Differential Expression of Gamma Interferon mRNA Induced by ...

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Jun 20, 2002 - Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center,1 and. Department of Statistics ...
INFECTION AND IMMUNITY, Jan. 2003, p. 354–364 0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.1.354–364.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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Differential Expression of Gamma Interferon mRNA Induced by Attenuated and Virulent Mycobacterium tuberculosis in Guinea Pig Cells after Mycobacterium bovis BCG Vaccination Amminikutty Jeevan,1* Teizo Yoshimura,2 Kyeong Eun Lee,3 and David N. McMurray1 Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center,1 and Department of Statistics, Texas A&M University,3 College Station, Texas 77843, and Laboratory of Molecular Immunoregulation, National Cancer Institute—Frederick, Frederick, Maryland 217022 Received 20 June 2002/Returned for modification 11 September 2002/Accepted 18 September 2002

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-␥) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-␥ from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-␥, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a 32P-labeled probe derived from the cDNA clone. Compared to the IFN-␥ mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-␥ mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-␥ mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4ⴙ T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-␥ mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-␥ mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-␥ mRNA accumulation is currently under investigation. Infection with Mycobacterium tuberculosis remains a major public health problem in many countries, including the United States. Recent reports indicate that one-third of the world’s population is infected with M. tuberculosis (20). Tuberculosis (TB) has become a serious concern mainly because of its occurrence in AIDS patients and also due to the emergence of drug-resistant strains of mycobacteria (49). The only TB vaccine currently available is Mycobacterium bovis BCG, although a wide variability in the efficacy of this vaccine against adult TB has been reported in clinical trials (5). Macrophages and lymphocytes are critical players in the immune response against mycobacteria (25). Both CD4⫹- and CD8⫹-T-cell subsets have been shown to contribute to antimycobacterial immunity (12, 33). In order to mediate an effective immune response against M. tuberculosis, macrophages and T lymphocytes act in concert with the help of costimulatory molecules as well as molecular mediators such as cytokines and chemokines (1). Among various cytokines, both gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) act at the effector level of resistance to mycobacteria (7). It has been demonstrated that TNF-␣ plays an important role in the formation and maintenance of the granuloma and,

along with IFN-␥, activates macrophages to produce effector molecules such as toxic oxygen and nitrogen intermediates (21). Interference with the production of appropriate mediators by lymphocytes and macrophages might be one of the pathogenic mechanisms employed by some mycobacteria (32). Considerable evidence from both in vitro and in vivo studies indicates that the activation of monocytes and macrophages by IFN-␥ plays an important role in the effective restriction as well as the clearance of mycobacteria (11). Culture of human monocytes in the presence of recombinant IFN-␥ reduced the growth of Mycobacterium avium (40). The role of IFN-␥ in macrophage activation and resistance to intracellular pathogens has been demonstrated by using gene knockout mice. Disruption of the IFN-␥ gene in mice infected with M. tuberculosis resulted in exacerbation of disease, progressive and widespread tissue destruction and necrosis with numerous bacteria (11), or reduced expression of class II antigens on macrophages (8). Similarly, targeted disruption of the IFN-␥ receptor gene in mice made them susceptible to lethal M. bovis BCG infection, reduced TNF-␣ production, and decreased production of nitric oxide by macrophages (17–19). There is also evidence that humans with a mutation in the IFN-␥ receptor or the IFN-␥ receptor signal-transducing chain develop disseminated mycobacterial infections, demonstrating the important role of IFN-␥ in the human immune response to mycobacteria (10, 35). Experimental data demonstrate that IFN-␥ has considerable potential in the treatment of multidrug-resistant TB (6).

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Texas A&M System Health Science Center, 407 Reynolds Medical Building, College Station, TX 778431114. Phone: (979) 862-2436. Fax: (979) 845-3479. E-mail address: [email protected]. 354

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Despite these rather extensive studies on the macrophageactivating effects of IFN-␥, it is not yet clear whether human macrophages can be stimulated in vitro to be bactericidal or to produce effector molecules such as toxic nitrogen intermediates (9). Additionally, there is a paucity of information on the role of IFN-␥ in mycobacterial immunity in the well-established guinea pig model of low-dose pulmonary TB (29, 31). We have characterized the course of infection caused by virulent M. tuberculosis as well as the ability of BCG vaccination to protect against virulent challenge (27). The recent availability of guinea pig cytokine and chemokine cDNA clones (3, 42, 47) has made it possible to elucidate some of the mechanisms of BCG vaccine-induced resistance in guinea pigs. For example, our laboratory has reported that spleen cells and macrophages from BCG-vaccinated guinea pigs show enhanced interleukin-1␤ (IL-1␤) and RANTES mRNA responses compared to the cells from naïve animals when stimulated in vitro with living mycobacteria (15). In addition, cells exposed to virulent M. tuberculosis (H37Rv) had a significant reduction in cytokine response compared to those stimulated with the attenuated strain H37Ra (15). We have cloned the guinea pig IFN-␥ cDNA from concanavalin A (ConA)-stimulated spleen cells by using a human IFN-␥ cDNA probe. The cDNA was used to generate a probe for Northern blot analysis to assess mRNA expression in guinea pig cells stimulated in vitro with living mycobacteria. The purpose of the present study was to determine whether the IFN-␥ mRNA response is enhanced after BCG vaccination and whether the response is altered in cells stimulated in vitro with virulent M. tuberculosis. MATERIALS AND METHODS Screening of a guinea pig cDNA library. Construction of cDNA libraries from ConA-stimulated guinea pig spleen cells was previously described (47). A guinea pig genomic library was purchased from Stratagene, La Jolla, Calif. Cloning of cDNA. Approximately 5 ⫻ 105 recombinant phages from the guinea pig spleen cell cDNA library were screened by high-density plaque hybridization with a 32P-labeled human IFN-␥ cDNA probe. Hybridization to nitrocellulose filters was carried out overnight at 37°C in a solution containing 30% formamide, 5⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5⫻ Denhardt’s solution, 1% sodium dodecyl sulfate (SDS), 100 ␮g of heat-denatured sheared salmon sperm DNA/ml, and 106 dpm of the probe/ml. The filters were washed twice with 2⫻ SSC–0.1% SDS at room temperature for 15 min each time and once at 50°C for 30 min. The filters were dried and exposed overnight to XR-5 film (Kodak) with an intensifying screen at ⫺80°C. Phagemids carried within lambda ZAP II recombinants were rescued with helper phage. DNA sequencing was performed directly from double-stranded DNA by the dideoxynucleoside triphosphate chain termination method (38). Animals. Outbred Hartley strain guinea pigs weighing 200 to 300 g were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.). The animals were housed individually in polycarbonate cages in a temperatureand humidity-controlled environment; ambient lighting was automatically controlled to provide 12-h-light–12-h-dark cycles. Animals were given commercial chow (Ralston Purina, St. Louis, Mo.) and tap water ad libitum. All procedures were reviewed and approved by the Texas A&M University Lab Animal Care Committee. Antibodies. The monoclonal antibodies (MAb) to the guinea pig surface markers used in flow cytometry were purchased from Serotec Ltd., Oxford, England. MAb CT5, directed against guinea pig pan T cells (see below), MAb CT7, directed against guinea pig CD4, and MAb CT6, directed against guinea pig CD8, were produced in mice. BCG vaccination. Guinea pigs were vaccinated intradermally with 0.1 ml (103 CFU) of M. bovis BCG (Danish 1331 strain; Statens Seruminstitut, Copenhagen, Denmark) in the left and right inguinal regions. The lyophilized vaccine was reconstituted with Sauton’s medium (Statens Seruminstitut) just before use.

