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Mycobacterial Dissemination and Cellular Responses after 1-Lobe Restricted Tuberculosis Infection of Genetically Susceptible and Resistant Mice

  1. Vladimir V. Mischenko1,
  2. Marina A. Kapina1,
  3. Evgenyi B. Eruslanov1,
  4. Elena V. Kondratieva1,
  5. Irina V. Lyadova1,
  6. Douglas B. Young2 and
  7. Alexander S. Apt1
  1. 1Laboratory for Immunogenetics, Central Institute for Tuberculosis, Moscow, Russia
  2. 2Department of Infectious Diseases & Microbiology, Imperial College, London, United Kingdom
  1. Reprints or correspondence: Dr. Alexander Apt, Laboratory for Immunogenetics, Central Institute for Tuberculosis, Yauza Alley 2, Moscow 107564, Russia (asapt{at}aha.ru).
 
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Abstract

Background and methods. To study mycobacterial dissemination and immune-cell trafficking in tuberculosis, we developed a mouse model in which we introduced 1 εL of Mycobacterium tuberculosis directly into the middle lobe of the right lung. We investigated the kinetics of both mycobacterial spread to different anatomical sites and recruitment of phagocytes and activated lymphocytes.

Results. Mycobacterial dissemination was independent of susceptibility to infection and was identical in H-2- congenic mouse strains with high and low resistance to tuberculosis. In resistant mice, recruitment of phagocytic cells to the uninfected lung occurred before the appearance of mycobacteria and decreased shortly thereafter. In susceptible mice, this recruitment was delayed in both lungs but increased during a 10-week period. Recruitment of CD4+ and CD8+ lymphocytes to the contralateral lung was observed before mycobacterial dissemination in both strains, so mycobacterial seeding of secondary tissues occurred in the presence of immune lymphocytes. In resistant mice, more T cells expressed the CD44hi CD62lo activation phenotype, and higher levels of interferon-γ were produced.

Conclusions. Mycobacterial spread to lymphoid organs preceded spread to the initially uninfected contralateral lung. Genetic differences in susceptibility to tuberculosis are associated with differences in dynamics of the immune response, rather than differences in mycobacterial trafficking.

Primary pulmonary tuberculosis (TB) develops in the alveoli of the lung and then can spread to other organs and tissues [1]. Infection by very small clumps or single bacilli is almost always initiated in a single lobe of the lung and produces a lesion in only 1 area [2]. During the initial phase of infection, mycobacteria are ingested by resident alveolar macrophages [3] and/or by dendritic cells [4], or they actively invade the lung epithelium [5–8]. After crossing the epithelial layer, mycobacteria begin to multiply within interstitial lung phagocytes. From the results of animal studies, it is assumed that, after early replication within the initial lesion, mycobacteria subsequently disseminate to draining lymph nodes and then spread hematogenously throughout the body [9–11]. Although, in humans, the true initial lesion is probably never observed, a similar sequence of events is suggested by the common occurrence of a so-called primary lymph node TB complex and by far higher rates of extrapulmonary TB in children, compared with those in adults [12, 13]. If primary infection progresses, bacteria return via the bloodstream to infect lobes in both the primarily infected and the initially “sterile” contralateral lung [13, 14].

Little is known about the chain of events that immediately follows the initial infection of lung macrophages. For example, the mycobacterial factors involved in dissemination are poorly characterized. Although the heparin-binding hemagglutinin adhesin (HbhA) of Mycobacterium tuberculosis was reported recently to be an essential virulence factor involved in extrapulmonary dissemination [15], its effect is on virulence rather than on the degree of mycobacterial spread from the lung [16] (authors' unpublished observations). An open question is whether professional phagocytes are responsible for the hematogenous spread of mycobacteria, as has been demonstrated for other intracellular parasites, such as salmonella [17] and Leishmania major [18]. Also, it is not known whether hematogenous mycobacterial spread to distant organs—such as the spleen, liver, and contralateral lung—is directly from the affected lung or from pulmonary lymph nodes.

