]. Mice genetically lacking IL-2Rß [49 , 50 ], {gamma}c [51 ], or IL-15R{alpha} [31 ] do not have NK cells, indicating that IL-15 is a key cytokine in NK cell development. Exogenous IL-15 is reported to regulate NK cell survival and stimulate NK cells to accumulate, proliferate, produce IFN-{gamma}, and exhibit increased cytotoxicity [24 , 32 , 52 ]. The current results revealed that NK cells significantly increased in number in the peritoneal cavity of control mice at the early stage of M. bovis BCG infection, coincident with IL-15 production by the peritoneal macrophages. We have previously reported that neutralization of endogenous IL-15 by administration of anti-IL-15 mAb significantly inhibited the appearance of NK cells after Salmonella infection in mice [37 ]. Thus, it appears that IL-15 helps to protect against mycobacterial infection through the emergence of NK cells. The results of the present study revealed that IL-15 production was severely impaired and concurrently NK population rapidly disappeared in MAIDS mice after M. bovis BCG infection. IL-15 has been recently shown to protect not only the activation-independent apoptosis but also the activation-induced apoptosis of cells [53 , 54 ]. Thus, endogenous IL-15 may prevent NK cells from activation-independent or activation-induced cell death during the course of mycobacteria infection. Impaired IL-15 production in MAIDS mice may be responsible for accelerating the NK cell death, resulting in a sharp decrease in NK cells after mycobacterial infection. Further investigation is needed to elucidate the mechanisms for disappearance of NK cells in MAIDS mice infected with M. bovis BCG.

IL-15 is known to be less active in induction of IFN-{gamma} production [30 , 55 ] than IL-12, which is known to have the ability to induce IFN-{gamma} production by both resting and activated NK and T cells [56 ]. The present results revealed that IL-12 was produced in the peritoneal macrophages of MAIDS mice on day 14 but the production was impaired on day 28 after M. bovis BCG infection. IL-12 is produced by monocytes/macrophages in direct response to bacterial constitutes [57 ]. IL-12 production is also induced by the interaction between activated T cells and APC via CD40-CD40 ligand and IFN-{gamma} promotes the IL-12 production [58 , 59 ]. We speculate that IL-12 may be produced normally by macrophages of MAIDS mice in direct response to M. bovis BCG components at the early stage of the infection, whereas impaired generation of Th1 cells expressing CD40 ligand may affect the IL-12 production in MAIDS mice on day 28 after M. bovis BCG infection. TNF-{alpha}, together with IL-12, was shown to be essential for NK cells to produce IFN-{gamma} [60 ]. We found an impairment of TNF-{alpha} production in MAIDS mice on day 28 after M. bovis BCG infection, suggest that impaired TNF-{alpha} production is also linked to the impaired IFN-{gamma} production. Protective effect of TNF-{alpha} administration, lethal effect of depletion of TNF-{alpha}, TNF receptor by antibody treatment or gene mutation has been observed in mice with several intracellular pathogens [61 ]. Thus, TNF-{alpha} produced by macrophages is important for protection against mycobacterial infection. It has been reported that TNF-{alpha} and IL-12 synergistically stimulate NK and T cell proliferation [62 ]. Hence, it appears that impairment of NK and Th1 responses in MAIDS may also be attributable in part to a production of cytokines other than IL-15.

Mycobacteria sp. are capable of surviving within mammalian host cells and a Th1-type response is essential for the control of such intracellular pathogens [15 ]. Early IFN-{gamma} production by NK cells is thought to be important for CD4+ Th0 cells to develop into CD4+ Th1 cells [17 ]. IL-15 has been recently reported to up-regulate IL-12Rß1 expression on T cells [63 ]. Hence, IL-15 in synergy with IL-12 may also serve to induce a Th1 response by {alpha}ß T cells against mycobacterial infection. Although IL-12 production was detected in MAIDS mice on day 14 after M. bovis BCG infection, serum IFN-{gamma} and CD4+ Th1 cells were depressed at this stage. IL-15 production, which is responsible for response of NK cells to M. bovis BCG infection and for induction of IL-12Rß expression on CD4 Th1 cells, was severely impaired in MAIDS mice after M. bovis BCG infection. Impaired IL-15 production at this stage might underlie at least partly the impaired generation of Th1 cells in MAIDS mice infected with M. bovis BCG.

It remains to be determined why macrophages of MAIDS mice cannot produce IL-15 during mycobacterial infection. IL-15 production is regulated at both the transcriptional and translational level. We have previously reported that the 5’ upstream region of the mouse IL-15 gene contained several consensus motifs for transcription factor binding such as SP-1, NF-{kappa}B, NF-IL-6, gamma-activated site (GAS) and interferon-stimulated response element (ISRE), and that NF-{kappa}B/ISRE binding sites are essential for IL-15 gene transcription [64 65 66 ]. A short isoform of NF-IL-6 has been recently reported to inhibit NF-{kappa}B binding to the promoter sequence [67 ]. We found in the present study that the macrophages from MAIDS mice expressed IL-6 mRNA even before M. bovis BCG infection, suggesting that NF-IL-6 is spontaneously activated in macrophages in MAIDS mice, in which a Th2-biased response could be associated with the disease progression. Hence, it is possible that LP-BM5 infection impairs IL-15 production after M. bovis BCG infection via the inhibition of NF-{kappa}B activation.

In conclusion, we have provided evidence that LP-BM5 infection results in an increased susceptibility to mycobacteria infection. Although the reason for this still remains to be elucidated, an impairment of IL-15 production may be partly responsible for inhibiting the NK cell response and the generation of Th1 cells in MAIDS mice after mycobacterial infection.


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ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from Ministry of Education, Science and Culture of the Japanese Government, JSPS-RFTF97L00703, Ohyama Health Foundation, Inoue Foundation for Science, the Center of Excellence, and Core Research for Evolutional Science and Technology (CREST) Project. We thank Mrs. Itano, Ms. Yamada, Ms. Kato, and Ms.Nishikawa for providing excellent technical support.

Received March 8, 2000; revised August 14, 2000; accepted August 16, 2000.


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