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Preparation of spleen and lymph node cells. The procedures for preparation of spleen and lymph node cells have been described in detail before (15). Briefly, at 4 to 6 weeks postvaccination, guinea pigs from untreated and BCG-vaccinated groups were euthanized by the intraperitoneal injection of 100 mg of sodium pentobarbital (Sleepaway; Fort Dodge Laboratories Inc.)/kg of body weight. The spleens were removed aseptically for the preparation of a single-cell suspension in RPMI medium (Irvine Scientific, Santa Ana, Calif.). The inguinal lymph nodes from the right and left flanks draining the site of BCG vaccination were removed and processed in the same manner. The red blood cells were depleted by using ACK lysing buffer (0.14 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA [pH 7.2 to 7.4]), and the remaining cells were thoroughly washed and then suspended in RPMI medium supplemented with 2 ␮M glutamine (Irvine Scientific), 0.01 mM 2-mercaptoethanol (Sigma, St. Louis, Mo.), 100 U of penicillin (Irvine Scientific) per ml, 100 ␮g of streptomycin (Irvine Scientific) per ml, and 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, Ga.). After the cell viability was determined by staining with trypan blue, the cells were cultured in 50-ml polypropylene tubes at a concentration of 2 ⫻ 106 cells/ml in a total volume of 5 ml at 37°C in 5% CO2. Harvesting of resident peritoneal cells. Guinea pigs were anesthetized by the intramuscular injection of ketamine hydrochloride (30 mg/kg) and xylazine (2.5 mg/kg) and then euthanized by cardiac injection of sodium pentobarbital (100 mg/kg). The resident macrophages from the peritoneal cavity were harvested by flushing the cavity three times with 20 ml of RPMI medium containing 20 U of heparin. The cells were washed in RPMI medium, counted, and suspended at 5 ⫻ 106 cells/ml in RPMI medium containing 2-mercaptoethanol, glutamine, antibiotics, and 10% FBS. The cells (2 ⫻ 107) were allowed to adhere for 2 h in 100-mm-diameter plastic petri dishes. The nonadherent cells were removed, and the population of the monolayers thus obtained consisted predominantly of macrophages (more than 95%), as visualized by nonspecific esterase staining (46). Purification of T cells. Two methods were used to obtain cell populations with enhanced numbers of T cells. (i) Panning. The nonadherent cells from spleens were purified by panning on plastic plates (100 mm) coated with anti-guinea pig immunoglobulin G (IgG; Sigma) and were termed pan T cells. The panning plates were prepared by delivering 12 ml of 0.1 M Trizma buffer (pH 9.0; Sigma) onto each plate and adding 25 ␮l of the reconstituted antibody in physiological saline (2 mg/ml). The plates were shaken for 1 min and then incubated overnight at 4°C. Just before use, the plates were washed four times with cold 1⫻ phosphate-buffered saline (pH 7.2). The spleen cell suspension in complete RPMI medium was added at a concentration of 3 ⫻ 107 cells/plate and incubated at room temperature for 1 h, with the plates being swirled gently after 30 min. After incubation, the nonadherent cells were collected, washed with cold 1⫻ Hanks balanced salt solution (HBSS; pH 7.4) containing 1% FBS, and centrifuged at 250 ⫻ g for 10 min at 4°C. The pellet was then resuspended in HBSS containing 1% FBS, the number of viable cells was determined, and the cells were adjusted to a final concentration of 5 ⫻ 106 cells/ml. (ii) Nylon wool purification. The column was prepared by packing 0.5 g of scrubbed and combed ready-for-use nylon wool fiber (Polysciences Inc., Warrington, Pa.) into a 10-ml syringe and autoclaving for 15 min. The column was washed with RPMI medium containing 10% fetal calf serum and incubated at 37°C for 1 h, after which it was loaded with 1 ⫻ 108 to 2 ⫻ 108 viable cells in a volume of 2 ml. The loaded column was incubated for 1 h at 37°C, and the nonadherent cells were collected by using two 50-ml washes. The collected cells were centrifuged at 250 ⫻ g for 10 min, the cell pellet was resuspended in RPMI medium containing 10% fetal calf serum, and the viable cells were counted. The purity of cells obtained after panning or nylon wool purification was checked by fluorescence-activated cell sorter (FACS) analysis, and the percentage of T cells was found to be 60 to 70% and 90%, respectively. Flow cytometry. Cells were stained with MAb against guinea pig pan T cells and CD4⫹- and CD8⫹-T-cell phenotypic markers. For each MAb or control, 5 ⫻ 105 cells were placed in a small polypropylene tube and pelleted by centrifugation at 200 ⫻ g for 10 min at 4°C. The supernatant was removed, and the pellet was resuspended in 50 ␮l of the appropriate dilution of primary anti-guinea pig T cell antibody (1:500), anti-CD4 (1:500), or anti-CD8 (1:1,000) and incubated for 1 h in ice on a shaker. At the end of the incubation, the cells were washed three times in HBSS containing 10% FBS. The pellet was resuspended in a 1:10 dilution of the secondary antibody (fluorescein isothiocyanate [FITC]-conjugated AffiniPure goat anti-mouse IgG [heavy plus light chains]; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) and incubated for 1 h in ice on a shaker. The cells were then washed two times and finally suspended in 300 ␮l of HBSS containing 1% paraformaldehyde. The tubes were then covered with aluminum foil and kept in the cold overnight until FACS analysis. Just prior to analysis, the