There is insufficient information concerning the early establishment of acquired immunity to mycobacteria at different anatomical sites. It has been reported that, after aerosol challenge in mice, mycobacteria-specific T cells appear in the draining pulmonary lymph nodes no earlier than 2–3 days after the dissemination of mycobacteria to these organs [10]. However, given that T cells primed with an antigen in lymph nodes readily circulate through lung tissue [19–22], it remains unclear whether immune T cells enter the contralateral lung before or after mycobacteria have reached this site. This is an important question, because the conditions encountered by mycobacteria in primary (“preimmune”) and secondary (“immune”) lesions will have an important influence on subsequent pathological features, such as granuloma size and necrotization [11]. Information regarding possible links between the degree and speed of mycobacterial dissemination and the genetically determined severity of TB in different mouse strains [10] has indicated a nontrivial inverse correlation, and this issue warrants further study.

A major disadvantage of current mouse models of TB, with respect to the analysis of mycobacterial dissemination and immune- lymphocyte trafficking, is that the infection is initiated simultaneously in both lungs, irrespective of whether the aerogenic or intratracheal challenge route is chosen. This precludes any analysis of the sequential steps of bacterial spread and host cell trafficking that precede the development of bilateral pulmonary TB. Here, we report on the establishment of a new mouse model of TB based on administration of a low dose (<100 cfu) of single-cell mycobacteria, contained in an extremely small volume (1 εL), directly into the middle lobe of the right lung. We show that this 1-lobe infection model is easily reproduced and reasonably accurate (<10% variation in the size of inoculum) and that injected mycobacteria do not spread outside the infected lung during the first week after infection. Using this model, we describe the dynamics of mycobacterial spread to lymphoid organs and to the contralateral lung. We also characterize the accumulation of lymphoid cells, the kinetics of lymphocyte activation, and the development of immune responses in primarily infected and secondarily seeded lungs. We compare these parameters between mouse strains with well-defined genetic differences in TB susceptibility and severity.

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Materials and Methods

Mice. Inbred B10.SM/SnEgCit (B10.SM), B10.MBR/JDvCit (B10.MBR), and C57BL/6JCit (B6) mice were bred under conventional conditions at the Animal Facilities of the Central Institute for Tuberculosis (Moscow, Russia). Water and food were provided ad libitum. Female mice 2–3 months old were used. Animal experiments were conducted at the Central Institute for Tuberculosis, which is accredited by the National Institutes of Health Office of Laboratory Animal Welfare (assurance A5502-01) and in accordance with guidelines from the Russian Ministry of Health (guideline 755). All experimental procedures were approved by the Institutional Animal Care and Use Committee.

Bacteria and colony-forming unit counting. M. tuberculosis strain H37Rv (gift of G. Marchal, Institut Pasteur, Paris) was used. The preparation of clump-free midlog-phase mycobacterial suspensions has been described elsewhere [23, 24]. To assess mycobacterial loads in the spleen, lungs, liver, and pulmonary lymph nodes, 0.1 mL of serial 10-fold dilutions of sterile wholeorgan homogenates were plated onto Dubos agar (Difco), and colonies were counted after 18–20 days of incubation at 37°C (see the footnote to table 1 for the method of enumeration of extremely low numbers of mycobacteria in organs).

Experimental infection. The following protocol was used for the initiation of infection in the middle lobe of the right lung. The mycobacterial suspension was filtered through a 5- εm pore-size filter (Sigma). The concentration of bacilli (>90% single cells) was directly estimated by microscopy, by use of a cell cytometer (figure 1A), and was adjusted to 8 × 104 cells/ mL. The bacterial suspension was dispensed by use of a 50-εL syringe (Hamilton), and numbers of bacteria in serial 1-εL inocula were assessed in control experiments by plating inoculum that had been diluted 10 times (to 10 εL) onto Dubos agar and counting all microcolonies (figure 1B) under an inverted microscope after 3 days of incubation at 37°C. Consistent results with >10% variation were obtained in 6 independent experiments (figure 1B). Before mice were injected, the needle of the Hamilton syringe was supplied with a silicon overrun limiter 3 mm from its spike, to prevent overpenetration (figure 1C). Mice were lightly anesthetized by intraperitoneal (ip) injection of 100 mg/kg hexenalum (Farmsinteze). Inoculation was performed across the chest, from the right side, in the apex point of the costoclavicular perpendicular curve (figure 1D). To monitor the exact location of the site of injection and to estimate the volume of liquid that was stably contained within the lobe without rapid spreading to the contralateral lung via the bronchial tree, several control-injection experiments were performed by use of trypan blue dye in saline. As shown in figure 1E, the procedure described here led to the inoculation of liquid into the middle lobe of the right lung. The dye always appeared in the left lung within 10 min after injection if the amount of inoculum was <15 εL, but this was never observed with 1–3 mL inocula. In all experiments, mycobacteria were injected in a 1-εL volume. Placing the inoculum into the middle lobe was successful in >90% of cases.

