Differential Expression of Gamma Interferon mRNA Induced ... · Department of Medical Microbiology...

INFECTION AND IMMUNITY, Jan. 2003, p. 354–364 Vol. 71, No. 10019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.1.354–364.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Differential Expression of Gamma Interferon mRNA Induced byAttenuated and Virulent Mycobacterium tuberculosis in Guinea

Pig Cells after Mycobacterium bovis BCG VaccinationAmminikutty 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 andDepartment 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-�) mRNAexpression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pigIFN-� from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frameencoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human andmouse IFN-�, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs werestimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA wassubjected to Northern blot analysis with a 32P-labeled probe derived from the cDNA clone. Compared to theIFN-� mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to variousstimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-� mRNA levelswhen cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacteriumper 10 cells. The enhanced IFN-� mRNA response in BCG-vaccinated animals was associated with an increasein the proportions of CD4� 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 pigimmunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-� mRNA thanwhole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principallyresponsible for the upregulation of IFN-� mRNA expression following BCG vaccination. The mechanism bywhich virulent mycobacteria suppress IFN-� mRNA accumulation is currently under investigation.

Infection with Mycobacterium tuberculosis remains a majorpublic health problem in many countries, including the UnitedStates. Recent reports indicate that one-third of the world’spopulation is infected with M. tuberculosis (20). Tuberculosis(TB) has become a serious concern mainly because of its oc-currence in AIDS patients and also due to the emergence ofdrug-resistant strains of mycobacteria (49). The only TB vac-cine currently available is Mycobacterium bovis BCG, althougha wide variability in the efficacy of this vaccine against adult TBhas been reported in clinical trials (5).

Macrophages and lymphocytes are critical players in theimmune response against mycobacteria (25). Both CD4�- andCD8�-T-cell subsets have been shown to contribute to anti-mycobacterial immunity (12, 33). In order to mediate an ef-fective immune response against M. tuberculosis, macrophagesand T lymphocytes act in concert with the help of costimula-tory molecules as well as molecular mediators such as cyto-kines and chemokines (1). Among various cytokines, bothgamma 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 importantrole in the formation and maintenance of the granuloma and,

along with IFN-�, activates macrophages to produce effectormolecules such as toxic oxygen and nitrogen intermediates(21). Interference with the production of appropriate media-tors by lymphocytes and macrophages might be one of thepathogenic mechanisms employed by some mycobacteria (32).

Considerable evidence from both in vitro and in vivo studiesindicates that the activation of monocytes and macrophages byIFN-� plays an important role in the effective restriction aswell as the clearance of mycobacteria (11). Culture of humanmonocytes in the presence of recombinant IFN-� reduced thegrowth of Mycobacterium avium (40). The role of IFN-� inmacrophage activation and resistance to intracellular patho-gens has been demonstrated by using gene knockout mice.Disruption of the IFN-� gene in mice infected with M. tuber-culosis resulted in exacerbation of disease, progressive andwidespread tissue destruction and necrosis with numerous bac-teria (11), or reduced expression of class II antigens on mac-rophages (8). Similarly, targeted disruption of the IFN-� re-ceptor gene in mice made them susceptible to lethal M. bovisBCG infection, reduced TNF-� production, and decreasedproduction of nitric oxide by macrophages (17–19). There isalso evidence that humans with a mutation in the IFN-� re-ceptor or the IFN-� receptor signal-transducing chain developdisseminated mycobacterial infections, demonstrating the im-portant role of IFN-� in the human immune response to my-cobacteria (10, 35). Experimental data demonstrate that IFN-�has considerable potential in the treatment of multidrug-resis-tant TB (6).

* Corresponding author. Mailing address: Department of MedicalMicrobiology and Immunology, Texas A&M System Health ScienceCenter, 407 Reynolds Medical Building, College Station, TX 77843-1114. Phone: (979) 862-2436. Fax: (979) 845-3479. E-mail address:[email protected].

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Despite these rather extensive studies on the macrophage-activating effects of IFN-�, it is not yet clear whether humanmacrophages can be stimulated in vitro to be bactericidal or toproduce effector molecules such as toxic nitrogen intermedi-ates (9). Additionally, there is a paucity of information on therole of IFN-� in mycobacterial immunity in the well-estab-lished guinea pig model of low-dose pulmonary TB (29, 31).We have characterized the course of infection caused by viru-lent M. tuberculosis as well as the ability of BCG vaccination toprotect against virulent challenge (27). The recent availabilityof guinea pig cytokine and chemokine cDNA clones (3, 42, 47)has made it possible to elucidate some of the mechanisms ofBCG vaccine-induced resistance in guinea pigs. For example,our laboratory has reported that spleen cells and macrophagesfrom BCG-vaccinated guinea pigs show enhanced interleu-kin-1� (IL-1�) and RANTES mRNA responses compared tothe cells from naïve animals when stimulated in vitro withliving mycobacteria (15). In addition, cells exposed to virulentM. tuberculosis (H37Rv) had a significant reduction in cytokineresponse compared to those stimulated with the attenuatedstrain H37Ra (15).

