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Immunology and Infection Program, Department of Pathology, McMaster University, Hamilton, Ontario, Canada;
Pasteur Mérieux Connaught, North York, Ontario, Canada; and
Hoffmann-La Roche, Inc., Nutley, NJ 07110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-
, but not the type 2 cytokines IL-4 and granulocyte macrophage
(GM)-CSF, markedly increased in the lung and peripheral blood of wt
mice postinfection, which resulted in the development of intense
granulomatous responses and the effective control of mycobacterial
infection in the lung. In contrast, IL-12-/- mice
demonstrated a lack of both types 1 and 2 cytokines in the lung and
blood and a severely impaired tissue immune-inflammatory response
lacking not only macrophages and neutrophils but CD4 and CD8 T cells
and NK cells in the lung throughout the entire course of study. Total
lung mononuclear cells isolated from these mice, in contrast to wt
mice, had an impaired recall immune response to Ag challenge in vitro.
These impaired responses resulted in an uncontrolled local growth and
systemic spread of bacilli. Our findings reveal that IL-12 plays an
irreplaceable role in the initiation of Th1 responses, and the loss of
its function cannot be compensated for by alternative mechanisms in the
lung. This cytokine, together with IFN- and TNF-, and
granulomatous inflammation are critically required for the effective
control of pulmonary mycobacterial infection. Our results also indicate
that the absence of type 1 cytokines does not necessarily favor a Th2
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-,
and IL-18 and the type 2 counterparts such as IL-4 (3, 4, 5, 6). Adequate
activation of tissue macrophages is thought to be the key to the
ultimate eradication of intracellular mycobacteria. IL-12 induces Th1
differentiation and IFN- release from Th1 and NK cells. IFN-
plays a role in anti-mycobacterial immune responses likely via
activating macrophages. TNF- is capable of many proinflammatory
activities including macrophage activation (7). Much of our
understanding of the role of cytokines in host responses against
mycobacterial infection has derived from studies using cells isolated
from humans or systemically infected animals (8, 9, 10, 11). Recently, studies
by using "gain or loss of function" approaches have started to help
us understand the role of cytokines in vivo. The role of IFN-,
TNF-, or IL-12 in anti-mycobacterium immune responses has been
suggested from studies using gene knock-out mice, Abs, or recombinant
cytokines in models of systemic infection with Mycobacterium
tuberculosis or Mycobacterium bovis bacille
Calmette-Guérin (BCG)3
(12, 13, 14, 15, 16, 17, 18). However, the relative role of these cytokines still remains
to be elucidated. Very recently, IL-12-/- mice were shown
to have delayed IFN-/TNF- mRNA expression in the liver in
response to systemic mycobacterial infection, but, notably, expression
of these type 1 cytokines rebounded to levels similar to those in wt
mice at later stage of infection. Augmented IL-18 expression was held
as an alternative mechanism compensating for the loss of IL-12 function
in this model of systemic infection (19). Little is known to date about
the profiles of both types 1 and 2 cytokines at the protein level and
their relative contribution to regulating tissue immune-inflammatory
responses in the lung during pulmonary mycobacterial infection. The natural route of infection by many strains of mycobacteria is the respiratory tract, and the nature of local immune responses in the lung determines the outcome of disease, including the extent of systemic dissemination (1). Importantly, recent evidence has suggested that the nature of such responses in the lung may be different from that observed at other tissue sites. The same strain of mycobacteria was found to be much more virulent when inoculated via the airway than via the systemic route (20), and when infected via the airway, the mouse strain previously shown to be resistant to systemic infection proves susceptible (21). These findings suggest a necessity to understand immune-inflammatory responses in the lung during pulmonary mycobacterial infection.
The objective of our study was 1) to investigate the profiles of both
types 1 and 2 cytokine proteins in the lung and peripheral blood, and
tissue immune-inflammatory responses in immune-competent mice after
airway inoculation with live M. bovis BCG, and 2) to
determine the role of IL-12 in the induction of type 1 cytokines and
protective tissue immune responses during pulmonary mycobacterial
infection in IL-12-deficient mice. We have demonstrated that IL-12,
IFN-, and TNF-, and tissue granulomatous responses are required
for the effective control of pulmonary mycobacterial infection, and
IL-12 plays an irreplaceable role in the initiation of Th1
immune-inflammatory responses in the lung. We have also found that the
lack of type 1 cytokines does not result in Th2 responses in the lung
during mycobacterial infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both male and female mice at the age of 10 to 14 wk were used. The generation of IL-12p40-/- mice (C57BL/6 background) has previously been described, and these mice demonstrate a normal development of immune systems (22). These mice were bred in our central animal facility. C57BL/6 mice (Harlan, Indianapolis, IN) were used as wt controls for IL-12 p40-/- mice. All mice were housed in autoclaved cages with autoclaved bedding, food, water, and microfilter lids, in a pathogen-free Level B facility. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of McMaster University.