Figure 1.
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Figure 1.

One-lobe infection model (see Materials and Methods). Mycobacterial suspension was filtered, and the concentration of bacilli (>90% single cells, A) was estimated by microscopy. The no. of bacteria in 1 εL of inoculum was assessed by counting microcolonies under an inverted microscope (B; N, no. of colonies in individually seeded 1-εL portions in 1 of 6 similar independent experiments). The needle of a 50-εL Hamilton syringe was supplied with a silicon overrun limiter 3 mm from its spike (C). Inoculation was performed across the chest, from the right side, above the rib arc (arrow A), in the apex point (arrow B) of the costoclavicular perpendicular curve (D). Control experiments with trypan blue dye confirmed the localization of inocula within the middle lobe of the right lung (E).

Lung and spleen cell suspensions. At the time points after infection indicated, mice were killed by an ip injection of an overdose of thiopental (Biochemie), and cell suspensions from right and left lungs were prepared individually by use of methods described elsewhere [23, 25]. Briefly, blood vessels were washed out with 0.02% EDTA-Hanks' balanced salt solution. Right and left lungs were extracted from the chest and sliced with scissors into 1–3-mm3 pieces. The tissue was incubated for 1.5 h at 37°C in RPMI 1640 medium that contained a 10- mmol/L mixture of HEPES, l-glutamine, nonessential amino acids, sodium pyruvate, 2% fetal calf serum (FCS), antibiotics (all components, HyClone), as well as 200 U/mL collagenase and 50 U/mL DNase-I (Sigma). Cell suspensions obtained by vigorous pipetting were washed twice and resuspended in RPMI 1640 medium that contained 5% FCS. The viability of cells was >93%, according to trypan blue exclusion. Spleens were homogenized individually in glass homogenizers, cells were centrifuged, and erythrocytes were lysed with NH4Cl. Cells were washed twice and resuspended in RPMI 1640 medium that contained 5% FCS.

Staining of cell-surface molecules. The bulk lung-cell suspensions (3–5 × 105 cells/sample) were washed with PBS supplemented with 0.5% bovine serum albumin and 0.01% NaN3 and were incubated for 5 min at 4°C with anti-CD16/CD32 monoclonal antibodies (clone CT-17.1,17.2; Caltag), to block Fc receptors. Cells were then double-stained with directly conjugated antibodies, according to the manufacturer's instructions. The following antibodies were used in different combinations: fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled anti-CD4 (clone CT-CD4; Caltag), FITC- or PElabeled anti-CD8 (clone CT-CD8a; Caltag), PE-anti-F4/80 (clone CI:A3-1; Caltag), FITC-anti-CD11c (clone HL3; PharMingen), FITC-anti-Ly-6G (clone RB6–8C5; Caltag), FITC-anti-CD44 (clone IM7; PharMingen), or PE-anti-CD62L (clone MEL-14; Caltag). Stained cells were washed twice, fixed with 1% paraformaldehyde, and analyzed by use of a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo software (version 4.5.9; San Carlos). At least 104 cells from each sample were analyzed.