We have cloned the guinea pig IFN-� cDNA from con-canavalin A (ConA)-stimulated spleen cells by using a humanIFN-� cDNA probe. The cDNA was used to generate a probefor Northern blot analysis to assess mRNA expression inguinea pig cells stimulated in vitro with living mycobacteria.The purpose of the present study was to determine whether theIFN-� mRNA response is enhanced after BCG vaccinationand whether the response is altered in cells stimulated in vitrowith virulent M. tuberculosis.

MATERIALS AND METHODS

Screening of a guinea pig cDNA library. Construction of cDNA libraries fromConA-stimulated guinea pig spleen cells was previously described (47). A guineapig genomic library was purchased from Stratagene, La Jolla, Calif.

Cloning of cDNA. Approximately 5 � 105 recombinant phages from the guineapig spleen cell cDNA library were screened by high-density plaque hybridizationwith a 32P-labeled human IFN-� cDNA probe. Hybridization to nitrocellulosefilters 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’ssolution, 1% sodium dodecyl sulfate (SDS), 100 �g of heat-denatured shearedsalmon sperm DNA/ml, and 106 dpm of the probe/ml. The filters were washedtwice with 2� SSC–0.1% SDS at room temperature for 15 min each time andonce at 50°C for 30 min. The filters were dried and exposed overnight to XR-5film (Kodak) with an intensifying screen at �80°C. Phagemids carried withinlambda ZAP II recombinants were rescued with helper phage. DNA sequencingwas performed directly from double-stranded DNA by the dideoxynucleosidetriphosphate chain termination method (38).

Animals. Outbred Hartley strain guinea pigs weighing 200 to 300 g werepurchased from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.).The animals were housed individually in polycarbonate cages in a temperature-and humidity-controlled environment; ambient lighting was automatically con-trolled to provide 12-h-light–12-h-dark cycles. Animals were given commercialchow (Ralston Purina, St. Louis, Mo.) and tap water ad libitum. All procedureswere reviewed and approved by the Texas A&M University Lab Animal CareCommittee.

Antibodies. The monoclonal antibodies (MAb) to the guinea pig surface mark-ers 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 pigCD8, 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 wasreconstituted with Sauton’s medium (Statens Seruminstitut) just before use.

Preparation of spleen and lymph node cells. The procedures for preparationof 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-vaccinatedgroups were euthanized by the intraperitoneal injection of 100 mg of sodiumpentobarbital (Sleepaway; Fort Dodge Laboratories Inc.)/kg of body weight. Thespleens were removed aseptically for the preparation of a single-cell suspensionin RPMI medium (Irvine Scientific, Santa Ana, Calif.). The inguinal lymph nodesfrom the right and left flanks draining the site of BCG vaccination were removedand processed in the same manner. The red blood cells were depleted by usingACK lysing buffer (0.14 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA [pH 7.2to 7.4]), and the remaining cells were thoroughly washed and then suspended inRPMI medium supplemented with 2 �M glutamine (Irvine Scientific), 0.01 mM2-mercaptoethanol (Sigma, St. Louis, Mo.), 100 U of penicillin (Irvine Scientific)per ml, 100 �g of streptomycin (Irvine Scientific) per ml, and 10% heat-inacti-vated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, Ga.). After the cellviability was determined by staining with trypan blue, the cells were cultured in50-ml polypropylene tubes at a concentration of 2 � 106 cells/ml in a totalvolume of 5 ml at 37°C in 5% CO2.

Harvesting of resident peritoneal cells. Guinea pigs were anesthetized by theintramuscular injection of ketamine hydrochloride (30 mg/kg) and xylazine (2.5mg/kg) and then euthanized by cardiac injection of sodium pentobarbital (100mg/kg). The resident macrophages from the peritoneal cavity were harvested byflushing the cavity three times with 20 ml of RPMI medium containing 20 U ofheparin. The cells were washed in RPMI medium, counted, and suspended at5 � 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 in100-mm-diameter plastic petri dishes. The nonadherent cells were removed, andthe population of the monolayers thus obtained consisted predominantly ofmacrophages (more than 95%), as visualized by nonspecific esterase staining(46).

Purification of T cells. Two methods were used to obtain cell populations withenhanced numbers of T cells.

