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*
Department of Immunology, Central Institute for Tuberculosis of Russian Academy of Medical Sciences; and
Laboratory for Immunochemistry, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Moscow, Russia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and of activating macrophages for NO production and
mycobacterial growth inhibition. However, significantly more
CD8+ T cells were accumulated in the lungs of resistant
A/Sn and F1 compared with I/St mice. About 80% A/Sn and
F1 CD8+ cells expressed
CD44high/CD45RB+ phenotype, while about 40%
I/St CD8+ cells did not express CD45RB marker at week 5 of
infection. In contrast, in susceptible I/St mice lung CD4+
cells proliferated much more strongly in response to mycobacterial
sonicate, and a higher proportion of these cells expressed CD95 and
underwent apoptosis compared with A/Sn cells. Unseparated lung cells
and T cells of I/St origin produced more IL-5 and IL-10, respectively,
whereas their A/Sn and F1 counterparts produced more IFN- following
infection. F1 cells overall expressed an intermediate
phenotype between the two parental strains. Such a more balanced type
of immune reactivity could be linked to a better TB
defense. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 were
found to be essential for the development of protective immunity
(1, 2, 3, 4), confirming the concept that IFN--producing
CD4+ and CD8+ T cells are
crucial for the control of mycobacterial infections
(5, 6, 7, 8, 9, 10). These experimental data are in agreement with
recent findings in humans. Thus, it was shown that individuals lacking
functional IFN- receptors (11, 12) or IL-12 receptors
(13, 14) due to mutations in corresponding genes are
particularly susceptible to mycobacterial infections. The mechanism by
which IFN- mediates anti-mycobacterial activity is thought to be
macrophage activation for the production of NO, the metabolite that
plays a pivotal role in mycobacterial intracellular killing
(15). Another type 1 cytokine that is able to up-regulate
NO production by macrophages, TNF-
, apparently displays a dual role
in the course of M. tuberculosis-triggered disease. Besides
the host-protective anti-mycobacterial activity of TNF-
(16), its ability to cause tissue damage may contribute to
lung pathology and to a more rapid death of the host (17, 18). Although murine experimental systems based upon genetic disruption of genes encoding central elements of host defense have proven to be extremely useful analytical tools, they possess at least one major intrinsic disadvantage for modeling the spectrum of genetic and immune variability existing in the general population. Gene targeting that results in the complete abrogation of any key function of the immune system leads to a defect in protection against infection which is extreme. Given that such defects are normally rapidly eliminated from a population by natural selection, they hardly could account for the much more common, modestly susceptible phenotypes. Genetic and immunologic mechanisms underlying the expression of the latter could be more rationally studied by the traditional means of segregation genetic analysis and interstrain comparison of several parameters of the immune response. It should be emphasized, however, that quantitative interstrain differences concerning, for example, cytokine production or the degree of cell activation are in most cases under polygenic control. Thus, the interpretation of results obtained by comparison between conventional mouse strains is usually far more ambiguous than that of results from experiments with gene-targeted animals.
Nevertheless, using these traditional approaches we demonstrated the impact of several H-2 and non-H-2 genetic loci on the course of a lethal TB infection and clarified several aspects of the immune response to mycobacterial Ags following Calmette-Guérin bacillus vaccination and infection (19, 20, 21, 22). In particular, it was found that the severe course of the disease caused by i.v. M. tuberculosis H37Rv injection in mice of the I/St inbred strain contrasts sharply with its relatively mild development in mice of the A/Sn inbred strain (22, 23). Genome-wide linkage analysis of the severity of TB showed a significant linkage with microsatellite loci on distal chromosome 3 and proximal chromosome 9 in females and suggestive linkages for both sexes with loci on chromosomes 5, 8, 10, and 17 (23). Despite the fact that chromosome regions surrounding corresponding quantitative trait loci contain several genes that regulate the function of cells of the immune system (candidate genes), the physiologic basis for the difference in susceptibility to TB between I/St and A/Sn mouse strains remains unknown. Interestingly, it was found that in (I/St x A/Sn)F1 hybrids both mortality and body weight loss following tuberculous challenge were significantly postponed compared even to resistant A/Sn parental mice (20, 23, 24). Thus, the combination of resistant A/Sn, susceptible I/St, and hyperresistant F1 mice probably reflects the spectrum that exists in the general population and provides a useful tool to study functional aspects of TB control.
To address this issue, we have started to study the properties of immune T cells that reside in the lungs of infected mice, assuming that their responses to mycobacteria play the pivotal role in both protection against and pathology of M. tuberculosis-triggered disease. It was demonstrated that after the onset of infection lung T cells of susceptible I/St mice rapidly acquire the CD44+/CD45RB-/low activated surface phenotype. These cells readily proliferate in the presence of mycobacterial Ags and produce several type 1 and type 2 cytokines, with a bias toward IL-10 synthesis (25). Here we report on the comparative ex vivo study of T lymphocytes recovered from the lungs of susceptible I/St, resistant A/Sn, and hyperresistant (I/St x A/Sn)F1 mice following tuberculous challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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I/StYCit (I/St), A/JSNYCit (A/Sn), and (A/Sn x I/St) F1 mice were bred at the animal facilities of the Central Institute for Tuberculosis (Moscow, Russia) according to the rules of Russian Academy of Medical Sciences, with water and food provided ad libitum. Female mice, 2–4 mo of age, were used.
