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Tuberculosis (2001) 81(5/6), 327I334 ^ 2001 Harcourt Publishers Ltd doi: 10.1054/tube.2001.0306, available online at http://www.idealibrary.com.on
CD4ⴙ lymphocyte responses to pulmonary infection with Mycobacterium tuberculosis in naeK ve and vaccinated BALB/c mice C. M. Mason, E. Dobard, J. Shellito, S. Nelson Pulmonary/Critical Care Medicine, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA, USA
Summary Setting: Local CD4# lymphocyte responses are critical in determining the fate of infection with Mycobacterium tuberculosis. Objective: To evaluate the lung and regional lymph node CD4# lymphocyte responses to pulmonary infection with M. tuberculosis H37Rv in BALB/c mice, with and without prior vaccination with M. bovis Bacille Calmette-GueH rin (BCG). Design: Lung and lung-associated lymph node (LALN) CD4# lymphocytes were isolated from BALB/c mice infected with low dose M. tuberculosis H37Rv by the intratracheal route in BCG-vaccinated and unvaccinated animals, and cytokine responses assessed in vitro. Results: CD4#lymphocytes increased in both the lung and LALN by 21 days after infection, and elicited production of interferon (IFN)-c was upregulated in the lymphocytes of the infected animals. Only low levels of interleukin (IL)-4 and IL-10 were elicited from the lung. The IFN-c response from both sites was facilitated in the previously vaccinated animals. Conclusion: In this model of low dose inoculation of M. tuberculosis in the lung, BALB/c mice respond with a Th1-biased CD4# lymphocyte response, which is accentuated in animals previously vaccinated with BCG. ^ 2001 Harcourt Publishers Ltd INTRODUCTION Infection with Mycobacterium tuberculosis is at epidemic levels globally. It is the most prevalent infectious disease worldwide, and annually there are eight million people who acquire the infection, with three million succumbing due to it.1 M. tuberculosis is an intracellular organism that promotes the development of CD4#lymphocyte-orchestrated Th1 cell-mediated immunity. Th1 immunity is characterized by speci9c CD4#lymphocytes that elaborate the cytokines interleukin (IL)-2 and interferon (IFN)-c, which function to expand the CD4#lymphocyte population, activate local macrophages for mycobactericidal activity, and confer protective immunity.2, 3 This is a unique infection in that, even in the face of established immunity, the pathogen may not be eliminated, but remains present in a state of latency.4 In a small percentage of those with latent infection, the disease can reactivate, with development of overt infection. Appropriate treatCorrespondence to: C. M. Mason, MD, Pulmonary/Critical Care Medicine, 1901 Perdido C3205, New Orleans, LA 70112, USA. Accepted: 6 September 2001
ment with antimicrobial agents usually successfully eliminates the organism, but requires several months to do so. As the lung is the primary site of inoculation for most infections with M. tuberculosis, the immune response of lung cells is critical in containing the organism. We used a BALB/c murine model of M. tuberculosis pulmonary infection, and studied the lung and lung-associated lymph node (LALN) CD4#lymphocyte responses serially during the course of infection, in mice with and without prior vaccination with BCG. We found evidence that Th1 responses predominate in both the lung and the LALN, concurrently with growth restriction of the organism, con9rming the critical role of CD4#lymphocytes in this infection. With prior vaccination, the Th1 response was facilitated in the lung, accompanied by greater growth restriction.
MATERIALS AND METHODS Animals Speci9c pathogen-free BALB/c mice (Hilltop Labs, Scottdale, PA) were used for these experiments. Animals were
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housed in the LSUHSC Pulmonary/Critical Care Medicine Biocontainment Level-3 Laboratory and experiments were performed in the Biocontainment Level-3 Laboratory in accordance with appropriate safety precautions recommended by the CDC.5 All animal procedures were approved by the LSUHSC institutional Animal Care and Use Committee. All data represent groups of n"5 unless otherwise noted.
M. tuberculosis H37Rv Infection M. tuberculosis H37Rv was obtained from ATCC (Rockville, MD, catalog C27294), and was grown in Middlebrook 7H11 broth at 373C for 14 days. This culture was concentrated by centrifugation, gently sonicated, and stored in 0.1 ml aliquots at !803C. At the time of inoculation, an aliquot was thawed, gently sonicated, and diluted in endotoxin-free PBS to a concentration of 1;102 organisms/ml. Mice were lightly anesthetized with ketamine/xylazine (200 mg/kg/10 mg/kg) intraperitoneally (IP), the ventral surface of the neck swabbed with isopropyl alcohol, and a midline skin incision performed in a sterile fashion. The soft tissues of the neck were gently retracted laterally to expose the trachea. Mycobacteria were sonicated for 10 s prior to injection to achieve uniform single organism dispersion. Direct intratracheal (IT) injection was performed, injecting 100 ll of bacterial suspension (101 organisms) through a 30 gauge needle, followed by 0.3 ml of air. The incision was closed with a stainless steel surgical clip, and the animal allowed to recover. The TB group comprised animals inoculated IT with mycobacteria, and the Control group comprised those inoculated with PBS rather than mycobacteria. At serial time points (Days 7, 14, 21, 28, 35 and 42) after injection, animals were sacri9ced and lungs were homogenized, serially diluted, and plated in quadruplicate on Middlebrook 7H10 agar plates. The plates were incubated at 373C for 17 days. At the end of the incubation period, the number of colony-forming units (CFU) present on plates containing between 20 and 200 CFUs/quadrant was counted and multiplied by the appropriate dilution in order to determine the number of CFU in the initial tissue homogenate.
Vaccination with M. bovis BCG M. bovis BCG was obtained from ATCC (Rockville, MD, catalog C35734). Animals were administered M. bovis BCG subcutaneously (1;106 cfu) 28 days prior to IT inoculation with Mtb H37Rv,6 which is performed as described above. Serial cultures were performed as above. In this group, vehicle vaccinated animals comprise the Mtb group, and vaccinated animals the BCG/Mtb group. Tuberculosis (2001) 81(5/6), 327}334
Isolation of lung and lung-associated lymph node (LALN) lymphocytes Lung and LALN lymphocytes were isolated according to the following methods.7 Lungs were removed from mice in all groups at serial times after sacri9ce, and the conducting airways were removed. Lung tissue was minced, placed in RPMI-1640 containing collagenase (150 U/ml) and DNAse (50 U/ml) for enzymatic digestion. Ten ml of the enzymatic solution was used for every 600 mg lung tissue, and the mixture was incubated at 373C for 90 min with constant stirring. After incubation, the mixture was passed through a 70 lm nylon mesh, placed brie:y in NH4Cl lysis buffer, and resuspended in indomethacin (1 lg/ml) and catalase (250 U/ml). Cells were counted, viability checked, and then they were diluted to the desired concentration. LALN were isolated from hilar lymph nodes and mediastinal lymph nodes dissected after sacri9ce. Single cell suspensions were prepared by gently disrupting the tissue on a 70 lm nylon 9lter. Obtained cells were centrifuged prior to resuspending in NH4Cl lysis buffer, to which Hank’s BSS was added. Cells were centrifuged again, counted, and resuspended at 106/ml. CD4#lymphocytes were isolated from both lung cells and LALN cells by magnetic bead selection (Dynal, Inc., Lake Success, NY).8, 9 CD4#lymphocytes isolated by the magnetic bead technique were '95% pure for the LALN population and '90% pure for the lung population when checked by :ow cytometry (data not shown). By performing differential cell counts on cytospin monolayers of isolated lung and LALN cells, the total lymphocyte counts from each site were determined. Likewise, total CD4#lymphocyte counts were calculated by performing differential cell counts on cytospin monolayers from the cells resulting from magnetic bead isolation.