Cytokine assays. Lung cells were cultured at 2×106 cells/ mL in RPMI 1640 medium that contained 5% FCS and antibiotics. Cells were either stimulated with 10 εg/mL H37Rv sonicate or left unstimulated. Interleukin (IL)-4, IL-5, IL-10, IL-12, tumor necrosis factor (TNF)-α, and interferon (IFN)- γ were measured in 48-h culture supernatants by use of sandwich ELISAs. The following ELISA kits were purchased from PharMingen and were used according to the manufacturer's instructions: OptEIA mouse TNF-α (sensitivity, 31 pg/mL), OptEIA mouse IL-4 (sensitivity, 125 pg/mL), OptEIA mouse IL-5 (sensitivity, 75 pg/mL), OptEIA mouse IL-12 (sensitivity, 31 pg/mL), OptEIA mouse IL-10 (sensitivity, 63 pg/mL), and OptEIA mouse IFN-γ (sensitivity, 63 pg/mL). Results are expressed as the rate of antigen-specific cytokine production: ▲pg/mL = (pg/mLAg-stimulated-pg/mLnonstimulated).

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Results

Mycobacterial trafficking from the site of infection. We wished to answer 2 questions. First, does the hematogenous/lymphatic spread of mycobacteria to the lymph nodes and spleen precede their appearance in the contralateral lung? Second, does the speed and degree of dissemination differ between mouse strains with genetic differences in susceptibility to TB? To this end, we assessed the dynamics of the appearance and multiplication of mycobacteria in the contralateral lungs, spleens, and pulmonary lymph nodes after a single lung challenge of mice of 2 H-2-congenic mouse strains, B10.SM and B10.MBR, which have been characterized as being, respectively, highly and moderately susceptible to TB in terms of mortality [26].

As shown in figure 2, mycobacteria were initially contained within the infected right lung (figure 2A), and no spread to the left lung or lymphoid organs occurred during the first week after infection (figure 2B–2D). By the end of week 2, mycobacteria disseminated to the pulmonary lymph nodes (figure 2B), spleen (figure 2C), and liver (not shown); however, the contralateral left lung remained free of bacteria until week 3 (figure 2D). This dynamic picture indicates that infection of the initially intact lung is secondary to infection of the lymphoid organs and liver.

Figure 2.
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Figure 2.

Mycobacterial dissemination from infected right lung. Three independent experiments (4 mice analyzed individually in each experiment, n = 12) were performed. Then, 0.1 mL of serial 10-fold dilutions of right lung (A), pulmonary lymph node (B), spleen (C), and left lung (D) whole-organ homogenates were plated onto Dubos agar, and colonies were counted after 18-20 days of incubation at 37°C. Results are displayed as the mean no. of colony-forming units per organ ± SD (n = 12). Interstrain differences in colony-forming unit counts were significant (*P < .05–.01, Student's t test) in all organs at week 10 after infection (and at week 3 in the right lungs). White triangles, B10.MBR mice; black circles, B10.SM mice.

To more precisely define the time point when mycobacteria reach the left lung, we repeated the experiment and estimated numbers of colony-forming units in organs of mice on days 14, 17, and 21 after infection. Results obtained on days 14 and 21 (not shown) were in good agreement with those displayed in figure 2; results obtained on day 17 are presented in table 1. On day 17, mycobacteria were present in the spleens and livers of all mice in numbers very similar to those on day 14, which indicates the very beginning of their multiplication. No mycobacteria could be recovered from blood and distant lymph nodes not involved in the drainage of the lung. However, mycobacteria were recovered, although in very low numbers, from the left lungs of 3 of 4 B10.SM and from 2 of 4 B10.MBR mice, showing that dissemination to the contralateral lung occurs between days 16 and 18 after infection.

It is noteworthy that samples from the mouse strains under study did not differ in the size of the mycobacterial population detected in different tissues shortly after dissemination. Identical results were obtained when we applied the 1-lobe infection model to mice of the TB-resistant B6 strain (not shown). By week 10 after infection, however, the difference between B10.SM and B10.MBR mice in mycobacterial loads in organs reached ∼1 log (figure 2), which confirms the different susceptibility of these mouse strains to TB.