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cells were washed once and resuspended in 300 ␮l of HBSS containing 10% FBS. The proportions of positive cells were determined with a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, San Jose, Calif.). For analysis of CD4⫹- and CD8⫹-T-cell proportions, cells were gated on the lymphocytes by using the forward and the side-scatter parameters. Both unstained and secondary antibody controls were included, and the positive population was set so that less than 2% of the negative control was positive for green fluorescence. Stimulation of cells. For the coculture experiments, the nonadherent cells and macrophages were mixed at a ratio of 3:1. The whole spleen cells (2 ⫻ 107) and pan T cells or nylon wool-purified T cells (1.5 ⫻ 107) mixed with macrophages (5 ⫻ 106) were cultured with or without ConA (10 ␮g/ml; Sigma) or increasing doses of purified protein derivative (PPD; 5 to 25 ␮g/ml; Statens Seruminstitut) for 18 to 24 h. The amount of bacteria added to the cultures is expressed as a multiplicity of infection (MOI), and various MOIs were used to stimulate the spleen and lymph node cells. Attenuated M. tuberculosis H37Ra (ATCC 25177; American Type Culture Collection, Rockville, Md.) was used at MOIs of 0.05 to 10, and virulent M. tuberculosis H37Rv (ATCC 27294) was used at MOIs of 0.05 to 1.5. Both M. tuberculosis Erdman (ATCC 35801) and M. avium (ATCC 25291) were used at MOIs of 0.05. The MOI for BCG was 0.05. The mycobacterial suspension was briefly sonicated before the addition of an appropriate volume from a stock of 108 CFU per ml. All the cultures were stimulated for 24 h, and at the end of the incubation period, the medium was removed by centrifugation and the cells were used for RNA extraction. RNA isolation and Northern analysis. The methods used for RNA isolation and Northern analysis were described previously (15). Total RNA was extracted by using the Trizol reagent (Life Technologies, Grand Island, N.Y.) as per the manufacturer’s instructions. The RNA pellet was suspended in sterile 0.1% diethyl pyrocarbonate (Sigma)-treated distilled water and stored at ⫺80°C. For Northern analysis, 8 to 12 ␮g of denatured RNA from approximately 15 to 20 million cells was separated electrophoretically on 1.2% agarose–formaldehyde gels. The separated RNA was then transferred to nylon membranes, and the membrane was prehybridized in a solution containing 30% formamide, 5⫻ SSC, 50 ␮g of sheared denatured salmon sperm DNA/ml, 5⫻ Denhardt’s solution, and 0.5% SDS for 2 to 3 h at 37°C as described previously (15, 48). The IFN-␥ cDNA from the clone was 32P labeled by random priming (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) with 25 ng of DNA according to the manufacturer’s instructions. The unincorporated nucleotides were removed with G-50 Sephadex columns (5 Prime33 Prime Inc., Boulder, Colo.). The membrane was hybridized overnight in the prehybridizing solution that contained the guinea pig IFN-␥ cDNA probe. The filters were washed twice in 2⫻ SSC containing 0.5% SDS at room temperature for 15 min each time and once in 0.3⫻ SSC–0.5% SDS for 30 min at 50°C. The blots were analyzed with a phosphorimager. Following analysis, the blots were stripped and reprobed with 18S antisense RNA as an internal standard to ensure equal RNA loading. The sums of counts above the background were analyzed by using Imagequant software. Data are presented as the percentage of basal (unstimulated naïve) levels calculated by using the formula {[(stimulated IFN-␥ mRNA)/(18S mRNA)]/[(unstimulated IFN-␥ mRNA)/(18S mRNA)]} ⫻ 100. Each experiment was repeated at least three times. The results shown below (see Fig. 2 to 5 and 7 and 8) are RNA blots that are representative of the results from all replicate experiments and the combined densitometric analysis of each set of three or more independent experiments. The densitometric results are given relative to 100% of basal levels for unstimulated naïve or unstimulated BCG-vaccinated cultures. Statistics. The densitometric data are expressed as the means ⫾ the standard errors of the means (SEM). The main effect of vaccination was analyzed by analysis of variance (ANOVA). The significant differences between the naïve and BCG-vaccinated groups were determined by either the Bonferroni type F test or Hsu’s test (23). Nucleotide sequence accession number. The nucleotide sequence for guinea pig IFN-␥ clone 4b has been submitted to GenBank and assigned accession number AY151287.

RESULTS Cloning of guinea pig IFN-␥ cDNA. Approximately 5 ⫻ 105 phage clones from a ConA-stimulated spleen cell cDNA library were screened for guinea pig IFN-␥. After several rounds of screening with a 32P-labeled human IFN-␥ cDNA probe and DNA sequencing from denatured plasmids, two clones (desig-

nated 4b and 5) that appeared to code for guinea pig IFN-␥ were obtained. Figure 1 shows the complete nucleotide sequence of clone 4b. The open reading frame of the cDNA encoded a putative 166-amino-acid protein that showed 56 and 41% amino acid sequence similarity to human and mouse IFN-␥, respectively (Fig. 1). IFN-␥ mRNA response of spleen cells to attenuated and virulent mycobacteria. Whole spleen cells from naïve and BCG-vaccinated guinea pigs were stimulated in vitro for 24 h with various doses of viable attenuated and virulent M. tuberculosis strains. As high numbers of virulent M. tuberculosis bacteria are known to be cytotoxic, we used a lower range of doses of the virulent strain H37Rv (MOIs of 0.25 to 1.5) than of the attenuated strain H37Ra (MOIs of 1 to 10). Figure 2 illustrates the Northern analysis of IFN-␥ mRNA (Fig. 2A) and the densitometric evaluation of the blots (Fig. 2B). The spleen cells from naïve animals stimulated in vitro with the virulent or attenuated M. tuberculosis showed a very low level of IFN-␥ mRNA transcripts. In contrast, spleen cells from BCG-vaccinated animals exhibited a statistically significant (P ⬍ 0.0001) level of IFN-␥ mRNA expression after stimulation with either mycobacterial strain. However, the exposure of splenocytes to high doses of the virulent H37Rv strain of M. tuberculosis (MOI, 1 or 1.5) caused a significant (P ⬍ 0.0001) reduction in IFN-␥ mRNA expression (Fig. 2). The viability of the spleen cells treated with any dose of mycobacteria was more than 95% after 24 h in culture, as determined by the trypan blue exclusion assay or by the MTT [3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (13). IFN-␥ mRNA expression in spleen cells stimulated with identical doses of attenuated and virulent M. tuberculosis strains. In the previous experiment, the doses of attenuated and virulent strains of M. tuberculosis used for stimulation were not identical. Moreover, there was a dose-dependent reduction in the response to the H37Rv strain. In this experiment, spleen cells from BCG-vaccinated guinea pigs were stimulated with identical doses (MOIs of 0.15 to 0.675) of attenuated and virulent M. tuberculosis strains for 24 h. As evident from Northern blotting and densitometric analysis (Fig. 3), there was no clear dose response to the two strains. MOIs of M. tuberculosis H37Ra lower than 0.675 did not induce any statistically significant levels of IFN-␥ mRNA expression in the spleen cells during this short period in culture. In contrast, exposure of splenocytes to the same doses of the virulent H37Rv strain of M. tuberculosis induced a significant increase in the levels of mRNA expression in the spleen cells (P of ⬍0.01 to 0.0001). These results indicated that the induction of IFN-␥ mRNA in spleen cells is dependent on the doses of attenuated and virulent M. tuberculosis strains used for stimulation. IFN-␥ mRNA expression in lymph node cells. Cells from the inguinal lymph nodes draining the vaccination sites in BCGvaccinated and naïve guinea pigs were stimulated with a low dose (MOI, 0.05) of various strains of attenuated and virulent mycobacteria (BCG, M. avium, and M. tuberculosis strains H37Ra, H37Rv, and Erdman). This dose had induced IL-1␤ and RANTES mRNA expression in macrophages (15). Figure 4 shows the results of Northern blotting (Fig. 4A) and densitometric analysis (Fig. 4B). Lymph node cells from naïve animals expressed no IFN-␥ mRNA in response to mycobacteria