CD4ⴙ lymphocyte cytokine production Cytokine assays were performed by plating isolated CD4#lymphocytes from lung or LALN from the TB or Control group animals at a concentration of 1;105 cells/well, and stimulated with concanavalin A (5 lg/ml) for 48 h. Unstimulated cells from each animal were used as media controls. After the 48 h time period, supernatants were collected, centrifuged to remove cellular debris, frozen at !803C, and subsequently assayed for IFN-c, IL-4, and IL-10 by ELISA (R&D Systems, Minneapolis, MN). The lower limits of detection for these assays were for IFN-c: 2 pg/ml, for IL-4: 2 pg/ml, and for IL-10: 4 pg/ml. For the vaccinated animals, additional cytokine elicitation was performed in vitro with infected peritoneal macrophages.10 These cells were elicited from naive BALB/c mice with concanavalin A (100 lg, Type IV, Sigma, St. Louis, MO), injected IP 96 h prior to peritoneal lavage with ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 329
DMEM at 43C. Harvested cells were washed, counted (with a differential count), checked for viability, and the red blood cells were lysed. Cells were then placed in complete DMEM-10, adjusted to 2;106 cells/ml, and plated in 96 well plates at 2;105 cells/well and allowed to adhere for 2 h at 373C. Cells were then washed three times to remove nonadherent cells. Next, M. tuberculosis H37Rv was added to the macrophage monolayer at 1;106 cfu/well, and incubated for 18}24 h at 373C. Control wells were uninfected. After this time, extracellular bacteria were removed by aspiration with DMEM three times, complete DMEM-10 replaced, and the cell maintained in culture for an additional 24 h. Aliquots of isolated CD4#lymphocytes from LALN and lung of M. tuberculosis-infected animals were suspended in DMEM-10 at 2;106 cells/ml at 373C, and 1;105 cells added to each well of PM, and incubated at 373C for 48 h. After the incubation, supernatants were harvested, spun, and saved at !803C until cytokine assay.
RESULTS Lung infection with M. tuberculosis H37Rv Mice inoculated with M. tuberculosis H37Rv as described above were sacri9ced at Days 7, 14, 21, 28, 35, and 42 after inoculation. The IT inoculum was 2;101 CFU. Lungs were quantitatively cultured at each time point. Control animals inoculated with PBS had no growth of mycobacteria from their lungs at any time point. Growth of M. tuberculosis in the lungs of mice was exponential until approximately Day 21, when the growth rate slowed. Mycobacterial burden subsequently remained fairly constant at +106 cfu/lung for the remaining time points tested (data not shown), as has been previously reported.11
Lung infection with M. tuberculosis H37Rv after BCG vaccination Mice inoculated with M. tuberculosis H37Rv as described above 28 days after BCG vaccination were sacri9ced at Days 14, 28, and 42 after inoculation of M. tuberculosis. The IT inoculum was 2;101 CFU. Lungs were quantitatively cultured at each time point, and the results are shown in Figure 1. There was signi9cant reduction in lung growth of M. tuberculosis in the vaccinated animals at 28 and 42 days compared to the unvaccinated animals.
Lung and LALN CD4ⴙ lymphocytes CD4#lymphocytes were isolated from lung and LALN at serial time points after the M. tuberculosis inoculation in TB group and Control (uninfected) mice. Numbers of CD4#LALN and lung lymphocytes from each time point ^ 2001 Harcourt Publishers Ltd
Fig. 1 Total CFU in lungs of BALB/c mice at serial time points after IT inoculation on Day 0 with 2;101 CFU of M. tuberculosis H37Rv, which was 28 days after SC inoculation with 1;106 M. bovis BCG. Initially, the M. tuberculosis growth rate is similar between the two groups, but there is a significant reduction in M. tuberculosis cfu in the lungs of the BCG vaccinated mice at Day 28 and Day 42. *P(0.05 BCG/Mtb vs Mtb.
are shown in Figures 2A and 2B. The number of CD4#lymphocytes recovered from the LALN and the lung increased signi9cantly in the TB group animals as the infection progressed. LALN and lung CD4#lymphocytes from BCG vaccinated animals are shown in Figures 2C and 2D. There are no differences in the number of LALN CD4#lymphocytes at any time point, but signi9cantly more lung CD4#lymphocytes in the vaccinated animals at all time points after Day 7.
CD4ⴙ lymphocyte cytokine production The time course of lung and LALN CD4#lymphocyte IFN-c production from M. tuberculosis-infected or Control (uninfected) animals is shown in Figures 3A and 3B. The highest levels of IFN-c measured from the LALN CD4#cells occurred at Day 21 in the TB group, and then returned toward baseline by Day 28. In contrast, the lung CD4#lymphocyte IFN-c response was rising signi9cantly by Day 21 in the TB animals, and continued to increase at the Day 28 time point. LALN and lung lymphocytes without Con A stimulation (media controls) had no IFN-c production detectable at any time point studied. The time course of LALN and lung CD4#lymphocyte ConA-elicited IFN-c from BCG vaccinated animals is shown in Figures 3C and 3D. CD4#T cells from BCG-vaccinated animals (from both sites) have earlier peak IFN-c production compared to time-matched unvaccinated controls. Interestingly, the peak IFN-c production by the unvaccinated M. tuberculosis-infected animals’ lymphocytes is delayed until Day 28 for the LALN, and Day 35 for the lung, in contrast to the series presented in Figures 2A and 2B. This may be due to minor variations in the organism Tuberculosis (2001) 81(5/6), 327}334
330 Mason et al.
Fig. 2 CD4# LALN (A) and lung (B) lymphocytes isolated on Days 7, 14, 21, and 28 from mice infected IT on Day 0 with M. tuberculosis (TB group) or Control mice. There are significantly more lymphocytes recovered from the lungs of animals in the TB group at Days 21 and 28 compared to the Control animals. *P(0.05 TB vs Control. CD4#LALN (C) and lung (D) lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (BCG/Mtb group) or vehicle vaccination (Mtb group). There are significantly more CD4#lymphocytes recovered from the lungs of animals in the Mtb/BCG group at Days 14, 21, 28, and 35 compared to the Mtb group. *p(0.05 BCG/Mtb vs Mtb.
inoculation and/or growth curves between the two different experiments, or to the fact that no information is obtained regarding IFN-c production between the reported 7-day time points (due to reasons of practicality). This is the reason, however, that concurrent controls are performed for every experiment. When M. tuberculosisinfected peritoneal macrophages were used to stimulate IFN-c production from the lymphocytes, cells from both sites produced high levels of IFN-c (Figures 4A and 4B), con9rming an M. tuberculosis-speci9c response. There was no IL-4 detected at any time in the stimulated LALN CD4#lymphocytes, nor in the media control samples from either tissue site. Low levels of IL-4 ((300 pg/ml) were detectable in the stimulated lung CD4#lymphocytes at all time points assessed, but no signi9cant differences were noted between the TB and Control groups. IL-10 was also assessed in the supernatants from both lung and LALN stimulated CD4#lymphocytes, and low levels (4300 pg/ml) were detectable in the lung lymphocyte supernatants at all time points assessed. From these results, it is apparent that the magnitude of the IFN-c response is more that 40-fold greater than the levels of Tuberculosis (2001) 81(5/6), 327}334
IL-4 or IL-10, con9rming the Th1 response of the infected animals. In the BCG and vehicle vaccinated animals, lung CD4#lymphocytes from both groups produced low-level IL-4 (4500 pg/ml), and even lower levels of IL-10 (4250 pg/ml), again con9rming the predominant Th1 response induced by M. tuberculosis infection in these animals.