Infection
Mice were infected i.v. with 105, 104, or 103 CFUs of mid-log phase M. tuberculosis strain H37Rv (collection of the Central Institute for Tuberculosis) in 0.5 ml of saline. The method of establishment of clump-free, mid-log phase mycobacterial preparations was described previously in detail (22, 25). To assess mycobacterial load in spleens and lungs, 0.2 ml of serial 10-fold dilutions of organ homogenates was plated onto Dubos agar, and colonies were counted after 18–20 days of incubation at 37°C.
Lung, spleen, and lymph node cell suspensions
Two, 5, and 8 wk following challenge, mice were euthanized by injection of an overdose of thiopental (Biochemie, Vienna, Austria), and suspensions of spleen, auxiliary lymph nodes, and lung cells were prepared individually. Lung cells were isolated using the method described by Holt et al. (26) with our modifications (25). Briefly, blood vessels were washed out, and repeated bronchoalveolar lavage was performed using 0.02% EDTA-HBSS solution. Lung tissue was sliced into 1- to 2-mm3 pieces and incubated at 37°C in RPMI 1640 containing 5% FCS, antibiotics, 10 mM HEPES (all from HyClone, Carlington, The Netherlands), 200 U/ml collagenase, and 50 U/ml DNase (Sigma, St. Louis, MO). Single-cell suspensions were obtained by vigorous pipetting. Cells (hereafter referred to as unseparated cells) were washed and resuspended in culture medium, i.e., RPMI 1640 supplemented with 5% FCS, 10 mM HEPES, 2 mM L-glutamine, 1% nonessential amino acids, pyruvate, 5 x 10-5 2-ME, antibiotics (all from HyClone). T lymphocyte enrichment was achieved by sequential elimination of plastic-adherent and nylon wool-adherent cells, which resulted in approximately 75% CD3+ cell purity (25). The viability of cells, as determined by trypan blue exclusion, was >93%.
Cloning of lung T cells
To estimate the efficacy of lung T cell cloning, T-enriched lung cells were cloned by limiting dilutions as described previously (25). Briefly, lung T lymphocytes obtained at week 5 postinfection were enriched in mycobacteria-specific cells by stimulation in vitro with H37Rv sonicate; the latter were isolated by centrifugation on Lympholyte M gradient (Cedarlane, Ontario, Canada) and cloned in the presence of H37Rv sonicate, irradiated splenic APCs, and conditioned medium, as a source of cytokines. Positive wells were restimulated in situ or were split into new wells every 10–14 days. The efficacy of T cell cloning in response to Con A (2.5 µg/ml; Sigma) was estimated for freshly isolated T-enriched lung cells from either infected or naive mice. Cells were stimulated once with Con A, APC, and cytokines at the beginning of experiment, and the number of positive wells was estimated microscopically between days 7 and 12 of culture.
Proliferative response
T-enriched lung and spleen cells (105) mixed with 3 x 105 syngenic APC or 3 x 105 bulk lymph node cells were cultured in a well of 96-well flat-bottom plate (Costar, Badhoevedorp, The Netherlands) at 37°C in 5% CO2 in the presence of 10 µg/ml H37Rv sonicate. All cultures were performed in triplicate, and nonstimulated wells served as controls. Cultures were pulsed with 0.5 µCi of [3H]thymidine for the last 18 h of a 48- to 72-h incubation. The label uptake was measured in a liquid scintillation counter (Wallac, Turku, Finland) after harvesting the well’s contents onto fiberglass filters using a semiautomatic cell harvester (Scatron, Oslo, Norway).
Staining of cell surface molecules
Lung cells (3–5 x 105) were washed
twice in PBS containing 0.01% NaN3 and 0.5% BSA
and were incubated for 5 min at 4°C in the presence of CD16/CD32 mAbs
(clone 2.4G2, PharMingen, San Diego, CA) to block Fc receptors. Cells
were then double or triple stained with directly conjugated Abs
according to the manufacturer’s instructions. All Abs except
FITC-anti-CD11 (clone I21/7, Sigma) were purchased from
PharMingen: FITC-anti-CD4 (clone H129.19), PE-anti-CD8a (clone
53-6.7), PE-anti-CD44 (clone IM7), FITC-anti-CD45RB (clone
16A), PE-anti-CD28 (clone 37.51), FITC-anti-CD152 (CTLA-4,
clone UC10-4F10-11), FITC-anti-CD95 (clone Jo2), and
FITC-anti-CD95 ligand (anti-CD95L; clone MFL3). Stained cells
were washed twice, fixed with 1% paraformaldehyde, and analyzed by
flow cytometry.
Apoptosis evaluation
Unseparated lung cells recovered from the lungs of infected mice were cultured in 24-well plates (106/well) in the presence or the absence of 10 µg/ml H37Rv sonicate for 40 h. Cells were harvested and stained with PE-labeled anti-CD4 or anti-CD8 mAbs (Caltag, South San Francisco, CA), followed by staining with FITC-annexin V and 7-amino-actinomycin D (7AAD; PharMingen), according to the manufacturer’s instructions. 7AAD stain was used because, unlike propidium iodide, its fluorescent spectrum allows gating of PE-stained cell subsets.