(i) Panning. The nonadherent cells from spleens were purified by panning onplastic plates (100 mm) coated with anti-guinea pig immunoglobulin G (IgG;Sigma) and were termed pan T cells. The panning plates were prepared bydelivering 12 ml of 0.1 M Trizma buffer (pH 9.0; Sigma) onto each plate andadding 25 �l of the reconstituted antibody in physiological saline (2 mg/ml). Theplates were shaken for 1 min and then incubated overnight at 4°C. Just beforeuse, 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 aconcentration 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 nonad-herent 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 at4°C. The pellet was then resuspended in HBSS containing 1% FBS, the numberof viable cells was determined, and the cells were adjusted to a final concentra-tion of 5 � 106 cells/ml.

(ii) Nylon wool purification. The column was prepared by packing 0.5 g ofscrubbed and combed ready-for-use nylon wool fiber (Polysciences Inc., War-rington, Pa.) into a 10-ml syringe and autoclaving for 15 min. The column waswashed with RPMI medium containing 10% fetal calf serum and incubated at37°C for 1 h, after which it was loaded with 1 � 108 to 2 � 108 viable cells in avolume of 2 ml. The loaded column was incubated for 1 h at 37°C, and thenonadherent cells were collected by using two 50-ml washes. The collected cellswere centrifuged at 250 � g for 10 min, the cell pellet was resuspended in RPMImedium containing 10% fetal calf serum, and the viable cells were counted. Thepurity of cells obtained after panning or nylon wool purification was checked byfluorescence-activated cell sorter (FACS) analysis, and the percentage of T cellswas found to be 60 to 70% and 90%, respectively.

Flow cytometry. Cells were stained with MAb against guinea pig pan T cellsand 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 centrifugationat 200 � g for 10 min at 4°C. The supernatant was removed, and the pellet wasresuspended in 50 �l of the appropriate dilution of primary anti-guinea pig T cellantibody (1:500), anti-CD4 (1:500), or anti-CD8 (1:1,000) and incubated for 1 hin ice on a shaker. At the end of the incubation, the cells were washed three timesin HBSS containing 10% FBS. The pellet was resuspended in a 1:10 dilution ofthe secondary antibody (fluorescein isothiocyanate [FITC]-conjugated Affi-niPure goat anti-mouse IgG [heavy plus light chains]; Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) and incubated for 1 h in ice on a shaker. Thecells were then washed two times and finally suspended in 300 �l of HBSScontaining 1% paraformaldehyde. The tubes were then covered with aluminumfoil 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 flowcytometer and Cell Quest software (Becton Dickinson, San Jose, Calif.). Foranalysis of CD4�- and CD8�-T-cell proportions, cells were gated on the lym-phocytes by using the forward and the side-scatter parameters. Both unstainedand secondary antibody controls were included, and the positive population wasset so that less than 2% of the negative control was positive for green fluores-cence.

Stimulation of cells. For the coculture experiments, the nonadherent cells andmacrophages were mixed at a ratio of 3:1. The whole spleen cells (2 � 107) andpan 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 increasingdoses 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 amultiplicity of infection (MOI), and various MOIs were used to stimulate thespleen and lymph node cells. Attenuated M. tuberculosis H37Ra (ATCC 25177;American Type Culture Collection, Rockville, Md.) was used at MOIs of 0.05 to10, and virulent M. tuberculosis H37Rv (ATCC 27294) was used at MOIs of 0.05to 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 mycobacterialsuspension was briefly sonicated before the addition of an appropriate volumefrom a stock of 108 CFU per ml. All the cultures were stimulated for 24 h, andat the end of the incubation period, the medium was removed by centrifugationand the cells were used for RNA extraction.

RNA isolation and Northern analysis. The methods used for RNA isolationand Northern analysis were described previously (15). Total RNA was extractedby using the Trizol reagent (Life Technologies, Grand Island, N.Y.) as per themanufacturer’s instructions. The RNA pellet was suspended in sterile 0.1%diethyl pyrocarbonate (Sigma)-treated distilled water and stored at �80°C. ForNorthern analysis, 8 to 12 �g of denatured RNA from approximately 15 to 20million cells was separated electrophoretically on 1.2% agarose–formaldehydegels. The separated RNA was then transferred to nylon membranes, and themembrane was prehybridized in a solution containing 30% formamide, 5� SSC,50 �g of sheared denatured salmon sperm DNA/ml, 5� Denhardt’s solution, and0.5% SDS for 2 to 3 h at 37°C as described previously (15, 48). The IFN-� cDNAfrom the clone was 32P labeled by random priming (Amersham PharmaciaBiotech Inc., Piscataway, N.J.) with 25 ng of DNA according to the manufactur-er’s instructions. The unincorporated nucleotides were removed with G-50 Seph-adex columns (5 Prime33 Prime Inc., Boulder, Colo.). The membrane washybridized overnight in the prehybridizing solution that contained the guinea pigIFN-� 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% SDSfor 30 min at 50°C. The blots were analyzed with a phosphorimager. Followinganalysis, the blots were stripped and reprobed with 18S antisense RNA as aninternal standard to ensure equal RNA loading. The sums of counts above thebackground were analyzed by using Imagequant software. Data are presented asthe percentage of basal (unstimulated naïve) levels calculated by using theformula {[(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 resultsfrom all replicate experiments and the combined densitometric analysis of eachset of three or more independent experiments. The densitometric results aregiven relative to 100% of basal levels for unstimulated naïve or unstimulatedBCG-vaccinated cultures.