DISCUSSION The work presented here con9rms that pulmonary infection of normal BALB/c mice with a virulent strain of M. tuberculosis is accompanied by high level IFN-c production and lower levels of IL-4 and IL-10 production, signifying the development of Th1 immunity by the CD4#lymphocyte populations in both the lung and the regional lymph nodes. In previous reports, the BALB/c mouse is reported to respond to various antigenic challenges with a Th2 immunity bias,12, 13 but our work clearly shows that in response to challenge with M. tuberculosis, the lung and LALN CD4#lymphocyte immune responses are strongly Th1-biased over the initial 28 days following ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 331
Fig. 3 In vitro IFN-c production (after Con A stimulation) from LALN (A) and lung (B) CD4# lymphocytes isolated on Days 7, 14, 21, and 28 from mice infected IT on Day 0 with M. tuberculosis. There is significantly more IFN-c elicited by LALN CD4# lymphocytes (A) on Days 14 and 21 in the TB group compared to the Control group (*P(0.05 TB group vs Control group). The TB group at Day 14 (n"4) produced 22$14 pg/ml IFN-c, with no IFN-c detectable in the Control group at this time. At Day 21, the TB group produced 2,146$670 pg/ml IFN-c, with no IFN-c detectable in the Control group at this time. For the Day 7 values, n"3 for each group due to the low numbers of LALN CD4# cells. For the lung CD4# lymphocytes (B), there was significantly greater IFN-c elicited in the TB group at Day 7 (484$66 pg/ml TB vs 230$31 pg/ml Control), Day 14 (289$52 pg/ml TB vs 113$45 pg/ml Control), Day 21 (10,251$2,749 pg/ml TB vs 67$16 pg/ml Control), and Day 28 (14, 138$2,557 pg/ml TB vs 292$67 pg/ml Control) than the Control group. *P(0.05 TB group vs Control group. The TB group values of IFN-c (pg/ml) that were low, but statistically different from the Control group, are shown on the graphs, above the respective symbols. (C) shows in vitro IFN-c production (after Con A stimulation) from LALN and (D) shows in vitro IFN-c production (after Con A stimulation) from lung CD4#lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (Mtb/BCG) or vehicle vaccination (Mtb). There is an earlier peak of IFN-c production in the BCG/Mtb group at 21 days for both the LALN (1,308$246 pg/ml) and the lung (34,686$9,256 pg/ml) lymphocytes, compared to the Mtb group, in which peak IFN-c production from the LALN is seen at Day 28 (1,126$295 pg/ml), and from the lung, at Day 35 (22,932$3,232 pg/ml). *P(0.05 BCG/Mtb vs Mtb for each time point.
Fig. 4 In vitro IFN-c production (after stimulation by M. tuberculosis-infected peritoneal macrophages) from LALN (A) and lung (B) CD4# lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (Mtb/BCG) or vehicle vaccination (Mtb). No BCG/Mtb LALN values were obtained on Day 35. There is significantly more IFN-c from the LALN CD4# lymphocytes at Day 21 (A) in the BCG/Mtb group (733$199 pg/ml) compared to the Mtb group (7$5 pg/ml), and significantly more IFN-c from the lung CD4# lymphocytes at Days 14, 21, and 28 in the BCG/Mtb group (102$33 pg/ml Day 14; 8,920$3178 pg/ml Day 21; and 4,878$1,462 pg/ml Day 28) compared to the Mtb group (14$5 pg/ml Day 14; 42$20 pg/ml Day 21; and 83$24 pg/ml Day 28). N"2 for Day 7 and 14 for both groups LALN, and Day 7 for BCG/Mtb lung, due to low numbers of lymphocytes at these time points. *P(0.05 BCG/Mtb vs MTB at each time point.
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infection. Howard and Zwilling evaluated murine M. tuberculosis (strain Erdman) infection in BALB/c mice, and found similar cytokine patterns in the lungs of the infected animals.14 They did report a Th2 response at later time points in mediastinal lymph node cells, however. When our animals had been previously vaccinated with BCG, the IFN-c responses from LALN and lung CD4#lymphocytes were greater in magnitude that in the unvaccinated animals, and also occurred earlier than in timed controls, corresponding with the growth limitation of lung M. tuberculosis. IFN-c production was elicited in an identical pattern from the CD4#lymphocytes of the vaccinated animals with M. tuberculosis-infected peritoneal macrophages, con9rming the speci9c nature of the response. Immunity against the intracellular pathogen, M. tuberculosis, is of the CD4#lymphocyte mediated Th1 type, which results in a granulomatous cell-mediated response. The lung, as the site of initial inoculation of this infection acquired by aerosol exposure, is the location where immunity most frequently manifests. Among the features of Th1 immune reactions are the sensitization and clonal expansion of CD4#lymphocytes that produce IFN-c, resulting in the activation of alveolar macrophages to become mycobactericidal. IFN-c is critical to containment of the pathogen as demonstrated in murine models,15, 16 and humans with defective IFN-c receptors are exquisitely susceptible to mycobacterial infections.17, 18 Cells other than CD4#lymphocytes may also be a source of IFN-c production in the mycobacterium-infected host, including CD8# and cd lymphocytes19, 20 and, under appropriate conditions, alveolar macrophages.21 However, the CD4#lymphocyte is likely the primary source of IFN-c in the setting of mycobacterial infection. Human subjects with active pulmonary tuberculosis infection have increased numbers of activated CD4#lymphocytes recoverable from the involved areas of the lung by bronchoalveolar lavage, which produce IFN-c. These BAL 9ndings correlated with lymphocyte pro9les and activation in lung tissue determined by immunohistochemistry.22 A trial of aerosolized IFN-c in patients with drug-resistant pulmonary tuberculosis suggested that this therapy, in addition to antimicrobial therapy, may be bene9cial.23 Serbina and Flynn described activated T lymphocyte populations in the lungs of C57BL/6 mice infected intravenously with a virulent strain of M. tuberculosis.19 Activated CD4#lymphocytes appeared in the lungs of infected mice by 1 week, and persisted to at least 4 weeks post-infection. These cells demonstrated IFN-c producing ability upon in vitro stimulation by the second week of infection, which persisted at least until the fourth week after infection. No evidence of production of the Th2 cytokines, IL-4 or IL-10, could be detected during this time Tuberculosis (2001) 81(5/6), 327}334