Assessment of T cell proliferation by bromodeoxyuridine (BrdU) incorporation
At week 5 postinfection, mice were injected with 2 mg of BrdU (Sigma) i.p. Two hours later, auxiliary lymph nodes were extracted, single-cell suspensions were prepared individually, and BrdU incorporation was assessed as described previously by Esin et al. (27). Cells were washed in mouse tonicity HBSS supplemented with 5% FCS and 20 U/ml DNase I (Sigma). CD4 and CD8 membrane markers were stained with PE-labeled mAbs. Cells were washed in supplemented mouse tonicity HBSS and fixed with 1% paraformaldehyde in PBS containing 0.01% Tween 20. After 96 h of incubation at 4°C, cells were washed and incubated with DNase solution (4.2 mM MgCl2, 10 µM HCl, and 50 U/ml DNase in 0.15 M NaCl) at 37°C for 60–120 min. After washing, cells were stained with FITC-anti-BrdU mAbs (Becton Dickinson, San Jose, CA), washed in PBS/FBS/Tween 20 (PBS supplemented with 0.5% Tween 20 and 5% FBS), resuspended in PBS, and analyzed by flow cytometry.
Flow cytometry of stained cells
An EPICS Elite flow cytometer (Coulter, Miami, FL) equipped with a Cyonics argon laser (Uniphase, San Jose, CA) with excitation at 488 nm and 15-mW power, and barrier filters at 488BK, 550DL, 525BP, 625DL, and 575BP, was used throughout the experiments. At least 104 cells from each sample were analyzed, and the data were processed by means of MultiGraph software (Coulter). Unstained cell controls were analyzed at each time point.
Cytokine assays
ELISAs were used to detect IL-4, IL-5, IL-10, IL-12, TNF-,
and IFN- in 48-h culture supernatants. Capture and detecting
(biotinylated) mAbs specific for mouse cytokines were purchased from
PharMingen: for IFN-, clones R4-6A2 and XMG1.2 (sensitivity, 312
pg/ml); for IL-4, clones 11B11 and BVD6-24G2 (sensitivity, 62 pg/ml);
for IL-5, clones TRFK5 and TRFK4 (sensitivity, 24 pg/ml); for IL-10,
clones JES5-2A5 and JES5-16E3 (sensitivity, 312 pg/ml); for IL-12,
clones C 17.8 and C 15.6 (sensitivity, 250 pg/ml); and for TNF-,
clone MP6-XT22 and polyclonal Abs (sensitivity, 125 pg/ml). ELISAs were
performed following the manufacturer’s instructions. A standard curve
for each assay was generated with known concentrations of mouse rIL-4,
rIL-5, rIL-12, and rTNF- (all from PharMingen), rIL-10 (Sigma), and
rIFN- (Genzyme, Boston, MA).
Antimycobacterial activity of macrophages
To assess the anti-mycobacterial activity of macrophages, we
used the method described by Stach et al. (28) with
modifications (25). Briefly, peritoneal exudate cells
(15 x 106) were incubated for 1 h on
60-mm petri dishes (Costar) in 3 ml of antibiotic-free cultural medium
at 37°C. Plastic nonadherent cells were removed, and plastic adherent
cells were detached by incubation in 2 mM EDTA-PBS. Plastic adherent
cells (6 x 104; >90% of nonspecific
esterase-positive cells) were put in a well of a flat-bottom 96-well
plate (Costar) and 12 x 104 live-filtered
M. tuberculosis/well were added. Infected macrophages were
cocultured with 6 x 104/well lung or spleen
T cells freshly isolated from infected mice. T cell-free cultures of
mycobacteria-loaded macrophages, supplemented or not with 100 U/ml
recombinant murine IFN- (Genzyme), served as positive and negative
controls, respectively. Multiplication of mycobacteria was assessed by
[3H]uracile uptake exactly as previously
described (25, 28). Results are expressed as counts per
minute.
NO production by infected macrophages
NO production was assessed as described previously (29). Briefly, peritoneal macrophages were loaded with M. tuberculosis and cultured with or without addition of T cells as described above. Thirty-six hours later, 100 µl of Griess reagent was added to 100-µl supernatant aliquots in the wells of a round-bottom plate, and plates were incubated for 10 min at room temperature. Absorbance was measured at 550 nm in a micro-ELISA reader (Sigma), using a 620-nm reference filter.
Statistical analysis
The significance of the differences was estimated by Student’s t test, Wilcoxon test, and Mann-Whitney test. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Groups of I/St, A/Sn, and F1 female mice
were challenged i.v. with 105 M.
tuberculosis H37Rv CFUs, and individual whole-organ homogenates of
lungs and spleens were plated in serial dilutions onto agar dishes to
determine mycobacterial recovery. As shown in Fig. 1
, during the first 2 wk of infection
mycobacterial multiplication was prominent in spleens of all infected
mice; at least 20 times more mycobacteria were recovered compared with
the number injected. An approximately 5-fold difference between
susceptible I/St and two other mouse strains was observed (Fig. 1A). The size of the mycobacterial population residing in
lungs 2 wk postinfection was considerably smaller than that in spleens,
and mycobacterial burden was 10-fold higher in I/St compared with A/Sn
and F1 mice (Fig. 1B). At week 5
following challenge, the number of mycobacteria in spleens of
susceptible I/St mice remained constant, whereas in spleens of both
A/Sn and F1 mice mycobacterial load decreased to
a significantly lower (p = 0.03) level (Fig. 1A). In the lungs, the enlargement of the mycobacterial
population between weeks 2 and 5 was registered in all three mouse
strains (p < 0.01 compared with week 2).