Preparation of M. bovis BCG
M. bovis BCG (Connaught Laboratories, North York, Ontario, Canada) was grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook OADC enrichment (Life Technologies, Gaithersburg, MD), 0.002% glycerol, and 0.05% Tween 80. Lyophilized BCG was reconstituted in saline + 0.05% Tween 80 and used to inoculate a small (20-ml) starter culture. The culture was incubated at 37°C for 7 days with gentle aeration. The starter culture was subinoculated with 0.1 ml of fresh broth. After 4 days incubation, the culture was harvested by centrifugation, and the cell pellet was resuspended in 7H9 broth to an absorbance of 2.8 at 600 nm. This cell suspension was aliquoted and stored at -70°C until needed. After thawing, viable cell counts were determined by plating serial dilutions of the suspension on Middlebrook 7H11 agar plates (Life Technologies) and incubating at 37°C (23).
Establishment of pulmonary mycobacterial infection
An attenuated live strain of Mycobacterium tuberculosis bovis (BCG) was used to establish a model of pulmonary mycobacterial infection. The use of such mycobacteria requires only the Level II biohazard working conditions, which, in contrast to biohazard facilities for mycobacteria of higher virulence, allow detailed dissection of cellular and cytokine responses in noninactivated body fluids and freshly isolated tissue cells. Before infection, BCG stock solution was diluted in PBS, and the preparation was sonicated to ensure proper dispersion of mycobacteria. Mice were infected by intratracheal (i.t.) instillation of live BCG at a dose of 106 CFU in a total volume of 40 µl per mouse. We have found that a dose of 5 x 104 CFU elicits qualitatively similar differences in cellular and cytokine responses between C57BL/6 and IL-12p40-/- mice as compared with the 106 CFU dose, but the peak of such responses is delayed (data not shown). Intratracheal instillation was conducted following a previously described procedure with minor modifications (24). Briefly, anesthetized mice were maintained under anaesthesia using a nose cone device containing isofluorane, and an incision through the skin of the neck was made. Muscles and connective tissues overlying the trachea were gently separated to reveal the trachea. The trachea was elevated slightly using a 23-gauge needle to support it from the underside, while a 23-gauge needle attached to a 1-ml syringe was used to deliver the BCG. Following administration, the skin layers from the incision were stapled together using a MikRon 9 mm Autoclip Applier (Becton Dickinson, Sparks, MD).
Groups of four to five mice per time point, per mouse strain, were set up for each experiment. At days 7, 14, 27, 43, 57, and 71 postinfection, mice were anesthetized, bled retroorbitally for serum preparation, and then exsanguinated by bleeding the abdominal vessels. Lungs were removed and subjected to BAL, followed by perfusion with 10% formalin. Spleens were also removed, and half of each spleen was fixed in 10% formalin, with the other half subjected to the colony enumeration assay procedure. Fixed spleens and lungs were further processed for histologic analysis. With the dose of BCG we used in our studies, there was no death of IL-12-/- mice observed up to 71 days post-i.t. infection.
BAL and cytologic analysis
After retroorbital bleeding, anesthetized mice were bled via the abdominal vessels to exsanguinate, followed by removal of the lungs, with the heart and a portion of the trachea intact. To collect BAL fluid, a polyethylene tube (Becton Dickinson, Sparks, MD) was used to cannulate the trachea. Lungs were lavaged twice with PBS (0.25 and 0.20 ml), and approximately 0.4 ml of BAL fluid was consistently recovered (25). All BAL samples were kept on ice until processing. BAL samples were spun at 4000 rpm for 1 min at 4°C, and supernatants were removed and stored at -20°C for cytokine analysis. Cell pellets were resuspended in 300 to 500 µl of PBS, and total cells were determined on a hematocytometer. Cytospins were made in a cytospin machine (Shandon, Pittsburgh, PA), and stained using Diff-Quick stain (Baxter, McGraw Park, IL) for differential cell counting. Routinely, 300 to 500 cells per cytospin were differentiated in a random fashion.