course. The work presented here largely con9rms Serbina and Flynn’s report. More recently, these investigators described lung lymphocyte responses in C57BL/6 mice with speci9c antimycobacterial immunity, established by previous infection with M. tuberculosis and subsequent treatment with antimycobacterial agents.24 There are several similarities between their recent work and ours, including greater limitation of M. tuberculosis growth in the lungs of immune animals after the initial 14 days, earlier expansion of the number of CD4#lymphocytes in the lungs of immune animals (by 14 days) in response to aerosol M. tuberculosis challenge, and an earlier CD4#lymphocyte IFN-c producing capacity in immune mice compared to nonimmune mice (by 7}14 days after the aerosol challenge). Thus, our 9ndings regarding the earlier in:ux and activation of CD4#lymphocytes in immune mice are concordant, despite different murine strains and different means of induction of speci9c immunity. Our lung data are also largely in agreement with that of Howard and Zwilling,14 which demonstrated that BALB/c mice infected with virulent M. tuberculosis intranasally also develop Th1-cytokine-producing activated lymphocytes in the lungs early after infection. In contrast to our work, however, these authors did not 9nd Th-1 cytokine production from CD4#LALN lymphocytes after infection with M. tuberculosis, but describe Th2-cytokine production between 6 and 12 weeks after infection. The differences from our work may be explained on the basis of variations in the model used, or the assay of intracellular versus elicited cytokines. Our work is among the 9rst to describe the local CD4#lymphocyte responses to pulmonary M. tuberculosis infection in the setting of prior BCG vaccination. We found increased lung CD4#lymphocyte numbers and accentuated IFN-c responses from both the lung and LALN lymphocytes compared to the unvaccinated animals. The infected peritoneal macrophage studies con9rm the speci9city of the IFN-c response. We speculate that some of the lung CD4#lymphocytes in the vaccinated animals may represent cells of the memory phenotype lo (CD44highCD45RB ), which are primed for activation upon reencountering speci9c antigen.25 Grif9n and Orme have demonstrated that this memory phenotype of CD4#T lymphocytes expand during the initial 21 days of primary mycobacterial infection, and that this expansion occurs locally, more rapidly, and to a greater extent upon rechallenge in the host in whom speci9c immunity has previously been induced.26 The accumulation of these cells during both the primary and recall immune reactions to mycobacteria implies that they play a central role in the more rapid mycobacterial growth restriction that occurs after rechallenge in the setting of established speci9c immunity. Current studies in our lab are investigating this possibility. ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 333
Several quali9cations of the work presented here merit discussion. Our methodology of in vitro stimulation may not exactly re:ect in vivo conditions in the infected animal.27, 28 We initially employed a nonspeci9c stimulus of the isolated cells (concanavalin A), although when others have used nonspeci9c (though different) stimuli, similar results were obtained.19 Concanavalin A is a lymphocyte mitogen, which also elicits cytokine production from the cells.29}32 However, our peritoneal macrophage stimulation studies con9rm the speci9city of this response, though levels of IFN-c stimulated by the macrophages were lower than those obtained with ConA stimulation, the time course of the two responses were identical. Interestingly, little IFN-c could be detected from the lymphocytes from the unvaccinated control M. tuberculosis-infected animals when stimulated with infected macrophages. This may represent too short a time for in vitro stimulation, suboptimal antigen-presenting cell function,19 or may imply other alterations in the CD4#lymphocytes from the vaccinated animals, such as optimal upregulation of costimulatory molecule functions.33, 34 These possibilities will be explored in the near future. We also con9ned our present study exclusively to CD4#lymphocyte responses, not other lymphocyte subpopulations, as the CD4#lymphocyte is the primary effector cell for defense against tuberculosis.35, 36 Thus, our work con9rms that a murine model of M. tuberculosis pulmonary infection utilizing BALB/c mice demonstrates the development of a predominantly Th1biased CD4#lymphocyte-mediated immunity in both the regional lymph nodes and the lung. This response is facilitated in animals with speci9c immunity conferred by prior vaccination with BCG. Traditional concepts of the adaptive immune response hold that antigen is processed initially after infection in the regional lymph nodes (LALN), where speci9c CD4#lymphocyte clonal expansion occurs. Then the speci9c CD4#lymphocytes selectively migrate to the site of infection (here, the lung), and produce IFN-c at that site,37 as IFN-c is a critical cytokine for containing M. tuberculosis. In previously vaccinated animals, antigen-speci9c CD4#lymphocytes respond more rapidly to the infection in the lung, with accentuated levels of IFN-c upon restimulation. This is linked to the more effective growth containment of the lung M. tuberculosis in the vaccinated animals. We found only low levels of IL-4 and IL-10 from the lung CD4#lymphocytes of any of these animals, con9rming the Th1 nature of the response in this model. We speculate that the lowlevel IL-10 detected may play a role in counterbalancing or downregulating the proin:ammatory events induced by the infection, functions described for this cytokine.38}40 However, the lung and regional lymph node CD4#lymphocyte responses in this murine model of pulmonary tuberculosis show a clear Th1 bias upon ^ 2001 Harcourt Publishers Ltd
infection with M. tuberculosis, which is greatly accentuated in animals that were previously sensitized with M. bovis BCG.
ACKNOWLEDGEMENTS Supported by NIH CAA11760, LA BOR HEF+2000-05,-06, and The Wetmore Foundation.
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17. Jouanguy E, Altare F, Lamhamedi S et al. Interferon-gammareceptor de9ciency in an infant with fatal Bacille Calmette-Guerin infection. N Eng J Med 1996; 335: 1956}1961. 18. Newport M, Huxley C, Huston S et al. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996; 335: 1941}1949. 19. Serbina N, Flynn J. Early emergence of CD8#T cells primed for production of Type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect Immun 1999; 67: 3980}3988. 20. Follows G, Munk M, Gatrill A, Conradt P, Kaufmann S. Gamma interferon and interleukin 2, but not interleukin 4, are detectable in gamma-delta T-cell cultures after activation with bacteria. Infect Immun 1992; 60: 1229}1231. 21. Wang J, Wakeham J, Harkness R, Xing Z. Macrophages are a signi9cant source of type 1 cytokines during mycobacterial infection. J Clin Invest 1999; 103: 1023}1029. 22. Law K, Jagirdar J, Weiden M, Bodkin M, Rom W. Tuberculosis in HIV-positive patients: cellular response and immune activation in the lung. Am J Respir Crit Care Med 1996; 153: 1377}1384. 23. Condos R, Rom W, Schluger N. Treatment of multidrug resistant pulmonary tuberculosis with interferon-gamma aerosol. Lancet 1997; 349: 1513}1515. 24. Serbina N, Flynn J. CD8#T cells participated in the memory immune response to Mycobacterium tuberculosis. Infec Immun 2001; 69: 4320}4328. 25. Howard A, Trask O, Weisbrode S, Zwilling B. Phenotypic changes in T cell populations during the reactivation of tuberculosis in mice. Clin Exp Immunol 1998; 111: 309}315. 26. Grif9n J, Orme I. Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis. Infect Immun 1994; 62: 1683}1690. 27. Mustafa T, Phyu S, Nilsen R, Jonsson R, Bjune G. In situ expression of cytokines and cellular phenotypes in the lungs of mice with slowly progressive primary tuberculosis. Scand J Immunol 2000; 51: 548}556. 28. Fayyazi A, Eichmeyer B, Soruri A et al. Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis. J Pathol 2000; 191: 417}425.