Again, the mycobacterial population reached about 10-fold higher
numbers in I/St compared with A/Sn (p = 0.01)
and F1 (p = 0.02) mice
(Fig. 1B). At week 8 postinfection, the mycobacterial
population diminished in size in spleens (Fig. 1A) and
reduced the speed of growth in lungs (Fig. 1B) of A/Sn and
F1 mice. In contrast to the generally good health
of all resistant animals at week 8 postinfection, only 4 of 11 I/St
mice survived (one, one, and two in three independent experiments),
which did not allow us to reliably estimate the bacterial load in their
organs. These results indicate that our earlier conclusions concerning
interstrain differences in susceptibility to infection (20, 22) and the dominant inheritance of resistance by
F1 hybrids (24) are applicable to
the dynamics of mycobacterial growth in organs of infected
mice.
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The total cell yield from the lungs of naive and infected mice of
the three strains was estimated by microscopy of unseparated lung cell
suspensions. The proportions of CD3+,
CD4+, CD8+, and
NK-1.1+ cells were assessed in T-enriched cell
suspensions by flow cytometry (see the footnote to Table I).
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, the cellularity of lungs of all three strains was
markedly elevated (p < 0.05–0.01) throughout
the infectious course compared with that in uninfected syngenic
controls. Accumulation of bulk CD3+ T cells
following infection was more pronounced in the lungs of A/Sn and
F1 mice compared with that in I/St mice. The most
notable difference between susceptible I/St and resistant A/Sn and
F1 mice was that significantly higher numbers of
CD8+ T cells were recovered from lungs of the
latter two strains throughout infection (p <
0.05). In addition, in F1 hybrids the total lung
cellularity as well as the number of CD4+ T cells
increased earlier than in either parental strain (Table I, second week,
p < 0.05). NK-1.1+ cells were
scarcely present in the lungs of all mice (
1.5% positive cells;
data not shown). Proliferation of lung and spleen T cells in the presence of mycobacterial Ag
It was of interest to find out whether the proliferative capacity
of lung T cells is essential for the expression of the resistant
phenotype. We also wished to clarify whether a more pronounced
accumulation of these cells in resistant animals is the consequence of
their capacity to proliferate more vigorously in response to
mycobacterial Ags. Thus, we assessed the level of lung T cell
proliferation in the presence of syngenic splenic APCs and H37Rv
sonicate as a source of Ag. Unexpectedly, I/St lung T cells started to
proliferate earlier following challenge and retained a higher
proliferative capacity (p = 0.02) throughout
the infectious course than their A/Sn counterparts (Fig. 2A). In four of five
experiments A/Sn lung T cell did not proliferate at all at 2 wk
postinfection. Furthermore, lung T cells from F1
mice exhibited intermediate levels of proliferation. Similar results
were obtained when the proliferation of cells from auxiliary lymph
nodes at 5 wk postinfection was measured (Fig. 2B). Taken
together, these results suggest that neither a more pronounced
accumulation of T cells in the lungs nor the ability of the host
to better control the disease is due to Ag-induced local T cell
proliferation in the vicinity of tuberculous foci.
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or could be due to their intrinsic capacity to develop higher levels of
mycobacteria-specific and/or nonspecific T cell responses. To
distinguish between these two possibilities, groups of I/St mice were
infected with decreasing doses of mycobacteria and compared with A/Sn
mice infected with a standard dose (105 CFUs)
with respect to mycobacterial load in their lungs and capacity of lung
T cells to respond to the mycobacterial sonicate. As shown in Table II, I/St lung T cells responded to the Ag
almost regardless of the challenging dose of H37Rv. Moreover, when I/St
mice were infected with 103 CFUs and the
mycobacterial load in their lungs did not differ from that of A/Sn mice
challenged with 105 CFUs, a dramatic interstrain
difference in proliferative response was still present (Table II).
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and IV,
I/St and A/Sn lung T cells extracted at 5 wk postinfection showed
strikingly different patterns of growth under identical limiting
dilution conditions (described previously (25)). During
the initial three stimulation/rest cycles, the efficacy of cloning (the
number of positive wells) for I/St lung T cells was at least 10- fold
higher than that for A/Sn cells (Table III). Moreover, a few initially
established A/Sn T cell clones ceased to proliferate at the fifth to
sixth stimulation round, while several I/St clones were successfully
expanded.
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). However, the size of all proliferating cell
populations of A/Sn origin by day 12 of culture was limited to 20–40
cells, whereas the vast majority of I/St-positive wells contained
several hundred blast-like cells. The difference was even more striking
when T cells were obtained from infected mice. In addition to the
manyfold bigger size of individual I/St clones, a higher frequency of
positive wells in I/St cultures was apparent (Table IV). These results
suggest that I/St lung T cells are more readily activated for further
proliferation both in vivo by infection and in vitro by Con A
stimulation than their A/Sn counterparts.