Statistics. The densitometric data are expressed as the means � the standarderrors of the means (SEM). The main effect of vaccination was analyzed byanalysis of variance (ANOVA). The significant differences between the naïve andBCG-vaccinated groups were determined by either the Bonferroni type F test orHsu’s test (23).

Nucleotide sequence accession number. The nucleotide sequence for guineapig IFN-� clone 4b has been submitted to GenBank and assigned accessionnumber AY151287.

RESULTS

Cloning of guinea pig IFN-� cDNA. Approximately 5 � 105

phage clones from a ConA-stimulated spleen cell cDNA li-brary were screened for guinea pig IFN-�. After several roundsof screening with a 32P-labeled human IFN-� cDNA probe andDNA 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 clone4b. The open reading frame of the cDNA encoded a putative166-amino-acid protein that showed 56 and 41% amino acidsequence similarity to human and mouse IFN-�, respectively(Fig. 1).

IFN-� mRNA response of spleen cells to attenuated andvirulent mycobacteria. Whole spleen cells from naïve andBCG-vaccinated guinea pigs were stimulated in vitro for 24 hwith various doses of viable attenuated and virulent M. tuber-culosis strains. As high numbers of virulent M. tuberculosisbacteria are known to be cytotoxic, we used a lower range ofdoses of the virulent strain H37Rv (MOIs of 0.25 to 1.5) thanof the attenuated strain H37Ra (MOIs of 1 to 10). Figure 2illustrates the Northern analysis of IFN-� mRNA (Fig. 2A)and the densitometric evaluation of the blots (Fig. 2B). Thespleen cells from naïve animals stimulated in vitro with thevirulent or attenuated M. tuberculosis showed a very low levelof IFN-� mRNA transcripts. In contrast, spleen cells fromBCG-vaccinated animals exhibited a statistically significant(P 0.0001) level of IFN-� mRNA expression after stimula-tion with either mycobacterial strain. However, the exposure ofsplenocytes 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 ofthe spleen cells treated with any dose of mycobacteria wasmore than 95% after 24 h in culture, as determined by thetrypan blue exclusion assay or by the MTT [3-(4,5-cimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (13).

IFN-� mRNA expression in spleen cells stimulated withidentical doses of attenuated and virulent M. tuberculosisstrains. In the previous experiment, the doses of attenuatedand virulent strains of M. tuberculosis used for stimulation werenot identical. Moreover, there was a dose-dependent reductionin the response to the H37Rv strain. In this experiment, spleencells from BCG-vaccinated guinea pigs were stimulated withidentical doses (MOIs of 0.15 to 0.675) of attenuated andvirulent M. tuberculosis strains for 24 h. As evident from North-ern blotting and densitometric analysis (Fig. 3), there was noclear dose response to the two strains. MOIs of M. tuberculosisH37Ra lower than 0.675 did not induce any statistically signif-icant levels of IFN-� mRNA expression in the spleen cellsduring this short period in culture. In contrast, exposure ofsplenocytes to the same doses of the virulent H37Rv strain ofM. tuberculosis induced a significant increase in the levels ofmRNA expression in the spleen cells (P of 0.01 to 0.0001).These results indicated that the induction of IFN-� mRNA inspleen cells is dependent on the doses of attenuated and vir-ulent M. tuberculosis strains used for stimulation.

IFN-� mRNA expression in lymph node cells. Cells from theinguinal lymph nodes draining the vaccination sites in BCG-vaccinated and naïve guinea pigs were stimulated with a lowdose (MOI, 0.05) of various strains of attenuated and virulentmycobacteria (BCG, M. avium, and M. tuberculosis strainsH37Ra, H37Rv, and Erdman). This dose had induced IL-1�and RANTES mRNA expression in macrophages (15). Figure4 shows the results of Northern blotting (Fig. 4A) and densi-tometric analysis (Fig. 4B). Lymph node cells from naïve ani-mals expressed no IFN-� mRNA in response to mycobacteria

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(data not shown). Stimulation of lymph node cells from BCG-vaccinated guinea pigs with BCG or attenuated M. tuberculosisH37Ra induced low levels of IFN-� mRNA which were notstatistically different from those of the unstimulated cells.However, the IFN-� mRNA levels were significantly higherafter 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 anMOI of 0.05. PPD also induced strong IFN-� mRNA expres-sion in the lymph node cells of BCG-vaccinated guinea pigs,and the response was significantly higher in these cells at dosesof 5 and 10 �g (P of 0.003 to 0.004) than that in the unstimu-lated controls.