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29. Ayala A, Chung C, Xu X, Evans T, Redmond K, Chaudry I. Increased inducible apoptosis in CD4#T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin. Immunol 1999; 97: 45}55. 30. Watzl B, Bub A, Brandstetter B, Rechkemmer G. Modulation of human T-lymphocyte functions by the consumption of carotenoid-rich vegetables. Br J Nutr 1999; 82: 383}389. 31. Shellito J, Tate C, Ruan S, Kolls J. Murine CD4#T lymphocyte subsets and host defense against Pneumocystis carinii. J Infect Dis 2000; 181: 2011}2017. 32. Lukacs N, Chensue S, Karpus W et al. C-C chemokines differently alter interleukin-4 production from lymphocytes. Am J Path 1997; 150: 1861}1868. 33. McCoy K, LeGros G. The role of CTLA-4 in the regulation of T cell immune responses. Immunol Cell Biol 1999; 77: 1}10. 34. Henderson R, Watkins S, Flynn J. Activation of human dendritic cells following infection with Mycobacterium tuberculosis. J Immunol 1997; 159: 635}643. 35. Caruso A, Serbina N, Klein E, Triebold K, Bloom B, Flynn J. Mice de9cient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis. J Immunol 1999; 162: 5407}5416. 36. Scanga C, Mohan V, Yu K et al. Depletion of CD4#T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon gamma and nitric oxide synthase 2. J Exp Med 2000; 192: 347}358. 37. Andrian Uv, Mackay C. T-cell function and migration. N Engl J Med 2000; 343: 1020}1034. 38. Prete GD, DeCarli M, Almerigogna F, Giudizi M, Biagiotti R, Romagnani S. Human IL-10 is produced by both type 1 helper (TH1) and type 2 (TH2) T cell clones and inhibits their antigenspeci9c proliferation and cytokine production. J Immunol 1993; 150: 353}360. 39. Gerard C, Bruyns C, Marchant A et al. Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia. J Exp Med 1993; 177: 547}550. 40. Takenaka H, Maruo S, Yamamoto N et al. Regulation of T celldependent and-independent IL-12 production by the three Th2type cytokines IL-10, IL-6, and IL-4. J Leuk Biol 1997; 61: 80}87.
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CD4ⴙ lymphocyte responses to pulmonary infection with Mycobacterium tuberculosis in naeK ve and vaccinated BALB/c mice C. M. Mason, E. Dobard, J. Shellito, S. Nelson Pulmonary/Critical Care Medicine, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA, USA
Summary Setting: Local CD4# lymphocyte responses are critical in determining the fate of infection with Mycobacterium tuberculosis. Objective: To evaluate the lung and regional lymph node CD4# lymphocyte responses to pulmonary infection with M. tuberculosis H37Rv in BALB/c mice, with and without prior vaccination with M. bovis Bacille Calmette-GueH rin (BCG). Design: Lung and lung-associated lymph node (LALN) CD4# lymphocytes were isolated from BALB/c mice infected with low dose M. tuberculosis H37Rv by the intratracheal route in BCG-vaccinated and unvaccinated animals, and cytokine responses assessed in vitro. Results: CD4#lymphocytes increased in both the lung and LALN by 21 days after infection, and elicited production of interferon (IFN)-c was upregulated in the lymphocytes of the infected animals. Only low levels of interleukin (IL)-4 and IL-10 were elicited from the lung. The IFN-c response from both sites was facilitated in the previously vaccinated animals. Conclusion: In this model of low dose inoculation of M. tuberculosis in the lung, BALB/c mice respond with a Th1-biased CD4# lymphocyte response, which is accentuated in animals previously vaccinated with BCG. ^ 2001 Harcourt Publishers Ltd INTRODUCTION Infection with Mycobacterium tuberculosis is at epidemic levels globally. It is the most prevalent infectious disease worldwide, and annually there are eight million people who acquire the infection, with three million succumbing due to it.1 M. tuberculosis is an intracellular organism that promotes the development of CD4#lymphocyte-orchestrated Th1 cell-mediated immunity. Th1 immunity is characterized by speci9c CD4#lymphocytes that elaborate the cytokines interleukin (IL)-2 and interferon (IFN)-c, which function to expand the CD4#lymphocyte population, activate local macrophages for mycobactericidal activity, and confer protective immunity.2, 3 This is a unique infection in that, even in the face of established immunity, the pathogen may not be eliminated, but remains present in a state of latency.4 In a small percentage of those with latent infection, the disease can reactivate, with development of overt infection. Appropriate treatCorrespondence to: C. M. Mason, MD, Pulmonary/Critical Care Medicine, 1901 Perdido C3205, New Orleans, LA 70112, USA. Accepted: 6 September 2001
ment with antimicrobial agents usually successfully eliminates the organism, but requires several months to do so. As the lung is the primary site of inoculation for most infections with M. tuberculosis, the immune response of lung cells is critical in containing the organism. We used a BALB/c murine model of M. tuberculosis pulmonary infection, and studied the lung and lung-associated lymph node (LALN) CD4#lymphocyte responses serially during the course of infection, in mice with and without prior vaccination with BCG. We found evidence that Th1 responses predominate in both the lung and the LALN, concurrently with growth restriction of the organism, con9rming the critical role of CD4#lymphocytes in this infection. With prior vaccination, the Th1 response was facilitated in the lung, accompanied by greater growth restriction.
MATERIALS AND METHODS Animals Speci9c pathogen-free BALB/c mice (Hilltop Labs, Scottdale, PA) were used for these experiments. Animals were
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housed in the LSUHSC Pulmonary/Critical Care Medicine Biocontainment Level-3 Laboratory and experiments were performed in the Biocontainment Level-3 Laboratory in accordance with appropriate safety precautions recommended by the CDC.5 All animal procedures were approved by the LSUHSC institutional Animal Care and Use Committee. All data represent groups of n"5 unless otherwise noted.
M. tuberculosis H37Rv Infection M. tuberculosis H37Rv was obtained from ATCC (Rockville, MD, catalog C27294), and was grown in Middlebrook 7H11 broth at 373C for 14 days. This culture was concentrated by centrifugation, gently sonicated, and stored in 0.1 ml aliquots at !803C. At the time of inoculation, an aliquot was thawed, gently sonicated, and diluted in endotoxin-free PBS to a concentration of 1;102 organisms/ml. Mice were lightly anesthetized with ketamine/xylazine (200 mg/kg/10 mg/kg) intraperitoneally (IP), the ventral surface of the neck swabbed with isopropyl alcohol, and a midline skin incision performed in a sterile fashion. The soft tissues of the neck were gently retracted laterally to expose the trachea. Mycobacteria were sonicated for 10 s prior to injection to achieve uniform single organism dispersion. Direct intratracheal (IT) injection was performed, injecting 100 ll of bacterial suspension (101 organisms) through a 30 gauge needle, followed by 0.3 ml of air. The incision was closed with a stainless steel surgical clip, and the animal allowed to recover. The TB group comprised animals inoculated IT with mycobacteria, and the Control group comprised those inoculated with PBS rather than mycobacteria. At serial time points (Days 7, 14, 21, 28, 35 and 42) after injection, animals were sacri9ced and lungs were homogenized, serially diluted, and plated in quadruplicate on Middlebrook 7H10 agar plates. The plates were incubated at 373C for 17 days. At the end of the incubation period, the number of colony-forming units (CFU) present on plates containing between 20 and 200 CFUs/quadrant was counted and multiplied by the appropriate dilution in order to determine the number of CFU in the initial tissue homogenate.