To confirm that the interstrain difference in the proliferative
capacity of T cells was not restricted to the in vitro experimental
system, we assessed the proliferation of T cells in situ by BrdU
incorporation. At 5 wk postinfection, I/St and A/Sn mice were injected
i.p. with BrdU, and its incorporation into CD4+
and CD8+ lung and lymph node T cells was
individually analyzed by flow cytometry. As shown in Fig. 3, among lymph node cells extracted from
I/St mice, about twice as many (p < 0.05, by
Mann-Whitney t test; n1 =
4; n2 = 4) CD4+
and CD8+ cells incorporated BrdU compared with
A/Sn cells, thus confirming the results obtained in vitro. The
proportion of lung T cells incorporating BrdU was too low to draw
conclusions.
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Given the differences in the ability of I/St and A/Sn T cells to
proliferate in vitro in response to mycobacterial Ags, we wondered
whether the expression of T cell activation markers and of
costimulatory molecules also differ in the lungs of these mice.
T-enriched lung cell suspensions were triple stained with Abs against
either CD4 or CD8 subset marker in combination with anti-(CD44,
CD45RB) Abs, triple stained with the combination of anti-(CD4, CD8)
Abs with either anti-CD95 or anti-CD95L, or double stained with
anti-CD4 and anti-CD8 Abs in reciprocal combinations with
anti-CD28, anti-CD152, or anti-CD11- Abs.
No major interstrain differences were found in the expression of
CD44/CD45RB molecules on T cells before challenge (Fig. 4, plates 1–3). Regardless of
the mouse strain, 30–45% CD4+ cells and
15–30% CD8+ cells expressed the
CD44low/CD45RB+ phenotype
of resting/naive cells. Approximately 20–40%
CD4+ and 70–80% CD8+
cells expressed the phenotype of activated cells
(CD44+/CD45RBlow and
CD44+/CD45RB+,
respectively). The relative abundance of activated lung T cells before
challenge is most likely due to routine stimulation of the respiratory
tract with inhaled antigenic substances. Since the expression of CD44
and CD45 markers is known to vary naturally between mouse strains
(30), it was important to show that there was no major
intrinsic genetic variation with respect to this trait in the strains
under study. As early as 2 wk postinfection, the percentage of
CD44-positive activated lung T cells markedly increased in both major
subsets (data not shown) and remained remarkably high throughout
infection. At 5 wk only about 10–20% of the cells retained the
CD44low phenotype and up to 90% were expressing
CD44 activation marker (Fig. 4, plates 4–6 and
10–12).
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, in I/St lungs the total number of
CD45RB+ CD4+ cells did not
exceed 30% (Fig. 4, plate 4) compared with 50% in A/Sn
lungs (Fig. 4, plate 5). Cells from F1
mice occupied an intermediate position between those from parental
strains (Fig. 4, plate 6). Among CD8+,
the CD44+/CD45RB+ activated
phenotype was expressed by >70% A/Sn (Fig. 4, plate 11)
and F1 (Fig. 4, plate 12) cells, but
only by approximately 50% I/St (Fig. 4, plate 10) cells.
Making an inventory of the interstrain differences in total numbers of
lung CD8+ cells (Table I) following the
challenge, the number of CD8+
CD44+/CD45RB+ cells was
about 4-fold lower in I/St compared with A/Sn and
F1 mice (0.7, 2.7, and 2.0 million/mouse,
respectively).
Neither CD4+ nor CD8+ lung
T cells from all three mouse strains expressed CTLA-4 (CD152) molecule,
but both subsets readily and uniformly expressed CD28 molecule. In all
mice, the proportion of CD28-positive cells increased from 40–50% at
2 wk to 90–95% at 5 wk postinfection. Analogously, infection was
accompanied by a substantial increase in the proportion of cells
expressing the -chain of LFA-1 (CD11-) integrin (from 45–60%
before infection to 90–99% at 5 wk postinfection).
Since a significantly higher proliferative response of T cells in the
lungs of infected I/St mice (Fig. 2) did not lead to their accumulation
in the organ (Table I), we compared the expression of the most common
apoptotic marker, CD95 (Fas), on lung T cells, anticipating that more
I/St cells could undergo apoptosis during the infectious course. In all
mice CD95 was scarcely present on the surface of lung T cells at 2 wk
postinfection (data not shown). As shown in Fig. 5, at 5 wk following challenge, a
significantly higher proportion of I/St lung CD4+
T cells carried the CD95 marker compared with A/Sn (58 and 30%;
p < 0.05). Interestingly, in all three mouse strains
the proportion of CD8+ lung T cells expressing
CD95 was always higher than that of CD4+ cells,
although no interstrain differences were found (Fig. 5). To ensure
ourselves that Fas expression is not a simple consequence of cell
activation but is functionally linked to apoptosis, we have evaluated
the proportion of CD4+ and
CD8+ apoptotic cells from I/St and A/Sn infected
lungs. Unseparated lung cells were stained with annexinV/7AAD following
stimulation in vitro with mycobacterial sonicate. As shown in Fig. 6, fewer I/St CD4+
cells remained alive, and more underwent apoptosis compared with A/Sn
CD4+ cells (16 vs 26% and 83 vs 64%,
respectively). As with CD95 expression, a higher proportion of
CD8+ compared with CD4+
cells was subjected to apoptotic death, and no interstrain differences
were found in this T cell subset.