Processing and histologic assessment of lung and spleen tissues
Lungs and spleens were fixed in 10% formalin as described above. Both left and right lungs were sectioned from top to bottom, resulting in four to five cross-sectional pieces of tissue from each side. Tissues were then embedded in paraffin, cut into 4- to 5-µm sections and stained with hematoxylin and eosin (H&E). Tissue sections were also subjected to Ziehl-Neelson staining, a specific stain for mycobacteria.
Measurement of cytokines in BAL and sera
Cytokines were measured in BAL and sera by specific ELISA. All
ELISA kits were purchased from either R&D Systems (Minneapolis, MN;
IFN-, TNF-, IL-4, GM-CSF) or Biosource (Montréal,
Québec, Canada; IL-12). The sensitivity of detection for all of
these ELISA kits was 
5 pg/ml.
Mycobacterial colony enumeration
At days 27, 43, and 71 postinfection, lungs were removed and aseptically placed in 4.5 ml of PBS/0.05% Tween 80 on ice. Spleens were snap-frozen in liquid nitrogen and stored at -70°C for later use in the colony enumeration assay. Lungs were cut into small pieces (2–3 mm) under sterile conditions. Lung pieces or spleens (each in 4.5 ml PBS/0.05% Tween 80 buffer) were homogenized with a tissue homogenizer. Homogenates were allowed to settle on ice for 30 min, and 200 µl of properly diluted homogenates was plated onto each plate of Middlebrook 7H10 agar containing OADC enrichment (Difco) (23). Plates were incubated inside semisealed plastic bags at 37°C. Colonies were counted using a dissecting microscope, at day 14 for spleens and day 11 for lungs.
Lung tissue mononuclear cell isolation
At days 14 and 27 postinfection, two C57BL/6 and two IL-12 p40-/- mice were anesthetized, bled via the abdominal vessels, and then lungs were removed with the heart and a portion of the trachea intact. Pulmonary vasculature was perfused with 5 ml of warm (37°C) calcium and magnesium-free 1x HBSS containing 5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin via the right ventricle of the heart. After removing connective tissues, lungs were cut into small pieces (2–3 mm) in 2 to 3 ml of RPMI 1640 culture media. Lung pieces were collected and incubated with agitation in a solution containing 150 U/ml Type III collagenase (Life Technologies) at 37°C for 1 h. Lung pieces were then mashed through sterile metal screens in culture media, and the resultant cell suspension was filtered through two layers of nylon membrane (55 µm) and centrifuged at 1000 rpm for 8 min at 4°C. Pellets were resuspended and combined to make a total volume of 15 ml, which was layered onto a double Percoll (Pharmacia Biotech, Baie D’Urfé, Québec, Canada) gradient consisting of a bottom layer of 60% Percoll and an upper layer of 30% Percoll. After centrifugation at 2000 rpm for 25 min at room temperature, the interface containing mononuclear cells between the 30% and 60% Percoll gradients was collected. These cells were then washed three times with cold PBS by centrifugation at 1400 rpm for 8 min at 4°C, and cell pellets were resuspended in RPMI 1640 culture media. Cells from individual mice of the same strain were pooled. Total cell counts and viability were determined. The percentage of mononuclear cells in the final cell suspension was 80%.
Flow cytometric analysis
Total lung tissue-derived mononuclear cells obtained from mice at day 27 postinfection were subjected to FACS analysis. Mononuclear cells from two infected C57BL/6 and three IL-12 p40-/- mice, and three PBS-treated control C57BL/6 mice, were pooled and immunostained following a basic procedure previously described by us (24). Briefly, staining was conducted with a two color combination of anti-CD3 and anti-pan-NK surface marker (DX-5, rat IgM, PharMingen, Mississauga, Ontario, Canada) and a three color combination of anti-CD3, anti-CD4, and anti-CD8. For two color staining, cells were stained first with biotinylated DX-5 and FITC-conjugated anti-CD3 (purified from American Type Culture Collection (ATCC, Manassas, Va) No. CRL-1975, hamster IgG) followed by incubation with streptavidin-PerCP (Becton Dickinson, Mountain View, CA). For three-color staining, the biotinylated anti-CD3 (hamster IgG, clone 145–2C11, PharMingen), phycoerythrin-conjugated anti-CD4 (rat IgG2a, clone H129.19, Life Technologies), and fluorescein-conjugated anti-CD8 (rat IgG2a, clone 53–6.7, PharMingen) were used. All samples were analyzed on a FACScan analyzer (Becton Dickinson). CD4, CD8, and NK cell phenotypes were analyzed by gating in the lymphocyte region, and results were analyzed using Cell-Quest for acquisition of data and PC-Lysis software for final analysis (Becton Dickinson).