Vaccination with M. bovis BCG M. bovis BCG was obtained from ATCC (Rockville, MD, catalog C35734). Animals were administered M. bovis BCG subcutaneously (1;106 cfu) 28 days prior to IT inoculation with Mtb H37Rv,6 which is performed as described above. Serial cultures were performed as above. In this group, vehicle vaccinated animals comprise the Mtb group, and vaccinated animals the BCG/Mtb group. Tuberculosis (2001) 81(5/6), 327}334
Isolation of lung and lung-associated lymph node (LALN) lymphocytes Lung and LALN lymphocytes were isolated according to the following methods.7 Lungs were removed from mice in all groups at serial times after sacri9ce, and the conducting airways were removed. Lung tissue was minced, placed in RPMI-1640 containing collagenase (150 U/ml) and DNAse (50 U/ml) for enzymatic digestion. Ten ml of the enzymatic solution was used for every 600 mg lung tissue, and the mixture was incubated at 373C for 90 min with constant stirring. After incubation, the mixture was passed through a 70 lm nylon mesh, placed brie:y in NH4Cl lysis buffer, and resuspended in indomethacin (1 lg/ml) and catalase (250 U/ml). Cells were counted, viability checked, and then they were diluted to the desired concentration. LALN were isolated from hilar lymph nodes and mediastinal lymph nodes dissected after sacri9ce. Single cell suspensions were prepared by gently disrupting the tissue on a 70 lm nylon 9lter. Obtained cells were centrifuged prior to resuspending in NH4Cl lysis buffer, to which Hank’s BSS was added. Cells were centrifuged again, counted, and resuspended at 106/ml. CD4#lymphocytes were isolated from both lung cells and LALN cells by magnetic bead selection (Dynal, Inc., Lake Success, NY).8, 9 CD4#lymphocytes isolated by the magnetic bead technique were '95% pure for the LALN population and '90% pure for the lung population when checked by :ow cytometry (data not shown). By performing differential cell counts on cytospin monolayers of isolated lung and LALN cells, the total lymphocyte counts from each site were determined. Likewise, total CD4#lymphocyte counts were calculated by performing differential cell counts on cytospin monolayers from the cells resulting from magnetic bead isolation.
CD4ⴙ lymphocyte cytokine production Cytokine assays were performed by plating isolated CD4#lymphocytes from lung or LALN from the TB or Control group animals at a concentration of 1;105 cells/well, and stimulated with concanavalin A (5 lg/ml) for 48 h. Unstimulated cells from each animal were used as media controls. After the 48 h time period, supernatants were collected, centrifuged to remove cellular debris, frozen at !803C, and subsequently assayed for IFN-c, IL-4, and IL-10 by ELISA (R&D Systems, Minneapolis, MN). The lower limits of detection for these assays were for IFN-c: 2 pg/ml, for IL-4: 2 pg/ml, and for IL-10: 4 pg/ml. For the vaccinated animals, additional cytokine elicitation was performed in vitro with infected peritoneal macrophages.10 These cells were elicited from naive BALB/c mice with concanavalin A (100 lg, Type IV, Sigma, St. Louis, MO), injected IP 96 h prior to peritoneal lavage with ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 329
DMEM at 43C. Harvested cells were washed, counted (with a differential count), checked for viability, and the red blood cells were lysed. Cells were then placed in complete DMEM-10, adjusted to 2;106 cells/ml, and plated in 96 well plates at 2;105 cells/well and allowed to adhere for 2 h at 373C. Cells were then washed three times to remove nonadherent cells. Next, M. tuberculosis H37Rv was added to the macrophage monolayer at 1;106 cfu/well, and incubated for 18}24 h at 373C. Control wells were uninfected. After this time, extracellular bacteria were removed by aspiration with DMEM three times, complete DMEM-10 replaced, and the cell maintained in culture for an additional 24 h. Aliquots of isolated CD4#lymphocytes from LALN and lung of M. tuberculosis-infected animals were suspended in DMEM-10 at 2;106 cells/ml at 373C, and 1;105 cells added to each well of PM, and incubated at 373C for 48 h. After the incubation, supernatants were harvested, spun, and saved at !803C until cytokine assay.
RESULTS Lung infection with M. tuberculosis H37Rv Mice inoculated with M. tuberculosis H37Rv as described above were sacri9ced at Days 7, 14, 21, 28, 35, and 42 after inoculation. The IT inoculum was 2;101 CFU. Lungs were quantitatively cultured at each time point. Control animals inoculated with PBS had no growth of mycobacteria from their lungs at any time point. Growth of M. tuberculosis in the lungs of mice was exponential until approximately Day 21, when the growth rate slowed. Mycobacterial burden subsequently remained fairly constant at +106 cfu/lung for the remaining time points tested (data not shown), as has been previously reported.11
Lung infection with M. tuberculosis H37Rv after BCG vaccination Mice inoculated with M. tuberculosis H37Rv as described above 28 days after BCG vaccination were sacri9ced at Days 14, 28, and 42 after inoculation of M. tuberculosis. The IT inoculum was 2;101 CFU. Lungs were quantitatively cultured at each time point, and the results are shown in Figure 1. There was signi9cant reduction in lung growth of M. tuberculosis in the vaccinated animals at 28 and 42 days compared to the unvaccinated animals.
Lung and LALN CD4ⴙ lymphocytes CD4#lymphocytes were isolated from lung and LALN at serial time points after the M. tuberculosis inoculation in TB group and Control (uninfected) mice. Numbers of CD4#LALN and lung lymphocytes from each time point ^ 2001 Harcourt Publishers Ltd
Fig. 1 Total CFU in lungs of BALB/c mice at serial time points after IT inoculation on Day 0 with 2;101 CFU of M. tuberculosis H37Rv, which was 28 days after SC inoculation with 1;106 M. bovis BCG. Initially, the M. tuberculosis growth rate is similar between the two groups, but there is a significant reduction in M. tuberculosis cfu in the lungs of the BCG vaccinated mice at Day 28 and Day 42. *P(0.05 BCG/Mtb vs Mtb.
are shown in Figures 2A and 2B. The number of CD4#lymphocytes recovered from the LALN and the lung increased signi9cantly in the TB group animals as the infection progressed. LALN and lung CD4#lymphocytes from BCG vaccinated animals are shown in Figures 2C and 2D. There are no differences in the number of LALN CD4#lymphocytes at any time point, but signi9cantly more lung CD4#lymphocytes in the vaccinated animals at all time points after Day 7.