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FIG. 1. Nucleotide and deduced amino acid sequences of guinea pig IFN-␥. (A) Complete nucleotide and deduced amino acid sequences of clone 4b; (B) comparison of amino acid sequences of human (Hu), mouse (Mu), and guinea pig (GP) IFN-␥.

(data not shown). Stimulation of lymph node cells from BCGvaccinated guinea pigs with BCG or attenuated M. tuberculosis H37Ra induced low levels of IFN-␥ mRNA which were not statistically different from those of the unstimulated cells. However, the IFN-␥ mRNA levels were significantly higher after stimulation with M. avium (P ⬍ 0.004) or virulent M. tuberculosis H37Rv (P ⬍ 0.002) or Erdman (P ⬍ 0.002). Thus,

virulent strains of mycobacteria induced a significantly higher (P of ⬍0.01 to 0.005) response than the attenuated strains at an MOI of 0.05. PPD also induced strong IFN-␥ mRNA expression in the lymph node cells of BCG-vaccinated guinea pigs, and the response was significantly higher in these cells at doses of 5 and 10 ␮g (P of ⬍0.003 to 0.004) than that in the unstimulated controls.

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FIG. 2. Effect of BCG vaccination on IFN-␥ mRNA expression in spleen cells of guinea pigs. Whole spleen cells (2 ⫻ 107) from naïve and BCG-vaccinated guinea pigs were stimulated (4 to 6 weeks postvaccination) in vitro with various MOIs (0.25 to 5) of attenuated and virulent M. tuberculosis strains for 24 h. The total RNA extracted was subjected to Northern blot analysis, and the mRNA levels were analyzed with a phosphorimager. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of IFN-␥. The densitometric results for unstimulated cultures are given relative to 100% basal results. The results are expressed as the means ⫾ SEM of results from three experiments. The main treatment effect of vaccination was analyzed by ANOVA. The differences between the groups were analyzed by the Bonferroni type F test. **, P was ⬍0.0001 in comparisons between the stimulated groups and the unstimulated (cells alone) controls and between H37Ra- and H37Rv-stimulated groups. §, P was ⬍0.0001 when naïve and BCG-vaccinated groups were compared.

Kinetics of IFN-␥ mRNA induction. Spleen cells from BCGvaccinated guinea pigs were stimulated with PPD (15 ␮g/ml) or the virulent H37Rv strain of M. tuberculosis (MOI, 0.05) for various periods of time up to 48 h. Compared to controls, cells stimulated with PPD showed no significant levels of IFN-␥ mRNA until 18 h after stimulation (P ⬍ 0.0001), when the response peaked and then dropped by 24 h (P ⬍ 0.0009) and 48 h (P ⬍ 0.009) (Fig. 5). After stimulation with virulent M. tuberculosis, no significant IFN-␥ mRNA levels were seen at 4 h but a significant level was detected at 18 h (P ⬍ 0.0001). The mRNA levels had decreased by 48 h but were still higher (P ⬍ 0.0001) than those of the unstimulated cultures. The mRNA response induced in the spleen cells by the whole mycobacteria was significantly higher and lasted longer than the response to PPD after 18 h (P ⬍ 0.0001), 24 h (P ⬍ 0.0001), and 48 h (P ⬍ 0.0005) of culture. Phenotypic analysis of spleen cells. Cells from BCG-vaccinated guinea pigs, when stimulated in vitro with PPD or living mycobacteria, showed enhanced IFN-␥ mRNA expression compared to that of the cells from naïve animals. We hypothesized that this enhanced response was due to an alteration in the proportions of lymphoid cells in the spleens of BCG-vac-

cinated guinea pigs. The nonadherent cells from the spleens of both naïve and BCG-vaccinated animals were obtained by panning on anti-guinea pig IgG-coated plates. The proportions of T cells in these populations were determined by FACS analysis after staining the cells with MAb directed against T cells (antiguinea pig pan T cells) and their subsets (anti-CD4 and antiCD8). The binding of primary antibody was detected with FITC-labeled goat anti-mouse IgG. There was no difference in the proportions of T cells in the naïve and BCG-vaccinated guinea pigs (Fig. 6). However, the proportions of CD4⫹ T cells were significantly increased (P ⬍ 0.01) after BCG vaccination. CD8⫹-T-cell proportions remained unaltered statistically, although a slight drop (P ⬍ 0.054) was apparent in the BCGvaccinated group. IFN-␥ mRNA expression in purified T cells. In order to identify the splenic cells responsible for IFN-␥ mRNA expression, the nonadherent cells in the spleens of naïve and BCGvaccinated guinea pigs were enriched either by panning on anti-guinea pig IgG-coated plates or by purification on nylon wool columns. Whole spleen cells or enriched T cells cocultured with autologous, adherent peritoneal macrophages at a ratio of 3:1 were stimulated in the presence of ConA (10

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FIG. 3. IFN-␥ mRNA expression in spleen cells from BCG-vaccinated guinea pigs. Cells were stimulated with identical MOIs (0.15 to 0.675) of attenuated and virulent M. tuberculosis strains for 24 h. The RNA was processed as described above. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of the results from three experiments. In comparisons between stimulated groups and unstimulated (cells alone) controls, P was ⬍0.03 (†), ⬍0.003 to 0.002 (*), or ⬍0.0003 to 0.0001 (**).