In vitro Ag stimulation assay
Total lung tissue mononuclear cells isolated from mice at days 14 and 27 post-airway infection were subjected to an Ag stimulation assay for cytokine production. Briefly, lung cells were resuspended in RPMI 1640 and added to 96-well plates at a concentration of 2 x 105 cells/well. Cells were cultured without any stimulation, or with 10 µg/ml of PPD (BCG-derived purified protein derivative; Connaught Laboratories), or with 10 ng/ml PMA (Sigma, St. Louis, MO) for 72 h at 37°C. Supernatants were collected and stored at -20°C until cytokine assay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To quantitatively evaluate the cellular responses to mycobacterial
infection in the lung, we examined the cellular profiles in BAL fluids
recovered from the lungs of both C57BL/6 and IL-12
p40-/- mice at days 7, 14, 27, 43, 57, and 71 after
i.t. inoculation of live M. bovis BCG. The total
cell counts increased by day 7, reached a maximum by day 27, and
decreased thereafter in the lung of C57BL/6 mice. The number of
macrophages increased by day 7 and peaked by day 27, and significant
lymphocytic and neutrophilic responses were noticed by day 14, became
maximal by day 27, and decreased thereafter in the lung of these mice
(Fig. 1
, A, B, and
C). In contrast, cellular responses in the lung of
IL-12 p40-/- mice were severely impaired. Both total and
differential cell numbers were much smaller in the lung of
IL-12p40-/- mice at various time points. The most
striking difference was exhibited at day 27, with a total cell count
being 96.87 x 104 vs 32.68 x 104,
and differential counts of macrophages being 60.59 x
104 vs 23.12 x 104, lymphocytes
170.43 x 103 vs 29.9 x 103, and
neutrophils 428.70 x 103 vs 49.4 x
103 in C57BL/6 and IL-12 p40-/- mice,
respectively (Fig. 1, A, B, and
C).
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To examine tissue responses, histologic assessment of lung tissues
from mice sacrificed at days 7, 14, 27, 43, 57, and 71
post-intrapulmonary mycobacterial infection was conducted. At day 7
postinfection, few or no signs of inflammation were evident in the lung
tissue of either C57BL/6 or IL-12 p40-/- mice. By
day 14, the lungs of C57BL/6 mice demonstrated well-formed, distinct
granulomas comprising macrophages, epithelioid cells, and a small
number of lymphocytes (Fig. 2a). Perivascular and
peribronchial lymphocytic accumulation was evident. At days 27 and 43,
the immune-inflammatory tissue responses in C57BL/6 mouse lungs became
much more intense and diffuse with many granulomas densely packed with
epithelioid cells, lymphocytes, and neutrophils (Fig. 2c). From day 57 onward, inflammation started to
resolve (Fig. 2e), and, by day 71, only loose
inflammatory accumulation in the peribronchial and perivascular areas
with foamy macrophages in alveolar spaces was seen (Fig. 2g). There were no overt signs of tissue fibrotic
responses.
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, b, d, f, and
h). Few foamy macrophages were seen in alveolar
spaces in contrast to those seen in the lung of C57BL/6 mice (Fig. 2, e and f). Mycobacterial burden in tissue assessed by specific microbiologic staining
Having examined cellular and tissue responses to intrapulmonary
mycobacterial infection, we sought to determine the mycobacterial
burden in the lung and whether a weakened immune-inflammatory response
in the lung of IL-12 p40-/- mice led to an increased
mycobacterial burden. Since the immune-inflammatory response locally in
the lung is believed to control the extent of mycobacterial
dissemination from the lung to other tissue sites and subsequent
propagation, the mycobacterial burden in the spleen was also
determined. To this end, we first analyzed lung and spleen tissues
stained by Ziehl-Neelson staining specific for mycobacterial bacilli.