CD4ⴙ lymphocyte cytokine production The time course of lung and LALN CD4#lymphocyte IFN-c production from M. tuberculosis-infected or Control (uninfected) animals is shown in Figures 3A and 3B. The highest levels of IFN-c measured from the LALN CD4#cells occurred at Day 21 in the TB group, and then returned toward baseline by Day 28. In contrast, the lung CD4#lymphocyte IFN-c response was rising signi9cantly by Day 21 in the TB animals, and continued to increase at the Day 28 time point. LALN and lung lymphocytes without Con A stimulation (media controls) had no IFN-c production detectable at any time point studied. The time course of LALN and lung CD4#lymphocyte ConA-elicited IFN-c from BCG vaccinated animals is shown in Figures 3C and 3D. CD4#T cells from BCG-vaccinated animals (from both sites) have earlier peak IFN-c production compared to time-matched unvaccinated controls. Interestingly, the peak IFN-c production by the unvaccinated M. tuberculosis-infected animals’ lymphocytes is delayed until Day 28 for the LALN, and Day 35 for the lung, in contrast to the series presented in Figures 2A and 2B. This may be due to minor variations in the organism Tuberculosis (2001) 81(5/6), 327}334
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Fig. 2 CD4# LALN (A) and lung (B) lymphocytes isolated on Days 7, 14, 21, and 28 from mice infected IT on Day 0 with M. tuberculosis (TB group) or Control mice. There are significantly more lymphocytes recovered from the lungs of animals in the TB group at Days 21 and 28 compared to the Control animals. *P(0.05 TB vs Control. CD4#LALN (C) and lung (D) lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (BCG/Mtb group) or vehicle vaccination (Mtb group). There are significantly more CD4#lymphocytes recovered from the lungs of animals in the Mtb/BCG group at Days 14, 21, 28, and 35 compared to the Mtb group. *p(0.05 BCG/Mtb vs Mtb.
inoculation and/or growth curves between the two different experiments, or to the fact that no information is obtained regarding IFN-c production between the reported 7-day time points (due to reasons of practicality). This is the reason, however, that concurrent controls are performed for every experiment. When M. tuberculosisinfected peritoneal macrophages were used to stimulate IFN-c production from the lymphocytes, cells from both sites produced high levels of IFN-c (Figures 4A and 4B), con9rming an M. tuberculosis-speci9c response. There was no IL-4 detected at any time in the stimulated LALN CD4#lymphocytes, nor in the media control samples from either tissue site. Low levels of IL-4 ((300 pg/ml) were detectable in the stimulated lung CD4#lymphocytes at all time points assessed, but no signi9cant differences were noted between the TB and Control groups. IL-10 was also assessed in the supernatants from both lung and LALN stimulated CD4#lymphocytes, and low levels (4300 pg/ml) were detectable in the lung lymphocyte supernatants at all time points assessed. From these results, it is apparent that the magnitude of the IFN-c response is more that 40-fold greater than the levels of Tuberculosis (2001) 81(5/6), 327}334
IL-4 or IL-10, con9rming the Th1 response of the infected animals. In the BCG and vehicle vaccinated animals, lung CD4#lymphocytes from both groups produced low-level IL-4 (4500 pg/ml), and even lower levels of IL-10 (4250 pg/ml), again con9rming the predominant Th1 response induced by M. tuberculosis infection in these animals.
DISCUSSION The work presented here con9rms that pulmonary infection of normal BALB/c mice with a virulent strain of M. tuberculosis is accompanied by high level IFN-c production and lower levels of IL-4 and IL-10 production, signifying the development of Th1 immunity by the CD4#lymphocyte populations in both the lung and the regional lymph nodes. In previous reports, the BALB/c mouse is reported to respond to various antigenic challenges with a Th2 immunity bias,12, 13 but our work clearly shows that in response to challenge with M. tuberculosis, the lung and LALN CD4#lymphocyte immune responses are strongly Th1-biased over the initial 28 days following ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 331
Fig. 3 In vitro IFN-c production (after Con A stimulation) from LALN (A) and lung (B) CD4# lymphocytes isolated on Days 7, 14, 21, and 28 from mice infected IT on Day 0 with M. tuberculosis. There is significantly more IFN-c elicited by LALN CD4# lymphocytes (A) on Days 14 and 21 in the TB group compared to the Control group (*P(0.05 TB group vs Control group). The TB group at Day 14 (n"4) produced 22$14 pg/ml IFN-c, with no IFN-c detectable in the Control group at this time. At Day 21, the TB group produced 2,146$670 pg/ml IFN-c, with no IFN-c detectable in the Control group at this time. For the Day 7 values, n"3 for each group due to the low numbers of LALN CD4# cells. For the lung CD4# lymphocytes (B), there was significantly greater IFN-c elicited in the TB group at Day 7 (484$66 pg/ml TB vs 230$31 pg/ml Control), Day 14 (289$52 pg/ml TB vs 113$45 pg/ml Control), Day 21 (10,251$2,749 pg/ml TB vs 67$16 pg/ml Control), and Day 28 (14, 138$2,557 pg/ml TB vs 292$67 pg/ml Control) than the Control group. *P(0.05 TB group vs Control group. The TB group values of IFN-c (pg/ml) that were low, but statistically different from the Control group, are shown on the graphs, above the respective symbols. (C) shows in vitro IFN-c production (after Con A stimulation) from LALN and (D) shows in vitro IFN-c production (after Con A stimulation) from lung CD4#lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (Mtb/BCG) or vehicle vaccination (Mtb). There is an earlier peak of IFN-c production in the BCG/Mtb group at 21 days for both the LALN (1,308$246 pg/ml) and the lung (34,686$9,256 pg/ml) lymphocytes, compared to the Mtb group, in which peak IFN-c production from the LALN is seen at Day 28 (1,126$295 pg/ml), and from the lung, at Day 35 (22,932$3,232 pg/ml). *P(0.05 BCG/Mtb vs Mtb for each time point.
Fig. 4 In vitro IFN-c production (after stimulation by M. tuberculosis-infected peritoneal macrophages) from LALN (A) and lung (B) CD4# lymphocytes isolated on Days 7, 14, 21, 28, and 35 from mice infected IT on Day 0 with M. tuberculosis 28 days after vaccination with BCG (Mtb/BCG) or vehicle vaccination (Mtb). No BCG/Mtb LALN values were obtained on Day 35. There is significantly more IFN-c from the LALN CD4# lymphocytes at Day 21 (A) in the BCG/Mtb group (733$199 pg/ml) compared to the Mtb group (7$5 pg/ml), and significantly more IFN-c from the lung CD4# lymphocytes at Days 14, 21, and 28 in the BCG/Mtb group (102$33 pg/ml Day 14; 8,920$3178 pg/ml Day 21; and 4,878$1,462 pg/ml Day 28) compared to the Mtb group (14$5 pg/ml Day 14; 42$20 pg/ml Day 21; and 83$24 pg/ml Day 28). N"2 for Day 7 and 14 for both groups LALN, and Day 7 for BCG/Mtb lung, due to low numbers of lymphocytes at these time points. *P(0.05 BCG/Mtb vs MTB at each time point.