Dynamics of lung-tissue infiltration with inflammatory cells. To quantitatively characterize inflammatory reactions in primary-infected right lungs and secondary-infected spleens and left lungs, cell suspensions from the corresponding organs were obtained, the total number of cells was counted, and the content of neutrophils (Ly6G+), macrophages (F4/80+), dendritic cells (CD11c+), and CD4+ and CD8+ T cells was assessed by flow cytometry. These parameters were compared between B10.SM and B10.MBR mice throughout the course of infection. As shown in figure 3, all types of phagocytic cells peaked sharply in both lungs of the more-resistant B10.MBR mice as early as week 2 after infection but had decreased significantly in numbers by week 10 (figure 3D, 3E, and 3F). In contrast to this well-regulated response, the onset of inflammation in the lungs of B10.SM mice did not begin until week 3 but then progressed quickly: cells of all subsets, except neutrophils, accumulated in both lungs between weeks 3 and 10 after infection (figure 3). The pattern of T cell infiltration of the lungs was similar: CD4+ and CD8+ subsets peaked in infected right lungs of B10.MBR mice at weeks 2 and 3 after infection, respectively, and then decreased in number, whereas a gradual accumulation of these cells was found up to week 10 in B10.SM mice (figure 3B and 3C). In the spleen, ∼2 times more CD4+, F4/80+, and CD11c+ cells were recovered from B10.SM mice than from B10.MBR mice at week 10 after infection (data not shown).

Figure 3.
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Figure 3.

Dynamics of lung infiltration with inflammatory cells. The total no. of cells in right and left lungs of infected mice was counted (A), and the content of neutrophils (Ly6G+; D) macrophages (F4/80+; E), dendritic cells (CD11c+; F), and CD4+ (B) and CD8+ (C) T cells was assessed by flow cytometry and calculated. Three independent experiments were performed (n = 3), and, in each experiment, cells from 3 mice of each strain were mixed and analyzed (n = 9). Results are expressed as the mean no. of cells per organ ± SD (n = 3). *P <.05 and **P <.01 for interstrain differences, Mann-Whitney U test. White triangles, B10.MBR mice; black circles, B10.SM mice.

Lung T cell activation and cytokine production by lung cells. The mouse strains under study did not differ in the kinetics of mycobacterial spread from the initial lesion. On the other hand, interstrain differences were evident both in the ability to control mycobacterial multiplication in lungs and in the dynamics of the cellular infiltration of lung tissue. Because the main mechanism of antimycobacterial defense in the lungs is thought to be the activation of macrophages by immune T cells [27, 28], it was important to analyze the differences between B10.SM and B10.MBR mice in terms of the activation of lung T cells and/or the capacity to produce key cytokines. Lung cell suspensions from mice of each strain were prepared, and the proportion of T cells that expressed the CD44hiCD62Llo phenotype was determined. In parallel, the content of type 1 and type 2 cytokines was evaluated in cultural supernatants of lung cells stimulated with mycobacterial sonicate.

As shown in table 2, there was a significant difference in the activation status of lung T cells from B10.SM versus B10.MBR mice. By week 2 after infection, fully activated (CD44hi) cells constituted at least 80% of the whole T cell population of the lungs of B10.MBR mice, whereas >50% of B10.SM lung T cells remained CD44inter/lo. Although the proportion of activated T cells had decreased slightly in B10.MBR mice by week 3 after infection, in parallel with the population constriction (table 2 and figure 3B), it still remained significantly higher than the proportion in B10.SM mice. As illustrated in figure 4, approximately one-half of B10.SM lung T cells retained incomplete activation status. The assessment of T cell activation by downregulation of CD62L expression showed similar interstrain differences (data not shown).

Figure 4.
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Figure 4.

Interstrain differences in lung T cell activation after infection with tuberculosis. Expression of the CD44 marker was assessed by flow cytometry on gated lung T cells from B10.MBR (A and B, CD4+; E and F, CD8+) and B10.SM (C and D, CD4+; G and H, CD8+) mice. Shown are results of 1 of 2 similar experiments obtained with a mixture of cells from the right lungs from 3 mice of each strain at weeks 2 (A–D) and 3 (E–F) after infection. Similar T cell activation profiles characterized left lungs. In noninfected mice, the portion of lung T cells expressing the CD44 marker was never >30%.