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



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Kinetics of IFN-� mRNA induction. Spleen cells from BCG-vaccinated guinea pigs were stimulated with PPD (15 �g/ml) orthe virulent H37Rv strain of M. tuberculosis (MOI, 0.05) forvarious periods of time up to 48 h. Compared to controls, cellsstimulated with PPD showed no significant levels of IFN-�mRNA until 18 h after stimulation (P 0.0001), when theresponse peaked and then dropped by 24 h (P 0.0009) and48 h (P 0.009) (Fig. 5). After stimulation with virulent M.tuberculosis, no significant IFN-� mRNA levels were seen at4 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. ThemRNA response induced in the spleen cells by the wholemycobacteria was significantly higher and lasted longer thanthe 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-vacci-nated guinea pigs, when stimulated in vitro with PPD or livingmycobacteria, showed enhanced IFN-� mRNA expressioncompared to that of the cells from naïve animals. We hypoth-esized that this enhanced response was due to an alteration inthe proportions of lymphoid cells in the spleens of BCG-vac-

cinated guinea pigs. The nonadherent cells from the spleens ofboth naïve and BCG-vaccinated animals were obtained by pan-ning on anti-guinea pig IgG-coated plates. The proportions ofT cells in these populations were determined by FACS analysisafter staining the cells with MAb directed against T cells (anti-guinea pig pan T cells) and their subsets (anti-CD4 and anti-CD8). The binding of primary antibody was detected withFITC-labeled goat anti-mouse IgG. There was no difference inthe proportions of T cells in the naïve and BCG-vaccinatedguinea pigs (Fig. 6). However, the proportions of CD4� T cellswere significantly increased (P 0.01) after BCG vaccination.CD8�-T-cell proportions remained unaltered statistically, al-though a slight drop (P 0.054) was apparent in the BCG-vaccinated group.

IFN-� mRNA expression in purified T cells. In order toidentify the splenic cells responsible for IFN-� mRNA expres-sion, the nonadherent cells in the spleens of naïve and BCG-vaccinated guinea pigs were enriched either by panning onanti-guinea pig IgG-coated plates or by purification on nylonwool columns. Whole spleen cells or enriched T cells cocul-tured with autologous, adherent peritoneal macrophages at aratio of 3:1 were stimulated in the presence of ConA (10

FIG. 2. Effect of BCG vaccination on IFN-� mRNA expression in spleen cells of guinea pigs. Whole spleen cells (2 � 107) from naïve andBCG-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 aphosphorimager. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of IFN-�. The densitometric resultsfor 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 Ftest. **, P was 0.0001 in comparisons between the stimulated groups and the unstimulated (cells alone) controls and between H37Ra- andH37Rv-stimulated groups. §, P was 0.0001 when naïve and BCG-vaccinated groups were compared.



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�g/ml) or PPD (15 �g/ml) for 18 to 24 h. Figure 7 shows theIFN-� mRNA expression in whole spleen cells, panned T cells,and nylon wool-purified T cells in response to the two stimu-lants. The spleen cells from naïve guinea pigs, whether unsepa-rated (P 0.002) or purified by panning (P 0.0003) or witha 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 signifi-cant IFN-� mRNA expression in response to ConA (P of0.003 to 0.0001). The levels were increased significantly afterPPD stimulation only in pan T (P 0.01) and nylon wool-purified (P 0.0001) populations. The nylon wool-purified Tcells exhibited a significantly greater IFN-� mRNA responseboth to ConA (P of 0.004 to 0.0001) and to PPD (P 0.0001). These results indicate that T cells are principally re-sponsible for IFN-� mRNA expression in the spleens of BCG-vaccinated guinea pigs.