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332 Mason et al.
infection. Howard and Zwilling evaluated murine M. tuberculosis (strain Erdman) infection in BALB/c mice, and found similar cytokine patterns in the lungs of the infected animals.14 They did report a Th2 response at later time points in mediastinal lymph node cells, however. When our animals had been previously vaccinated with BCG, the IFN-c responses from LALN and lung CD4#lymphocytes were greater in magnitude that in the unvaccinated animals, and also occurred earlier than in timed controls, corresponding with the growth limitation of lung M. tuberculosis. IFN-c production was elicited in an identical pattern from the CD4#lymphocytes of the vaccinated animals with M. tuberculosis-infected peritoneal macrophages, con9rming the speci9c nature of the response. Immunity against the intracellular pathogen, M. tuberculosis, is of the CD4#lymphocyte mediated Th1 type, which results in a granulomatous cell-mediated response. The lung, as the site of initial inoculation of this infection acquired by aerosol exposure, is the location where immunity most frequently manifests. Among the features of Th1 immune reactions are the sensitization and clonal expansion of CD4#lymphocytes that produce IFN-c, resulting in the activation of alveolar macrophages to become mycobactericidal. IFN-c is critical to containment of the pathogen as demonstrated in murine models,15, 16 and humans with defective IFN-c receptors are exquisitely susceptible to mycobacterial infections.17, 18 Cells other than CD4#lymphocytes may also be a source of IFN-c production in the mycobacterium-infected host, including CD8# and cd lymphocytes19, 20 and, under appropriate conditions, alveolar macrophages.21 However, the CD4#lymphocyte is likely the primary source of IFN-c in the setting of mycobacterial infection. Human subjects with active pulmonary tuberculosis infection have increased numbers of activated CD4#lymphocytes recoverable from the involved areas of the lung by bronchoalveolar lavage, which produce IFN-c. These BAL 9ndings correlated with lymphocyte pro9les and activation in lung tissue determined by immunohistochemistry.22 A trial of aerosolized IFN-c in patients with drug-resistant pulmonary tuberculosis suggested that this therapy, in addition to antimicrobial therapy, may be bene9cial.23 Serbina and Flynn described activated T lymphocyte populations in the lungs of C57BL/6 mice infected intravenously with a virulent strain of M. tuberculosis.19 Activated CD4#lymphocytes appeared in the lungs of infected mice by 1 week, and persisted to at least 4 weeks post-infection. These cells demonstrated IFN-c producing ability upon in vitro stimulation by the second week of infection, which persisted at least until the fourth week after infection. No evidence of production of the Th2 cytokines, IL-4 or IL-10, could be detected during this time Tuberculosis (2001) 81(5/6), 327}334
course. The work presented here largely con9rms Serbina and Flynn’s report. More recently, these investigators described lung lymphocyte responses in C57BL/6 mice with speci9c antimycobacterial immunity, established by previous infection with M. tuberculosis and subsequent treatment with antimycobacterial agents.24 There are several similarities between their recent work and ours, including greater limitation of M. tuberculosis growth in the lungs of immune animals after the initial 14 days, earlier expansion of the number of CD4#lymphocytes in the lungs of immune animals (by 14 days) in response to aerosol M. tuberculosis challenge, and an earlier CD4#lymphocyte IFN-c producing capacity in immune mice compared to nonimmune mice (by 7}14 days after the aerosol challenge). Thus, our 9ndings regarding the earlier in:ux and activation of CD4#lymphocytes in immune mice are concordant, despite different murine strains and different means of induction of speci9c immunity. Our lung data are also largely in agreement with that of Howard and Zwilling,14 which demonstrated that BALB/c mice infected with virulent M. tuberculosis intranasally also develop Th1-cytokine-producing activated lymphocytes in the lungs early after infection. In contrast to our work, however, these authors did not 9nd Th-1 cytokine production from CD4#LALN lymphocytes after infection with M. tuberculosis, but describe Th2-cytokine production between 6 and 12 weeks after infection. The differences from our work may be explained on the basis of variations in the model used, or the assay of intracellular versus elicited cytokines. Our work is among the 9rst to describe the local CD4#lymphocyte responses to pulmonary M. tuberculosis infection in the setting of prior BCG vaccination. We found increased lung CD4#lymphocyte numbers and accentuated IFN-c responses from both the lung and LALN lymphocytes compared to the unvaccinated animals. The infected peritoneal macrophage studies con9rm the speci9city of the IFN-c response. We speculate that some of the lung CD4#lymphocytes in the vaccinated animals may represent cells of the memory phenotype lo (CD44highCD45RB ), which are primed for activation upon reencountering speci9c antigen.25 Grif9n and Orme have demonstrated that this memory phenotype of CD4#T lymphocytes expand during the initial 21 days of primary mycobacterial infection, and that this expansion occurs locally, more rapidly, and to a greater extent upon rechallenge in the host in whom speci9c immunity has previously been induced.26 The accumulation of these cells during both the primary and recall immune reactions to mycobacteria implies that they play a central role in the more rapid mycobacterial growth restriction that occurs after rechallenge in the setting of established speci9c immunity. Current studies in our lab are investigating this possibility. ^ 2001 Harcourt Publishers Ltd
CD4#responses to murine tuberculosis 333
Several quali9cations of the work presented here merit discussion. Our methodology of in vitro stimulation may not exactly re:ect in vivo conditions in the infected animal.27, 28 We initially employed a nonspeci9c stimulus of the isolated cells (concanavalin A), although when others have used nonspeci9c (though different) stimuli, similar results were obtained.19 Concanavalin A is a lymphocyte mitogen, which also elicits cytokine production from the cells.29}32 However, our peritoneal macrophage stimulation studies con9rm the speci9city of this response, though levels of IFN-c stimulated by the macrophages were lower than those obtained with ConA stimulation, the time course of the two responses were identical. Interestingly, little IFN-c could be detected from the lymphocytes from the unvaccinated control M. tuberculosis-infected animals when stimulated with infected macrophages. This may represent too short a time for in vitro stimulation, suboptimal antigen-presenting cell function,19 or may imply other alterations in the CD4#lymphocytes from the vaccinated animals, such as optimal upregulation of costimulatory molecule functions.33, 34 These possibilities will be explored in the near future. We also con9ned our present study exclusively to CD4#lymphocyte responses, not other lymphocyte subpopulations, as the CD4#lymphocyte is the primary effector cell for defense against tuberculosis.35, 36 Thus, our work con9rms that a murine model of M. tuberculosis pulmonary infection utilizing BALB/c mice demonstrates the development of a predominantly Th1biased CD4#lymphocyte-mediated immunity in both the regional lymph nodes and the lung. This response is facilitated in animals with speci9c immunity conferred by prior vaccination with BCG. Traditional concepts of the adaptive immune response hold that antigen is processed initially after infection in the regional lymph nodes (LALN), where speci9c CD4#lymphocyte clonal expansion occurs. Then the speci9c CD4#lymphocytes selectively migrate to the site of infection (here, the lung), and produce IFN-c at that site,37 as IFN-c is a critical cytokine for containing M. tuberculosis. In previously vaccinated animals, antigen-speci9c CD4#lymphocytes respond more rapidly to the infection in the lung, with accentuated levels of IFN-c upon restimulation. This is linked to the more effective growth containment of the lung M. tuberculosis in the vaccinated animals. We found only low levels of IL-4 and IL-10 from the lung CD4#lymphocytes of any of these animals, con9rming the Th1 nature of the response in this model. We speculate that the lowlevel IL-10 detected may play a role in counterbalancing or downregulating the proin:ammatory events induced by the infection, functions described for this cytokine.38}40 However, the lung and regional lymph node CD4#lymphocyte responses in this murine model of pulmonary tuberculosis show a clear Th1 bias upon ^ 2001 Harcourt Publishers Ltd
infection with M. tuberculosis, which is greatly accentuated in animals that were previously sensitized with M. bovis BCG.
ACKNOWLEDGEMENTS Supported by NIH CAA11760, LA BOR HEF+2000-05,-06, and The Wetmore Foundation.
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