We also assessed whether B10.SM and B10.MBR mice differed with respect to the pattern of cytokine production. As shown in figure 5, there was a substantial difference in the ability to produce 2 major effector type 1 cytokines in response to mycobacterial sonicate between lung cells from B10.MBR and those from B10.SM mice. IFN-γ responses developed slowly in our low-dose infection model, but, by week 10 after infection, B10.MBR lung cells and splenocytes produced, respectively, ∼10- and 3-fold more IFN-γ than their B10.SM counterparts (figure 5A). This was not due to an impaired IFN-γ- inducing IL-12 response in the latter mice: the level of IL- 12 production was similar in the 2 mouse strains. In both strains, IL-12 production peaked much earlier than that of IFNg and then decreased (figure 5B). Antigen-specific TNF-α production was significantly higher in B10.MBR mice at week 3 after infection but decreased by week 10, whereas B10.SM lung cells increased TNF-a synthesis throughout infection. This pattern may reflect a more severe course of disease in B10.SM lungs, with greater lung-tissue damage, especially given the lack of interstrain differences in TNF-a production in spleens (figure 5C). In both mouse strains, production of type 2 cytokines by lung cells was either low (IL-5 and IL-10) or undetectable (IL-4), and no significant interstrain differences were observed (data not shown).

Figure 5.
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Figure 5.

Cytokine production in vitro. Cells from right lungs, left lungs, and spleens were cultured at 2 × 106 cells/mL for 48 h in the presence or absence of 10 εg/mL H37Rv sonicate. The content of interferon (IFN)-γ (A), tumor necrosis factor (TNF)-α (B), interleukin (IL)- 12 (C), and IL-10 (D) was measured in 48-h culture supernatants by use of ELISA kits (PharMingen) in triplicate wells. Results (in picograms per milliliter; mean ± SD for triplicate results) of 1 of 2 similar experiments are expressed as antigen-specific cytokine production: Dpg/mLp(pg/ mLAg-stimulated−pg/mLnonstimulated). *P < .05 and **P < .01 for interstrain differences, Mann-Whitney U test. White triangles, B10.MBR mice; black circles, B10.SM mice.

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Discussion

In the present study, we have created an experimental TB model that is particularly suitable for studying mycobacterial spread from a primary lung lesion to distant anatomical sites—the lymphoid organs, liver, and contralateral lung. Using this 1- lobe infection model, we have demonstrated that mycobacterial spread to the initially unaffected lung starts between 16 and 18 days after infection, subsequent to dissemination to the spleen and draining lymph nodes, which occurs before day 14 (table 1 and figure 2). The latter is in a good agreement with the results of Chackerian et al. [10], who demonstrated mycobacterial spread from the lungs to the spleen and pulmonary lymph nodes at day 9–10 after aerogenic infection. However, when addressing another aspect of mycobacterial dissemination—its dependence on genetic susceptibility of a murine host—we obtained results different from those of Chackerian et al. [10]. Their results indicate that mycobacteria disseminate earlier and more readily from the lungs of TB-resistant B6 mice than from the lungs of TB-susceptible C3H mice. They speculated that this may lead to an earlier onset of acquired immunity in lymphoid organs, which results in better protection. In our system, dissemination of mycobacteria from the initial lesion occurred simultaneously in susceptible B10.SM, resistant B10.MBR (table 1 and figure 2), and B6 (data not shown) mice. The difference in susceptibility between the 2 strains with the B10 genetic background was reflected in a difference of 1 log in mycobacterial burden at week 10 after infection. One possible reason for the discrepancy between the 2 studies is the choice of mouse strains. In our H-2-congenic system, genetic variability was substantially narrower, and all mice could simply have carried identical alleles of unknown loci that are involved in the control of dissemination. Nevertheless, our results argue against the hypothesis that the speed and/or degree of mycobacterial dissemination from the initial lesion are major determinants of phenotypic differences expressed late during the course of infection, such as degree of lung pathology or time to death.

Table 1.
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Table 1.

Mycobacterial spreading from the right lung to distant organs on day 17 after infection.