IFN-� mRNA expression in T cell–macrophage coculturesinfected with M. tuberculosis. T cells obtained from the spleensof BCG-vaccinated guinea pigs by panning on anti-IgG-coatedplates were cocultured with autologous peritoneal macro-phages at a T cell/macrophage ratio of 3:1. The cocultures wereexposed 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 combi-nation were stimulated with PPD (15 �g/ml). Figure 8 illus-trates that T cells alone (P 0.0003) or in combination with

macrophages (P 0.03), when stimulated with PPD, showedsignificantly higher levels of IFN-� mRNA expression than theunstimulated cultures. Macrophages alone after stimulationwith PPD produced no IFN-� mRNA, in contrast to the un-stimulated cultures. As observed with the whole spleen cells,the attenuated M. tuberculosis H37Ra strain at the doses em-ployed was incapable of inducing IFN-� mRNA transcripts inthe cocultures of T cells and macrophages. However, the samedoses of the virulent H37Rv strain of M. tuberculosis induced ahighly significant (P 0.0001) mRNA response in these cul-tures. These results indicate that the virulent strain of M.tuberculosis induces IFN-� mRNA expression in whole spleencells or in purified T cells when used at a ratio of 1 bacteriumto 10 or fewer cells.

DISCUSSION

IFN-� is a T-cell-derived cytokine with broad macrophage-activating effects which is known to play a critical role in an-timycobacterial immunity (11, 33). The guinea pig model oflow-dose pulmonary TB has contributed much to our general

FIG. 3. IFN-� mRNA expression in spleen cells from BCG-vacci-nated guinea pigs. Cells were stimulated with identical MOIs (0.15 to0.675) of attenuated and virulent M. tuberculosis strains for 24 h. TheRNA was processed as described above. (A) RNA blot representativeof results from several experiments; (B) densitometric analysis of theresults from three experiments. In comparisons between stimulatedgroups and unstimulated (cells alone) controls, P was 0.03 (†),0.003 to 0.002 (*), or 0.0003 to 0.0001 (**).

FIG. 4. Effect of in vitro stimulation on IFN-� mRNA expression inthe lymph node cells of BCG-vaccinated guinea pigs. Cells from theinguinal lymph nodes (2 � 107) in both flanks draining the site of BCGvaccination were stimulated in vitro for 24 h with an MOI of 0.05 of thefollowing strains of the attenuated and virulent mycobacteria: BCG, M.avium, and M. tuberculosis strains H37Ra, H37Rv, and Erdman. Thecells 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 was0.005 to 0.002 in comparison with results for unstimulated (cellsalone) cultures.



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understanding of the mechanisms of vaccine-induced resis-tance (29, 31). To enhance the value of this model, we havecloned the guinea pig IFN-� cDNA from ConA-stimulatedspleen cells by using a human IFN-� cDNA probe. Guinea pigIFN-� cDNA is composed of 1,110 bp, with an open readingframe that encodes a putative 166-amino-acid protein. Thenucleotide sequences are nearly identical to the sequences(positions 40 through 1167) submitted by others for guinea pigIFN-� (GenBank accession number E25787) except for onesubstitution of C for T (base 303 in our sequence); however,this substitution does not alter the amino acid sequence. Weused the cDNA clone to generate a probe for Northern blotanalysis to assess IFN-� mRNA expression in the lymphoidcells of BCG-vaccinated and nonvaccinated guinea pigs. Theresults presented here indicate that both vaccination and invitro stimulation affected the level of IFN-� mRNA expressionin whole lymphoid cell populations and purified T cells.

Previously, our laboratory reported that cell-mediated im-mune responses in guinea pigs as measured by delayed-typehypersensitivity and the ability of lymphocytes to proliferateand produce IL-2 in response to PPD were augmented afterBCG vaccination (29, 30). These responses were consistentwith the ability of animals to develop protective responsesagainst aerosol infection with virulent M. tuberculosis (28).

Recently, it has been reported that IL-1� and RANTESmRNA responses were enhanced in BCG-vaccinated guineapigs after the stimulation of spleen cells or macrophages invitro with PPD or living mycobacteria (15). Because IFN-� isconsidered to play a pivotal role in antimycobacterial immunity(11, 33), we studied whether BCG vaccination would increasethe IFN-� mRNA response in spleen cells. The results areconsistent with our hypothesis that IFN-� mRNA expression inspleen cells is enhanced after BCG vaccination in response toliving mycobacteria and their protein antigens. In clinical trialsof BCG vaccination, there is ample evidence to demonstratethat cell-mediated immune responses in BCG recipients, in-cluding the production of IFN-�, are enhanced compared tothose in control subjects (14, 39).

Earlier, it was demonstrated that the exposure of spleen cellsor macrophages to virulent M. tuberculosis decreased the cy-tokine mRNA responses for both IL-1� and RANTES com-pared to those induced by the exposure of the same cells to theattenuated strain (15). The results illustrated in Fig. 2 indicatethat there was a dose-dependent and significant decrease in theIFN-� mRNA response in spleen cells after stimulation withhigh doses of the virulent M. tuberculosis strain. The reductionin the mRNA response occurred in the absence of cytotoxicity,as the viability of the cells exposed to mycobacteria was morethan 95%, a fact confirmed by standard cell staining methods.