␮g/ml) or PPD (15 ␮g/ml) for 18 to 24 h. Figure 7 shows the IFN-␥ mRNA expression in whole spleen cells, panned T cells, and nylon wool-purified T cells in response to the two stimulants. The spleen cells from naïve guinea pigs, whether unseparated (P ⬍ 0.002) or purified by panning (P ⬍ 0.0003) or with a nylon wool column (P ⬍ 0.0001), showed significant IFN-␥ mRNA expression when stimulated with ConA, not with PPD. After BCG vaccination, all three cell cultures showed significant IFN-␥ mRNA expression in response to ConA (P of ⬍0.003 to 0.0001). The levels were increased significantly after PPD stimulation only in pan T (P ⬍ 0.01) and nylon woolpurified (P ⬍ 0.0001) populations. The nylon wool-purified T cells exhibited a significantly greater IFN-␥ mRNA response both to ConA (P of ⬍0.004 to 0.0001) and to PPD (P ⬍ 0.0001). These results indicate that T cells are principally responsible for IFN-␥ mRNA expression in the spleens of BCGvaccinated guinea pigs. IFN-␥ mRNA expression in T cell–macrophage cocultures infected with M. tuberculosis. T cells obtained from the spleens of BCG-vaccinated guinea pigs by panning on anti-IgG-coated plates were cocultured with autologous peritoneal macrophages at a T cell/macrophage ratio of 3:1. The cocultures were exposed to MOIs of 0.15 to 0.675 of attenuated and virulent M. tuberculosis strains, as in the experiments represented in Fig. 3. Additionally, T cells or macrophages alone or in a 3:1 combination were stimulated with PPD (15 ␮g/ml). Figure 8 illustrates that T cells alone (P ⬍ 0.0003) or in combination with

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FIG. 4. Effect of in vitro stimulation on IFN-␥ mRNA expression in the lymph node cells of BCG-vaccinated guinea pigs. Cells from the inguinal lymph nodes (2 ⫻ 107) in both flanks draining the site of BCG vaccination were stimulated in vitro for 24 h with an MOI of 0.05 of the following strains of the attenuated and virulent mycobacteria: BCG, M. avium, and M. tuberculosis strains H37Ra, H37Rv, and Erdman. The cells were processed for RNA as described in the legend to Fig. 2. (A) RNA blot representative of the results from several experiments; (B) densitometric analysis of the results of three experiments. *, P was ⬍0.005 to 0.002 in comparison with results for unstimulated (cells alone) cultures.

macrophages (P ⬍ 0.03), when stimulated with PPD, showed significantly higher levels of IFN-␥ mRNA expression than the unstimulated cultures. Macrophages alone after stimulation with PPD produced no IFN-␥ mRNA, in contrast to the unstimulated cultures. As observed with the whole spleen cells, the attenuated M. tuberculosis H37Ra strain at the doses employed was incapable of inducing IFN-␥ mRNA transcripts in the cocultures of T cells and macrophages. However, the same doses of the virulent H37Rv strain of M. tuberculosis induced a highly significant (P ⬍ 0.0001) mRNA response in these cultures. These results indicate that the virulent strain of M. tuberculosis induces IFN-␥ mRNA expression in whole spleen cells or in purified T cells when used at a ratio of 1 bacterium to 10 or fewer cells. DISCUSSION IFN-␥ is a T-cell-derived cytokine with broad macrophageactivating effects which is known to play a critical role in antimycobacterial immunity (11, 33). The guinea pig model of low-dose pulmonary TB has contributed much to our general

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FIG. 5. Kinetics of IFN-␥ mRNA induction in spleen cells after BCG vaccination. Spleen cells from BCG-vaccinated guinea pigs were cultured with or without PPD (15 ␮g/ml) or M. tuberculosis H37Rv (MOI, 0.05) for various periods of time. At regular intervals, cells were processed for RNA analysis. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of results from three experiments. The results are expressed as the means ⫾ SEM of results from three experiments. The differences between the results for unstimulated and stimulated cultures were compared by ANOVA. Hsu’s test was used for finding the best treatment for IFN-␥ mRNA expression. In comparisons between stimulated groups and unstimulated (cells alone) controls, P was ⬍0.009 (*) or ⬍0.0009 to 0.0001 (**).

understanding of the mechanisms of vaccine-induced resistance (29, 31). To enhance the value of this model, we have cloned the guinea pig IFN-␥ cDNA from ConA-stimulated spleen cells by using a human IFN-␥ cDNA probe. Guinea pig IFN-␥ cDNA is composed of 1,110 bp, with an open reading frame that encodes a putative 166-amino-acid protein. The nucleotide sequences are nearly identical to the sequences (positions 40 through 1167) submitted by others for guinea pig IFN-␥ (GenBank accession number E25787) except for one substitution of C for T (base 303 in our sequence); however, this substitution does not alter the amino acid sequence. We used the cDNA clone to generate a probe for Northern blot analysis to assess IFN-␥ mRNA expression in the lymphoid cells of BCG-vaccinated and nonvaccinated guinea pigs. The results presented here indicate that both vaccination and in vitro stimulation affected the level of IFN-␥ mRNA expression in whole lymphoid cell populations and purified T cells. Previously, our laboratory reported that cell-mediated immune responses in guinea pigs as measured by delayed-type hypersensitivity and the ability of lymphocytes to proliferate and produce IL-2 in response to PPD were augmented after BCG vaccination (29, 30). These responses were consistent with the ability of animals to develop protective responses against aerosol infection with virulent M. tuberculosis (28).

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FIG. 6. Proportions of T cells and T-cell subsets in the spleens of guinea pigs. Spleen cells from naïve and BCG-vaccinated guinea pigs were purified by panning on anti-guinea pig IgG-coated plates and staining for red fluorescence with MAb directed against the surface markers of T cells (CT5), CD4⫹ T cells (CT7), and CD8⫹ T cells (CT6). The binding of primary antibody was detected with FITCconjugated goat anti-mouse IgG. The proportions of positive cells were determined with a FACSCalibur flow cytometer and Cell Quest software. The significant differences between the naïve and BCGvaccinated groups were determined by Student’s t test. ⴱ, P was ⬍0.01 when the proportions of CD4⫹ cells from naïve and BCG-vaccinated groups were compared.

Recently, it has been reported that IL-1␤ and RANTES mRNA responses were enhanced in BCG-vaccinated guinea pigs after the stimulation of spleen cells or macrophages in vitro with PPD or living mycobacteria (15). Because IFN-␥ is considered to play a pivotal role in antimycobacterial immunity (11, 33), we studied whether BCG vaccination would increase the IFN-␥ mRNA response in spleen cells. The results are consistent with our hypothesis that IFN-␥ mRNA expression in spleen cells is enhanced after BCG vaccination in response to living mycobacteria and their protein antigens. In clinical trials of BCG vaccination, there is ample evidence to demonstrate that cell-mediated immune responses in BCG recipients, including the production of IFN-␥, are enhanced compared to those in control subjects (14, 39). Earlier, it was demonstrated that the exposure of spleen cells or macrophages to virulent M. tuberculosis decreased the cytokine mRNA responses for both IL-1␤ and RANTES compared to those induced by the exposure of the same cells to the attenuated strain (15). The results illustrated in Fig. 2 indicate that there was a dose-dependent and significant decrease in the IFN-␥ mRNA response in spleen cells after stimulation with high doses of the virulent M. tuberculosis strain. The reduction in the mRNA response occurred in the absence of cytotoxicity, as the viability of the cells exposed to mycobacteria was more than 95%, a fact confirmed by standard cell staining methods. There is considerable evidence to indicate that virulent and attenuated mycobacteria induce differential levels of cytokines in macrophages. The levels of TNF-␣ or IL-1␤ produced after mycobacterial stimulation seem to depend on the virulence of the organism, and it has been postulated that suppression of