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Cytokine production was determined in lung, spleen, and lymph node
cell culture supernatants of two different types. First, unseparated
cells alone were cultured in the presence of H37Rv sonicate, and the
contents of six different cytokines in supernatants were determined.
Second, T-enriched lung or spleen cells were cocultured with irradiated
syngenic splenic APC and the Ag, and the contents of IFN-, IL-4,
IL-5, and IL-10 were assessed. Since TNF- and IL-12 are synthesized
predominantly by macrophage-like cells, we did not measure the contents
of these two products in the second type of cultures.
At week 2 postinfection, I/St bulk lung cells produced significantly
(p < 0.05) less IFN- than their A/Sn and
F1 counterparts. At this time purified lung T
cells from all mice produced similar levels of IFN-, but always
lower levels compared with unseparated cells, suggesting that non-T
lung cells contribute much to IFN- production/induction, especially
in resistant mice (Table V). The most
potent IFN- inducer, IL-12, was also synthesized by unseparated lung
cells of all three mouse strains. At week 2 postinfection I/St and
F1 cells exhibited identical IL-12 production,
whereas at week 5 postinfection I/St cells synthesized lower amounts of
IL-12. A 2-fold difference in TNF- levels occurred at weeks 2 and 5
postinfection between I/St and resistant mice. Thus, the production
of three major type 1 cytokines by the lung cells was moderately
higher in resistant mice.
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). These
differences, however, were not statistically significant between
parental strains due to variations in the levels of cytokine production
in distinct experiments. Thus, in four experiments IL-10 levels in I/St
cultures varied between 300 and 3500 pg/ml. A level of 600 pg/ml was
detected in A/Sn supernatant in one experiment, but only trace
quantities were spotted in three others. However, the difference
between I/St and F1 mice was significant
(p < 0.01). In agreement with the results
obtained in lungs, IL-10 was synthesized by auxiliary lymph node cells
from infected I/St mice (500–800 pg/ml; three mice were assessed
individually at week 5), but was not produced by A/Sn lymph node cells.
That I/St lung cells more readily produce type 2 cytokines, as
suggested by IL-10 data, was confirmed by IL-5 assessment. Unseparated
lung cells from I/St mice produced measurable amounts of IL-5 at weeks
2 and 5, whereas only trace amounts were found in A/Sn supernatants
(Table V). F1 mice showed intermediate
inheritance of this phenotype. Interestingly, no production of IL-5 was
detected in cultures of T-enriched lung cells, suggesting that non-T
cells residing in infected lungs are responsible for IL-5 production
and/or induction.
Taken together, these results indicate that there is a moderate bias
toward activation of a type 2-like cell response in the lungs of
TB-susceptible I/St mice. However, polarization of response is
incomplete (i.e., the absence of IL-4 and readily detectable levels of
IFN- in I/St supernatants) and is masked when unseparated lung cells
are studied, apparently due to abundant production of several cytokines
by non-T lung cells.
Stimulation of anti-mycobacterial activity of macrophages by lung and spleen cells from infected mice
Given that IFN- production by I/St lung T cells was somewhat
lower than that by A/Sn and F1 cells and
anticipating that not only IFN- potentiates macrophage activation,
we examined whether T cells from these mice differ with respect to
their capacity to activate anti-mycobacterial macrophage function.
To address this issue, peritoneal macrophages from I/St, A/Sn, and
F1 mice were cocultured in vitro with live H37Rv
mycobacteria and syngenic T-enriched lung cells obtained from infected
mice. Mycobacterial growth was assessed by
[3H]uracil incorporation (28).
When cultured in antibiotic-free culture medium, alone or within
macrophages, mycobacteria readily incorporate
[3H]uracil (25). As shown in Fig. 7, addition of exogenous rIFN- to
macrophage/mycobacteria cocultures (positive control) caused a profound
inhibition (85–95%) of mycobacterial growth regardless of the mouse
strain. Addition of lung T cells extracted 2 wk postinfection from
I/St, A/Sn, and F1 mice moderately and equally
inhibited mycobacterial multiplication (20–30% inhibition in
different experiments). Five weeks after challenge the ability of T
cells of all three strains to stimulate anti-mycobacterial activity
of syngeneic macrophages significantly increased; up to 90% inhibition
of mycobacterial growth was registered. Thus, no major interstrain
differences in the ability of T cells to stimulate the restriction of
mycobacterial growth in vitro were found.
|
and with cells obtained from all three mouse
strains) were plotted against the levels of
[3H]uracil incorporation (counts per minute) in
corresponding cultures. As shown in Fig. 8, there was a good inverse correlation
between the two measurements (r = -0.781), suggesting
the pivotal role of RNI in mycobacterial killing in our system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following most notable interstrain differences were found. First,
the mycobacteria-specific proliferative response of lung T cells was
much higher in susceptible I/St mice (Fig. 2 and Table II).