There is considerable evidence to indicate that virulent andattenuated mycobacteria induce differential levels of cytokinesin macrophages. The levels of TNF-� or IL-1� produced aftermycobacterial stimulation seem to depend on the virulence ofthe organism, and it has been postulated that suppression of

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

FIG. 6. Proportions of T cells and T-cell subsets in the spleens ofguinea pigs. Spleen cells from naïve and BCG-vaccinated guinea pigswere purified by panning on anti-guinea pig IgG-coated plates andstaining for red fluorescence with MAb directed against the surfacemarkers of T cells (CT5), CD4� T cells (CT7), and CD8� T cells(CT6). The binding of primary antibody was detected with FITC-conjugated goat anti-mouse IgG. The proportions of positive cellswere determined with a FACSCalibur flow cytometer and Cell Questsoftware. The significant differences between the naïve and BCG-vaccinated groups were determined by Student’s t test. �, P was 0.01when the proportions of CD4� cells from naïve and BCG-vaccinatedgroups were compared.



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protective host cytokines might be related to pathogenesis. Forexample, the attenuated H37Ra strain of M. tuberculosis in-duced a larger amount of NO in cultured human peripheralblood mononuclear cells than did the virulent H37Rv strain(24). In both control and AIDS patients, more IL-1� wasreleased after the infection of monocytes with the less virulentM. avium (16). Similarly, infection of human monocytes ormacrophages with virulent M. avium downregulated the pro-duction of proinflammatory cytokines (IL-1�, TNF-�, IL-6,and granulocyte-macrophage colony-stimulating factor) com-pared to that by cells infected with the less virulent strain (32).Studies with mice revealed that lipoarabinomannan derivedfrom an attenuated strain of M. tuberculosis induced macro-phage activation and TNF-� production, whereas lipoarabino-mannan from the virulent Erdman strain was less stimulatory(2). The inability to induce the appropriate mediators in lym-phocytes and macrophages might be one of the mechanisms bywhich some mycobacteria evade or suppress the immune re-sponse (32). It is not clear whether virulent and attenuated M.tuberculosis strains induce differential IFN-� responses in hu-man or animal systems. However, human monocytes cocul-

tured with the virulent smooth-transparent M. avium strainproduced lower concentrations of IFN-� and IL-18, a potentIFN-� inducer, in the culture supernatants than did thosecocultured with the avirulent smooth-domed M. avium strain(43). Similarly, mice infected with highly virulent or avirulentM. avium demonstrated that the control of infection with thelow-virulence strain was associated with an increased expres-sion of IFN-� and IL-2 compared to that in mice infected withthe more virulent strain (4). The present results clearly dem-onstrate that attenuated and virulent M. tuberculosis strains failto induce a significant IFN-� mRNA response in guinea pigcells unless the cells are stimulated with appropriate doses ofbacteria and that the virulent M. tuberculosis H37Rv induces adose-dependent inhibition of the IFN-� mRNA response inspleen cells. It is well documented that IFN-� does not activatehuman macrophages to kill virulent M. tuberculosis. It wasfound that M. tuberculosis inhibited transcriptional responseswithout inhibiting activation of STAT-1. This was due to amarked decrease in the IFN-�-induced association of STAT-1with the transcriptional coactivators in M. tuberculosis-infectedmacrophages (45).

Humans with active TB or with multidrug-resistant TB areknown to have diminished IFN-� responses. In patients withactive pulmonary TB, the frequency of IFN-�-secreting CD4�

T cells was lower than that in healthy PPD-positive contacts orsubjects with minimal disease and low bacterial burdens (34).Similarly, multidrug-resistant TB patients with low CD4� Tcells from peripheral blood mononuclear cells had impairedIFN-� responses to M. tuberculosis, PPD, or mitogens com-pared to those of healthy PPD-positive and PPD-negative in-dividuals (26).

The attenuated H37Ra strain of M. tuberculosis was efficientin inducing a significant level of IFN-� mRNA expression onlyat high doses at which virulent M. tuberculosis induced a sig-nificantly weaker response (Fig. 2). M. tuberculosis H37Rv in-duces IFN-� mRNA expression in whole spleen cells or inpurified 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. tuber-culosis did not induce any significant IFN-� mRNA expressionin these cells at the same doses (Fig. 3 and 8). A significantlevel of IFN-� mRNA expression was observed only when cellswere stimulated with the attenuated strain at a bacterium-to-cell ratio of more than 1 (Fig. 2). Either no expression or lowlevels of IFN-� mRNA expression were seen when these cellswere stimulated with a smaller dose (Fig. 3, 4, and 8).