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FIG. 7. IFN-␥ mRNA expression by purified T-cell–macrophage cocultures. Whole spleen cells (2 ⫻ 107) or cocultures of panned T cells or nylon wool-purified T cells plus peritoneal macrophages (Mø) at a ratio of 3:1 were examined in naïve and BCG-vaccinated guinea pigs. T cells were purified either by panning on anti-guinea pig IgGcoated plates or on nylon wool columns. Resident peritoneal macrophages were prepared by allowing adherence to plastic for 2 h at 37°C. The cells were cultured in the presence of ConA (10 ␮g/ml) or PPD (15 ␮g/ml) for 18 to 24 h and processed for RNA analysis as described in the text. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of results from three experiments. The main treatment effect of vaccination was analyzed by ANOVA. The differences between the groups were analyzed by the Bonferroni type F test. In comparisons between stimulated groups and unstimulated (cells alone) controls, P was ⬍0.01 (†), ⬍0.002 (*), or ⬍0.0003 to 0.0001 (**).

protective host cytokines might be related to pathogenesis. For example, the attenuated H37Ra strain of M. tuberculosis induced a larger amount of NO in cultured human peripheral blood mononuclear cells than did the virulent H37Rv strain (24). In both control and AIDS patients, more IL-1␤ was released after the infection of monocytes with the less virulent M. avium (16). Similarly, infection of human monocytes or macrophages with virulent M. avium downregulated the production of proinflammatory cytokines (IL-1␤, TNF-␣, IL-6, and granulocyte-macrophage colony-stimulating factor) compared to that by cells infected with the less virulent strain (32). Studies with mice revealed that lipoarabinomannan derived from an attenuated strain of M. tuberculosis induced macrophage activation and TNF-␣ production, whereas lipoarabinomannan from the virulent Erdman strain was less stimulatory (2). The inability to induce the appropriate mediators in lymphocytes and macrophages might be one of the mechanisms by which some mycobacteria evade or suppress the immune response (32). It is not clear whether virulent and attenuated M. tuberculosis strains induce differential IFN-␥ responses in human or animal systems. However, human monocytes cocul-

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tured with the virulent smooth-transparent M. avium strain produced lower concentrations of IFN-␥ and IL-18, a potent IFN-␥ inducer, in the culture supernatants than did those cocultured with the avirulent smooth-domed M. avium strain (43). Similarly, mice infected with highly virulent or avirulent M. avium demonstrated that the control of infection with the low-virulence strain was associated with an increased expression of IFN-␥ and IL-2 compared to that in mice infected with the more virulent strain (4). The present results clearly demonstrate that attenuated and virulent M. tuberculosis strains fail to induce a significant IFN-␥ mRNA response in guinea pig cells unless the cells are stimulated with appropriate doses of bacteria and that the virulent M. tuberculosis H37Rv induces a dose-dependent inhibition of the IFN-␥ mRNA response in spleen cells. It is well documented that IFN-␥ does not activate human macrophages to kill virulent M. tuberculosis. It was found that M. tuberculosis inhibited transcriptional responses without inhibiting activation of STAT-1. This was due to a marked decrease in the IFN-␥-induced association of STAT-1 with the transcriptional coactivators in M. tuberculosis-infected macrophages (45). Humans with active TB or with multidrug-resistant TB are known to have diminished IFN-␥ responses. In patients with active pulmonary TB, the frequency of IFN-␥-secreting CD4⫹ T cells was lower than that in healthy PPD-positive contacts or subjects with minimal disease and low bacterial burdens (34). Similarly, multidrug-resistant TB patients with low CD4⫹ T cells from peripheral blood mononuclear cells had impaired IFN-␥ responses to M. tuberculosis, PPD, or mitogens compared to those of healthy PPD-positive and PPD-negative individuals (26). The attenuated H37Ra strain of M. tuberculosis was efficient in inducing a significant level of IFN-␥ mRNA expression only at high doses at which virulent M. tuberculosis induced a significantly weaker response (Fig. 2). M. tuberculosis H37Rv induces IFN-␥ mRNA expression in whole spleen cells or in purified T cells when used at a bacterium-to-cell ratio of 1:10. When stimulated with the virulent strain at an MOI of 1, guinea pig cells expressed significantly less IFN-␥ mRNA (P ⬍ 0.0001). In contrast, the attenuated H37Ra strain of M. tuberculosis did not induce any significant IFN-␥ mRNA expression in these cells at the same doses (Fig. 3 and 8). A significant level of IFN-␥ mRNA expression was observed only when cells were stimulated with the attenuated strain at a bacterium-tocell ratio of more than 1 (Fig. 2). Either no expression or low levels of IFN-␥ mRNA expression were seen when these cells were stimulated with a smaller dose (Fig. 3, 4, and 8). In order to investigate whether an alteration in the proportions of lymphoid cells in the spleen after BCG vaccination was responsible for enhanced IFN-␥ mRNA expression, the cells that were nonadherent after panning were used for phenotypic examination by FACS analysis. The T cells and their subsets were stained with MAb directed against the cells’ surface markers. The proportions of CD4⫹ cells were significantly increased in the BCG-vaccinated guinea pigs (Fig. 6), while total T-cell and CD8⫹-T-cell profiles remained unaltered. It is known that immune responses against M. tuberculosis are mediated by CD4⫹ T cells, although recent evidence indicates that CD8⫹ cells also contribute to antimycobacterial immunity (12, 33). It is quite likely that an increase in the CD4⫹ T cells

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FIG. 8. IFN-␥ mRNA expression induced by virulent and attenuated mycobacteria in cocultures of panned T cells plus peritoneal macrophages (Mø). T cells enriched from the spleen by panning and resident peritoneal macrophages from BCG-vaccinated guinea pigs were stimulated with PPD (15 ␮g/ml) or various doses (MOIs of 0.15 to 0.675) of attenuated or virulent M. tuberculosis for 24 h. The cells were processed as described in the text. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of results from three experiments (the means ⫾ SEM). The differences between unstimulated (T cells and macrophages alone) and stimulated cultures were determined by ANOVA (*, P ⬍ 0.003; **, P ⬍0.0003 to 0.0001).