Paradoxically, this was accompanied by a diminished T cell accumulation
in the lungs; significantly more bulk T cells and particularly
CD8+ T cells were recovered from resistant mice
following challenge (Table I). Moreover, a higher proportion of A/Sn
and F1 CD8+ lung T cells
expressed activated
CD44+/CD45RB+ phenotype
compared with I/St (Fig. 4). Secondly, in I/St mice a higher proportion
of lung CD4+ cells expressed CD95 (Fas) receptor
(Fig. 5) and underwent apoptosis (Fig. 6). This suggests that a more
active lung T cell proliferation in these mice is counterbalanced with
a more rapid apoptotic elimination. Thirdly, I/St lung cells produce
more IL-5 and IL-10 and less IFN- and TNF- in response to
mycobacterial Ags (Table V), indicating a moderate bias toward a
Th2-like response in susceptible mice.
Expansion of CD4 and CD8 T cells in murine lungs in response to mycobacterial infections was recently reported in the model of intratracheal infection with M. bovis Calmette-Guérin bacillus (37) and in the models of aerosol (38, 39), i.v. (25, 40), and i.p. (41) challenge with M. tuberculosis. In this study we have extended these observations by demonstrating that in the lungs of TB-resistant A/Sn and F1 mice accumulation of CD8+ cells reaches significantly higher levels than in susceptible I/St mice. These results are in line with numerous data indicating a protective role of CD8+ T cells in mycobacterial infections (8, 10, 42, 43), particularly evident in the lungs (42, 44).
There was a clear interstrain difference in the capacity of T cells
from infected mice to proliferate in response to mycobacterial Ags.
I/St T cells demonstrated a significantly higher proliferative response
both in vitro (Fig. 2 and Tables III and IV) and in vivo (Fig. 3) than
their A/Sn counterparts, and F1 T cells showed an
intermediate response. This superior responsiveness of T cells from
I/St mice was not due to a higher bacterial load in their lungs (Table II). Since the course of the disease is extremely severe in I/St mice,
it could be argued that the T cell proliferative response to
mycobacteria is nonprotective. On the other hand, the fact that the
most resistant F1 mice show not the lowest,
but an intermediate, level of T cell proliferation (Fig. 2) precludes
ascribing a predominantly pathological, lung tissue-damaging role to T
cell proliferation itself.
The capacity of I/St lung T cells to more readily proliferate in
response to mycobacteria is in sharp contrast with their low
accumulation in the infected organ. It is noteworthy that a higher
proportion of I/St lung CD4+ cells express CD95
apoptotic receptor (Fig. 5), ligation of which results in
activation-induced cell death (45, 46), and are subjected
to apoptosis (Fig. 6). One can speculate that an early onset and a high
degree of T cell proliferation combined with their apoptotic
elimination, i.e., high T cell turnover in lungs, lead to an
unfavorable disease course. Since the expression of CD95L on lung T
cells was virtually lacking (data not shown), elimination of
Fas-positive T cells from the infected lung most likely involves non-T
cells.
As infection progressed, both CD4 and CD8 lung T cells acquired
activated
CD44+CD28+CD11-+
phenotype. This is in agreement with the observations of other authors.
Thus, Serbina and Flynn (40) reported the expression of a
high density CD44 molecule by >85% of both CD4+
and CD8+ lung T cells at 2 wk following TB onset.
Feng et al. (39) have shown that the CD62L endothelium
adhesion molecule is down-regulated, while the CD44 activation marker
and CD11-, CD49d integrins are up-regulated in the lung and lymph
node CD4+ and CD8+ cells
following aerosol M. tuberculosis challenge.
Earlier, Griffin and Orme (47) described the increase in
CD44 expression along with the gradual decrease in CD45RB expression on
CD4+ splenocytes of infected mice. The shift in
CD45RB expression following activation of
CD45RBhigh naive/resting T lymphocytes occurs
differently in CD4 and CD8 subsets. While this marker is uniformly
down-regulated in activated CD4+ cells (31, 47), several lines of evidence indicate that its expression is
retained in the majority of activated CD8+ cells
(32, 33, 39, 48). In our study by week 5 postinfection the
loss of CD45RB expression by CD4-positive cells, indicating their high
activation, was more pronounced in I/St mice. In contrast, among their
CD8-positive lung cells a significantly smaller population continued to
express the CD44+/CD45RB+
double-positive phenotype by this time point compared with A/Sn and
F1 mice (Fig. 4). Although it is presently
unclear what the functional difference is between
CD44+/CD45RB-/low and
CD44+/CD45RB+ cells in the
CD8+ subset, some recent findings in humans
indicate its possible prominence. Thus, it was reported that mature
CD8-positive CTLs, possessing perforin and producing IFN- and
TNF-, reside within the CD45RA+ population,
whereas Ag-specific CTL precursors, which need to be restimulated to
acquire cytotoxic function, belong to the
CD45RA- subpopulation (49, 50). In
our system it is possible that in I/St mice a stronger activation of
CD4+ (more
CD44+/CD45RBlow cells) is
accompanied by a weaker activation of effector
CD8+ (fewer
CD44+/CD45RB+ cells) lung T
lymphocytes.