Another question that we tried to resolve is whether infection of the initially unaffected lung occurs in the presence or absence of activated immune cells in this organ. There is substantial evidence that secondary TB lesions, which, in humans, are heavily concentrated in the apical regions of the lung, arise from hematogenous mycobacterial reseeding [1, 11]. Although it has been suggested [12] that mycobacteria return to the lung from extrapulmonary locations relatively early during the course of infection, the estimated time to reseeding (3–5 weeks) seems to exceed the period of 1–2 weeks reported by other authors [29] for the development of T cell immunity to infection and the migration of both activated CD4+ and CD8+ T cells to the lungs. It is possible that the early appearance of activated T cells in the latter study was due to the intravenous route of infection, because numerous observations have indicated that, when infection occurs via the respiratory tract, the arrival of activated T cells to the lungs is significantly postponed [30– 32]. In the present study, there was no difference in the activation status of T cells homing to the infected right and uninfected left lung at week 2 or 3 after infection (table 2). These results suggest that, shortly after the infectious process has been developed at 1 site in the host, nonlymphoid tissues are under surveillance by already activated T cells, which penetrate into parenchymal tissues, irrespective of the presence of the infectious agent. This conclusion is supported by the results seen in nonmycobacterial infectious models. Thus, mice infected with influenza virus, vesicular stomatitis virus, or ovalbuminexpressing Listeria monocytogenes were seen to demonstrate substantial migration of CD8+ T cells to pathogen-free peripheral tissues [19, 33].

Table 2.
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Table 2.

The content of fully activated (CD44hi) CD4+ and CD8+ lymphocytes in the lungs of B10.SM and B10.MBR mice.

In contrast to the lack of influence of anatomic location, the activation status of T cells is strongly dependent on the genetics of the host. In the relatively resistant B10.MBR mice, a significantly larger proportion of T cells expressed the “full activation” CD44hiCD62lo phenotype (table 2 and figure 3). This was reflected in a much higher production of IFN-γ during the later stages of disease (figure 4) and corresponds well to data concerning activation status and memory establishment in TB-susceptible and -resistant mice that have been seen in other strain combinations [34].

In susceptible B10.SM mice, infiltration of left lungs with inflammatory cells was negligible before the beginning of infection at week 3. Remarkably, in resistant B10.MBR mice, populations of all phagocytic cells increased several-fold, not only in the infected right but also in the uninfected left lung, between weeks 1 and 2 after infection (figure 3D–3F). One possible explanation for this unexpected result could be a marked increase in the level of macrophage inflammatory protein-2 and IL-6 production by murine lung cells between days 0 and 7 after infection (authors' unpublished observation). These powerful inflammatory factors may cause a rapid extravasation of phagocytic cells and their influx into the lung tissue. However, it remains to be determined whether B10.MBR lung cells differ in this regard from their B10.SM counterparts.

Although the mouse strains differed in terms of the dynamics of lung infiltration, the accumulation of inflammatory cells in primary-infected right and secondary-seeded left lungs followed essentially identical patterns within each strain (figure 3). Given that the right lung is ∼2.5-fold bigger than the left one (this is the coefficient that we used to normalize cellularity per organ), one may conclude that populations of cells that infiltrate the originally infected and the uninfected lung during early phases of infection are the same in terms of subset composition and cell numbers. On the other hand, the dynamics of inflammation depend strongly on the genetics of the host, and it is attractive to speculate that, in resistant animals, the initiation of infection causes a temporary increase in phagocyte emigration from the bloodstream, enhancing “nonspecific” surveillance of parenchymal organs. A rapid down-regulation of the cell influx (figure 2) and potentially hazardous TNF-a production (figure 4) occur once infection has been brought under control. Delayed initiation of phagocyte recruitment and a failure to control tissue-damaging reactions in the lungs lead to a more severe course of disease in susceptible animals.

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Footnotes

  • Financial support: Wellcome Trust (Cooperative Research Initiative Grant to D.B.Y. and A.S.A.); Howard Hughes Medical Institute (grant 75301564101 to A.S.A. as a Howard Hughes International Research Scholar); US National Institutes of Health (R01 grant HL 68532-02).

  • Received May 4, 2004.
  • Accepted June 24, 2004.
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  1. J Infect Dis. (2004) 190 (12): 2137-2145. doi: 10.1086/425909
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