might contribute to the enhanced expression of IFN-␥ mRNA in spleen cells and lymph node cells after BCG vaccination. The type of cells that might be responsible for the induction of IFN-␥ mRNA was investigated by purifying T cells from whole splenocytes either by panning or passing the splenocytes through nylon wool columns. As evident from the FACS analysis, it was clear that there was a higher percentage of T cells in the nylon wool-purified population than among the cells obtained after panning. The nylon wool-purified T cells expressed highly significant levels of IFN-␥ mRNA in response to ConA and PPD (Fig. 7). Similarly, the panned T cells had much higher levels of IFN-␥ mRNA transcripts in response to M. tuberculosis than the whole spleen cells (Fig. 2 and 8). Although IFN-␥ is mainly produced by CD4⫹ T cells, CD8⫹ T cells, macrophages, and NK cells are also known to produce this cytokine after mycobacterial stimulation (41). Macrophages from bronchoalveolar lavage fluid of TB patients showed IFN-␥ protein mRNA expression; however, the majority of IFN-␥ mRNA was detected in lymphocytes after lavage (37). In other studies, in vitro infection of alveolar macrophages with M. tuberculosis induced IFN-␥ mRNA expression and the release of IFN-␥ protein (44). High levels of IFN-␥ and TNF-␣ are found in the pleural fluid of TB patients, and, in fact, IFN-␥ is considered to be a useful marker for diagnosing tuberculous pleurisy (36). Despite the vast literature on the macrophage-activating effects of IFN-␥ in humans and mice, relatively few studies

have addressed this question in guinea pigs. BCG vaccination of inbred guinea pigs induced RANTES and IFN-␥ mRNA in their spleens, as detected by reverse transcription-PCR; this was also observed in naïve guinea pigs. Surprisingly, only CD8⫹ T cells from the lymph nodes of vaccinated animals expressed RANTES and IFN-␥ mRNA, and not CD4⫹ cells (22). Unlike our studies, the earlier study used strain 2 guinea pigs that were vaccinated with a much higher dose of BCG (2 ⫻ 107 CFU) and investigated the IFN-␥ mRNA expression 8 days postvaccination by reverse transcriptase PCR. Furthermore, the authors of that study analyzed the cytokine mRNA expression directly from spleen and lymph node cells without further in vitro stimulation and, therefore, could not determine how much of the IFN-␥ response was actually specific to mycobacteria. We observed an enhanced IFN-␥ mRNA response in the spleen which was associated with a significant increase in the proportions of CD4⫹ T cells. We have also provided evidence which suggests that T cells in the spleen are responsible primarily for IFN-␥ mRNA induction. Studies are already under way to address the IFN-␥ mRNA responses in BCG-vaccinated guinea pigs following low-dose aerosol infection with virulent M. tuberculosis. With the development of recombinant guinea pig IFN-␥ and anti-guinea pig IFN-␥ antibodies, we will begin to elucidate the contributions of this important cytokine to the complex interaction between mycobacteria and the host’s protective immune response.

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IFN-␥ mRNA IN GUINEA PIGS ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant RO1 AI 15495 to D.N.M. We are indebted to Jane Miller for her help and expertise in FACS analysis and Gregory Foster for the critical evaluation of the manuscript. REFERENCES 1. Barnes, P. F., S. J. Fong, P. J. Brennan, P. E. Twomey, A. Mazumder, and R. L. Modlin. 1990. Local production of tumor necrosis factor and IFNgamma in tuberculous pleuritis. J. Immunol. 145:149–154. 2. Brown, M. C., and S. M. Taffet. 1995. Lipoarabinomannans derived from different strains of Mycobacterium tuberculosis differentially stimulate the activation of NF-␬B and KBF1 in murine macrophages. Infect. Immun. 63:1960–1968. 3. Campbell, E. M., A. E. Proudfoot, T. Yoshimura, B. Allet, T. N. Wells, A. M. White, J. Westwick, and M. L. Watson. 1997. Recombinant guinea pig and human RANTES activate macrophages but not eosinophils in the guinea pig. J. Immunol. 159:1482–1489. 4. Castro, A. G., P. Minoprio, and R. Appelberg. 1995. The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice. Immunology 85:556–561. 5. Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, and F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 271:698– 702. 6. Condos, R., W. N. Rom, and N. W. Schluger. 1997. Treatment of multidrugresistant pulmonary tuberculosis with interferon-gamma via aerosol. Lancet 349:1513–1515. 7. Cooper, A. M., D. K. Dalton, T. A. Stewart, J. P. Griffin, D. G. Russell, and I. M. Orme. 1993. Disseminated tuberculosis in interferon gamma genedisrupted mice. J. Exp. Med. 178:2243–2247. 8. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, and T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 259:1739–1742. 9. Denis, M., E. O. Gregg, and E. Ghandirian. 1990. Cytokine modulation of Mycobacterium tuberculosis growth in human macrophages. Int. J. Immunopharmacol. 12:721–727. 10. Dorman, S. E., and S. M. Holland. 1998. Mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection. J. Clin. Investig. 101:2364–2369. 11. Flynn, J. L., J. Chan, K. J. Triebold, D. K. Dalton, T. A. Stewart, and B. R. Bloom. 1993. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249–2254. 12. Flynn, J. L., M. M. Goldstein, K. J. Triebold, B. Koller, and B. R. Bloom. 2017. 1992. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA 89:12013–12017. 13. Hansen, M. B., S. E. Nielsen, and K. Berg. 1989. Re-examination and further development of a precise and rapid dye method for measuring cell growth/ cell kill. J. Immunol. Methods 119:203–210. 14. Hoft, D. F., E. B. Kemp, M. Marinaro, O. Cruz, H. Kiyono, J. R. McGhee, J. T. Belisle, T. W. Milligan, J. P. Miller, and R. B. Belshe. 1999. A doubleblind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guerin vaccination. J. Lab. Clin. Med. 134:244–252. 15. Jeevan, A., T. Yoshimura, G. Foster, and D. N. McMurray. 2002. Effect of Mycobacterium bovis BCG vaccination on interleukin-1␤ and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria. Infect. Immun. 70:1245–1253. 16. Johnson, J. L., H. Shiratsuchi, Z. Toossi, and J. J. Ellner. 1997. Altered IL-1 expression and compartmentalization in monocytes from patients with AIDS stimulated with Mycobacterium avium complex. J. Clin. Immunol. 17:387–395. 17. Kamijo, R., J. Gerecitano, D. Shapiro, S. J. Green, M. Aguet, J. Le, and J. Vilcek. 1995. Generation of nitric oxide and clearance of interferon-gamma after BCG infection are impaired in mice that lack the interferon-gamma receptor. J. Inflamm. 46:23–31. 18. Kamijo, R., J. Le, D. Shapiro, E. A. Havell, S. Huang, M. Aguet, M. Bosland, and J. Vilcek. 1993. Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guerin and subsequent challenge with lipopolysaccharide. J. Exp. Med. 178:1435–1440. 19. Kamijo, R., D. Shapiro, J. Le, S. Huang, M. Aguet, and J. Vilcek. 1993. Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor. Proc. Natl. Acad. Sci. USA 90:6626–6630. 20. Kaufmann, S. H. E. 1987. Towards new leprosy and tuberculosis vaccines. Microbiol. Sci. 4:324–328. 21. Kindler, V., A. P. Sappino, G. E. Grau, P. F. Piguet, and P. Vassalli. 1989. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56:731–740.