In order to investigate whether an alteration in the propor-tions of lymphoid cells in the spleen after BCG vaccination wasresponsible for enhanced IFN-� mRNA expression, the cellsthat were nonadherent after panning were used for phenotypicexamination by FACS analysis. The T cells and their subsetswere stained with MAb directed against the cells’ surfacemarkers. The proportions of CD4� cells were significantly in-creased in the BCG-vaccinated guinea pigs (Fig. 6), while totalT-cell and CD8�-T-cell profiles remained unaltered. It isknown that immune responses against M. tuberculosis are me-diated by CD4� T cells, although recent evidence indicatesthat CD8� cells also contribute to antimycobacterial immunity(12, 33). It is quite likely that an increase in the CD4� T cells

FIG. 7. IFN-� mRNA expression by purified T-cell–macrophagecocultures. Whole spleen cells (2 � 107) or cocultures of panned Tcells or nylon wool-purified T cells plus peritoneal macrophages (Mø)at a ratio of 3:1 were examined in naïve and BCG-vaccinated guineapigs. T cells were purified either by panning on anti-guinea pig IgG-coated plates or on nylon wool columns. Resident peritoneal macro-phages 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 describedin the text. (A) RNA blot representative of results from several exper-iments; (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 Bonferronitype F test. In comparisons between stimulated groups and unstimu-lated (cells alone) controls, P was 0.01 (†), 0.002 (*), or 0.0003 to0.0001 (**).



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might contribute to the enhanced expression of IFN-� mRNAin spleen cells and lymph node cells after BCG vaccination.

The type of cells that might be responsible for the inductionof IFN-� mRNA was investigated by purifying T cells fromwhole splenocytes either by panning or passing the splenocytesthrough nylon wool columns. As evident from the FACS anal-ysis, it was clear that there was a higher percentage of T cellsin the nylon wool-purified population than among the cellsobtained after panning. The nylon wool-purified T cells ex-pressed highly significant levels of IFN-� mRNA in response toConA and PPD (Fig. 7). Similarly, the panned T cells hadmuch higher levels of IFN-� mRNA transcripts in response toM. tuberculosis than the whole spleen cells (Fig. 2 and 8).Although IFN-� is mainly produced by CD4� T cells, CD8� Tcells, macrophages, and NK cells are also known to producethis cytokine after mycobacterial stimulation (41). Macro-phages from bronchoalveolar lavage fluid of TB patientsshowed IFN-� protein mRNA expression; however, the major-ity of IFN-� mRNA was detected in lymphocytes after lavage(37). In other studies, in vitro infection of alveolar macro-phages with M. tuberculosis induced IFN-� mRNA expressionand the release of IFN-� protein (44). High levels of IFN-� andTNF-� are found in the pleural fluid of TB patients, and, infact, IFN-� is considered to be a useful marker for diagnosingtuberculous pleurisy (36).

Despite the vast literature on the macrophage-activatingeffects of IFN-� in humans and mice, relatively few studies

have addressed this question in guinea pigs. BCG vaccinationof inbred guinea pigs induced RANTES and IFN-� mRNA intheir spleens, as detected by reverse transcription-PCR; thiswas also observed in naïve guinea pigs. Surprisingly, onlyCD8� T cells from the lymph nodes of vaccinated animalsexpressed RANTES and IFN-� mRNA, and not CD4� cells(22). Unlike our studies, the earlier study used strain 2 guineapigs that were vaccinated with a much higher dose of BCG (2� 107 CFU) and investigated the IFN-� mRNA expression 8days postvaccination by reverse transcriptase PCR. Further-more, the authors of that study analyzed the cytokine mRNAexpression directly from spleen and lymph node cells withoutfurther in vitro stimulation and, therefore, could not determinehow much of the IFN-� response was actually specific to my-cobacteria.

We observed an enhanced IFN-� mRNA response in thespleen which was associated with a significant increase in theproportions of CD4� T cells. We have also provided evidencewhich suggests that T cells in the spleen are responsible pri-marily for IFN-� mRNA induction. Studies are already underway to address the IFN-� mRNA responses in BCG-vaccinatedguinea pigs following low-dose aerosol infection with virulentM. tuberculosis. With the development of recombinant guineapig IFN-� and anti-guinea pig IFN-� antibodies, we will beginto elucidate the contributions of this important cytokine to thecomplex interaction between mycobacteria and the host’s pro-tective immune response.

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 withPPD (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 describedin the text. (A) RNA blot representative of results from several experiments; (B) densitometric analysis of results from three experiments (themeans � 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).



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ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant RO1AI 15495 to D.N.M.

We are indebted to Jane Miller for her help and expertise in FACSanalysis and Gregory Foster for the critical evaluation of the manu-script.

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