Assessment of cytokine profiles has shown that lung cells from infected
A/Sn and F1 mice produced more type 1 cytokines.
In contrast, in I/St cells there was a moderate bias toward the type 2
profile: somewhat lower IFN- and TNF- and higher IL-10 and IL-5
production (Table V). An interesting feature of I/St lung cell response
was that a lower IFN- was evident as early as 2 wk following
challenge. The significance of the early IFN- production by CD4 T
cells for TB control was recently shown by Caruso et al.
(51). Thus, a bias toward a type 2 response in I/St lungs
at the initial stages of the disease might contribute to its more
severe course.
Among lung T cells, a high level of IL-10 synthesis was
characteristic for I/St mice. This is in agreement with our previous
finding that the majority of lung-derived T cell clones of I/St origin
produce IL-10, even if IFN- is simultaneously produced
(25). Interestingly, Gerosa et al. (52) have
found that the majority of T cell clones derived from bronchoalveolar
lavage of patients with active TB also produced both IFN- and IL-10.
Thus, in mice and humans the activity of IFN-/IL-10-producing
(Th0-like?) T cells in the lungs is prominent when the disease rapidly
progresses. In view of the hypothesis that simultaneous synthesis of
type 1 and type 2 cytokines during the TB course has a tissue-damaging
effect (17, 18), conjunct production of these two, usually
antagonist, cytokines in the lung may be considered an immunological
correlate of the unfavorable development of pulmonary TB.
IL-10 is known to impair protective immune response to several infections by down-regulating Th1 function (53, 54). On the other hand, it probably regulates the balance between protective and pathologic immune responses during intracellular parasitic infections (55) and autoimmune disorders (56, 57). For example, IL-10 gene targeting results in systemic overproduction of proinflammatory cytokines and the development of lethal pathology (55). The role of IL-10 in tuberculosis is not completely understood. North (58) reported that IL-10 knockout mice and wild-type control mice display similar levels of TB protection. In our system IL-10 was produced in higher amounts by lung and lymph node T cells of susceptible mice. Another type 2 cytokine, IL-5, was produced almost exclusively by lung cells of I/St susceptible mice. Analogous results were recently obtained in other experimental infection models. Following infection with Chlamydia, a significantly greater amount of IL-10 was found in the lungs of susceptible compared with resistant mice (59). Huffnagle et al. (60) have reported IL-5 expression in the lungs of susceptible, but not of resistant, mice during pulmonary Cryptococcus neoformans infection. Thus, overproduction of IL-5 and IL-10 in the lung seems to be associated with susceptibility to pulmonary infections, although causality remains obscure.
Lung T cells from all three mouse strains exhibited a similar capacity
to stimulate anti-mycobacterial activity of peritoneal syngenic
macrophages in vitro, and there was a strong correlation between NO
production and mycobacteria growth inhibition (Fig. 8). The lack of
interstrain differences raises the question of the reason for the
severity of M. tuberculosis-triggered disease in I/St mice,
given that their lung T cells effectively promote macrophage-mediated
inhibition of mycobacteria growth. At least two explanations are
possible: 1) anti-mycobacterial activity of lung macrophages
differs prominently from that of peritoneal ones and is selectively
affected in I/St mice; and 2) the severe TB course in I/St mice is due
to lung pathology rather than to impaired restriction of mycobacterial
growth. Experiments are in progress to distinguish (or to combine)
these two possibilities.
In conclusion, there is an agreement between the results on immune reactivity in the lungs of TB-susceptible and resistant mice presented here and our genetic mapping studies. A genome-wide scan in segregating backcross (I/StxA/Sn)F1xI/St TB-infected mice demonstrated that the severity of tuberculosis is inherited as the oligogeneic quantitative trait and is controlled by more than two nonlinked quantitative trait loci (23). Analogously, interstrain differences in the lung T cell response to mycobacteria are quantitative, and the expression of major integrative phenotypes (survival, pathology, cachexia, etc.) depends, presumably, upon particular combinations of minor shifts in immune reactivity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Irina V. Lyadova, Laboratory for Immunogenetics, Central Institute for Tuberculosis, Yauza Alley 2, Moscow 107564, Russia.
3 Abbreviations used in this paper: TB, tuberculosis; 7AAD, 7-amino-actinomycin D; BrdU, bromodeoxyuridine; RNI, reactive nitrogen intermediates; CD95L, CD95 ligand.
Received for publication December 20, 1999. Accepted for publication August 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in resistance to M. tuberculosis infection. J. Exp. Med. 178:2249. gene-disrupted mice. J. Exp. Med. 178:2243.-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941.-receptor deficiency in an infant with fatal bacille Calmette-Guérin infection. N. Engl. J. Med. 335:1956. is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
in T cell-mediated inflammation depends on the Th1/Th2 cytokine balance. Immunology 82:591.[Medline]
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-crystalline homolog by exposure to nitric oxide donors. Infect. Immun. 67:460. interferon-secreting CD8+, as well as CD4+, T cells in lungs following aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67:3242., yet succumb to tuberculosis. J. Immunol. 165:5407.
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,
I/St; 
, A/Sn; 
, F1.
, macrophages, H37Rv, and lung T cells.