363

22. Klu ¨nner, T., T. Bartels, M. Vordermeier, R. Burger, and H. Scha ¨fer. 2001. Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin. Vaccine 19:1968–1977. 23. Kuehl, R. O. 1994. Statistical principles of research design and analysis. Duxbury Press, Pacific Grove, Calif. 24. Kwon, O. J. 1997. The role of nitric oxide in the immune response of tuberculosis. J. Korean Med. Sci. 12:481–487. 25. Mackaness, G. B. 1968. The immunology of antituberculous immunity. Am. Rev. Respir. Dis. 97:337–344. 26. McDyer, J. F., M. N. Hackley, T. E. Walsh, J. L. Cook, and R. A. Seder. 1997. Patients with multidrug-resistant tuberculosis with low CD4⫹ T cell counts have impaired Th1 responses. J. Immunol. 158:492–500. 27. McMurray, D. N., M. A. Carlomagno, and P. A. Cumberland. 1983. Respiratory infection with attenuated Mycobacterium tuberculosis H37Ra in malnourished guinea pigs. Infect. Immun. 39:793–799. 28. McMurray, D. N., M. A. Carlomagno, C. L. Mintzer, and C. L. Tetzlaff. 1985. Mycobacterium bovis BCG vaccine fails to protect protein-deficient guinea pigs against respiratory challenge with virulent Mycobacterium tuberculosis. Infect. Immun. 50:555–559. 29. McMurray, D. N., G. Dai, and S. Phalen. 1999. Mechanisms of vaccineinduced resistance in a guinea pig model of pulmonary tuberculosis. Tuber. Lung Dis. 79:261–266. 30. McMurray, D. N., C. L. Mintzer, R. A. Bartow, and R. L. Parr. 1989. Dietary protein deficiency and Mycobacterium bovis BCG affect interleukin-2 activity in experimental pulmonary tuberculosis. Infect. Immun. 57:2606–2611. 31. McMurray, D. N., and E. A. Yetley. 1983. Response to Mycobacterium bovis BCG vaccination in protein- and zinc-deficient guinea pigs. Infect. Immun. 39:755–761. 32. Michelini-Norris, M. B., D. K. Blanchard, C. A. Pearson, and J. Y. Djeu. 1992. Differential release of interleukin (IL)-1 alpha, IL-1 beta, and IL-6 from normal human monocytes stimulated with a virulent and an avirulent isogenic variant of Mycobacterium avium-intracellulare complex. J. Infect. Dis. 165:702–709. 33. Orme, I. M., E. S. Miller, A. D. Roberts, S. K. Furney, J. P. Griffin, K. M. Dobos, D. Chi, B. Rivoire, and P. J. Brennan. 1992. T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens. J. Immunol. 148:189–196. 34. Pathan, A. A., K. A. Wilkinson, P. Klenerman, H. McShane, R. N. Davidson, G. Pasvol, A. V. Hill, and A. Lalvani. 2001. Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J. Immunol. 167:5217–5225. 35. Pierre-Audigier, C., E. Jouanguy, S. Lamhamedi, F. Altare, J. Rauzier, V. Vincent, D. Canioni, J. F. Emile, A. Fischer, S. Blanche, J. L. Gaillard, and J. L. Casanova. 1997. Fatal disseminated Mycobacterium smegmatis infection in a child with inherited interferon gamma receptor deficiency. Clin. Infect. Dis. 24:982–984. 36. Ribera, E., I. Ocana, J. M. Martinez-Vazquez, M. Rossell, T. Espanol, and A. Ruibal. 1988. High level of interferon gamma in tuberculous pleural effusion. Chest 93:308–311. 37. Robinson, D. S., S. Ying, I. K. Taylor, A. Wangoo, D. M. Mitchell, A. B. Kay, Q. Hamid, and R. J. Shaw. 1994. Evidence for a Th1-like bronchoalveolar T-cell subset and predominance of interferon-gamma gene activation in pulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 149:989–993. 38. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 39. Sander, B., U. Skansen-Saphir, O. Damm, L. Hakansson, J. Andersson, and U. Andersson. 1995. Sequential production of Th1 and Th2 cytokines in response to live bacillus Calmette-Guerin. Immunology 86:512–518. 40. Sato, K., T. Akaki, and H. Tomioka. 1998. Differential potentiation of antimycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNF-alpha). Clin. Exp. Immunol. 112:63–68. 41. Serbina, N. V., and J. L. Flynn. 2001. CD8⫹ T cells participate in the memory immune response to Mycobacterium tuberculosis. Infect. Immun. 69:4320– 4328. 42. Shiratori, I., M. Matsumoto, S. Tsuji, M. Nomura, K. Toyoshima, and T. Seya. 2001. Molecular cloning and functional characterization of guinea pig IL-12. Int. Immunol. 13:1129–1139. 43. Shiratsuchi, H., and J. J. Ellner. 2001. Expression of IL-18 by Mycobacterium avium-infected human monocytes; association with M. avium virulence. Clin. Exp. Immunol. 123:203–209. 44. Soderblom, T., P. Nyberg, A. M. Teppo, M. Klockars, H. Riska, and T. Pettersson. 1996. Pleural fluid interferon-gamma and tumour necrosis factor-alpha in tuberculous and rheumatoid pleurisy. Eur. Respir. J. 9:1652– 1655. 45. Ting, L. M., A. C. Kim, A. Cattamanchi, and J. D. Ernst. 1999. Mycobacte-

364

JEEVAN ET AL.

rium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1. J. Immunol. 163:3898–3906. 46. Yam, L. T., C. Y. Li, and W. H. Crosby. 1971. Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55:283–290. 47. Yoshimura, T., and D. G. Johnson. 1993. cDNA cloning and expression of guinea pig neutrophil attractant protein-1 (NAP-1). NAP-1 is highly conserved in guinea pig. J. Immunol. 151:6225–6236.

Editor: W. A. Petri, Jr.

INFECT. IMMUN. 48. Yoshimura, T., M. Takeya, H. Ogata, S. Yamashiro, W. S. Modi, and R. Gillitzer. 1999. Molecular cloning of the guinea pig GRO gene and its rapid expression in the tissues of lipopolysaccharide-injected guinea pigs. Int. Arch. Allergy Immunol. 119:101–111. 49. Young, L. S., C. B. Inderlied, O. G. Berlin, and M. S. Gottlieb. 1986. Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex. Rev. Infect. Dis. 8:1024–1033.