Bacilli were not evident in either C57BL/6 or IL-12
p40-/- mouse lung tissues until day 14 postinfection. At
this time point, bacilli were localized in macrophages, and no
significant difference was observed in both mouse strains. By days 27
and 43, however, macrophages in IL-12 p40-/- mouse lungs
contained much more bacilli than in the lungs of C57BL/6 mice. By days
57 and 71, while only a very small number of intact or fragmented
bacilli could be detected in the lung of C57BL/6 mice, abundant intact
bacilli were noticed in macrophages in IL-12 p40-/- mouse
lungs (Fig. 3, a and
b). In the spleen, few bacilli were detected by days
7 and 14 post-intrapulmonary infection in both mouse strains. From day
27, particularly at day 57 or 71, whereas no bacilli, or very few
fragmented bacilli, were detected in the spleens of C57BL/6 mice, the
spleens from IL-12 p40-/- mice contained abundant bacilli
within areas of macrophage accumulation (Fig. 3, c and
d).
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For quantitative measurement, we examined mycobacterial burden in
the lung and spleen post-intrapulmonary infection by a colony
enumeration assay. We chose to focus on days 27, 43, and 71. In
agreement with the histologic assessment, the number of bacilli was
about 13 and 95 times higher in the lungs of IL-12
p40-/- mice than in C57BL/6 mice at days 43 and 71,
respectively (Fig. 4A).
In fact, by day 71, the lungs of most C57BL/6 mice contained either
undetectable or very few bacilli. Similarly, the number of bacilli was
also much higher in the spleen of IL-12 p40-/- mice than
in C57BL/6, being approximately 35, 100, and 1135 times higher at days
27, 43, and 71, respectively (Fig. 4B). Of note,
almost no bacilli were recovered in the spleen of C57BL/6 mice by days
43 and 71. The overall lower enumeration of mycobacteria in the spleen
than in the lung suggests that the appearance of bacilli in the spleen
resulted from lung dissemination.
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We next characterized the in vivo cytokine responses both locally
in the lung and systemically in the peripheral blood during pulmonary
mycobacterial infection in C57BL/6 and IL-12 p40-/-
mice to investigate the potential molecular mechanisms involved in
tissue immune-inflammatory responses. Cytokine contents in the BAL and
sera collected at various times were assessed by ELISA. A panel of
cytokines was examined, including the type 1 cytokines IL-12, IFN-,
and TNF-, and the type 2 cytokines IL-4 and GM-CSF. IL-4 is known to
drive Th2 immune-inflammatory responses whereas GM-CSF is often
coinduced with IL-4 during Th2 allergic responses (26).
IL-12 levels were low in the lung at day 7 in C57BL/6 mice, markedly
increased at day 14, peaked (888 pg/ml) at day 27, and decreased
thereafter (Fig. 5A).
The kinetics of IFN- responses closely paralleled those of IL-12 in
the lung of C57BL/6 mice at various time points with a peak release
(1490 pg/ml) at day 27 (Fig. 5C). Similarly, the
level of TNF- was not measurable at day 7 but became detectable at
day 14, then rose to a peak (109.67 pg/ml) at day 27, and declined
thereafter (Fig. 5E). In contrast, both IL -12 and
IFN- were not detected in the lung of IL-12 p40-/-
mice (Fig. 5, A and C), and the amounts of
TNF- detected were also minimal in these mice (Fig. 5E). The level of IFN- or TNF- never increased
in the lung throughout the entire course of observation (up to 71 days)
despite an ever-increasing number of mycobacterial bacilli in the lung.
Interestingly, while the level of IL-4 in BAL from C57BL/6 mice was
very low throughout the entire course of study, it was not increased in
the lung of IL-12 p40-/- mice despite the absence of type
1 cytokines (Table I). GM-CSF was present
in minimal amounts in BAL, and no difference was found in both C57BL/6
and IL-12 p40-/- mice (Table I).
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, B,
D, and F, while overall cytokine concentrations
in sera were much lower than those measured in the lung in C57BL/6
mice, the kinetics in these two compartments were similar. At the peak
time of day 27, a concentration of 235 pg/ml of IL-12 or 86 pg/ml of
IFN- was measured in C57BL/6 mice whereas there was no IL-12 or
IFN- detected at any time in IL-12 p40-/- mice. The
amounts of TNF-, GM-CSF, and IL-4 were very small in sera from both
C57BL/6 and IL-12 p40-/- mice (Table I). Phenotypes of immune cells in lung tissue by FACS
To investigate the nature of cell types, in addition to
macrophages and neutrophils, involved in controlling pulmonary
mycobacterial infection, total lung tissue-derived mononuclear cells
isolated from lungs of C57BL/6 and IL-12 p40-/- mice
at day 27 post-airway infection were phenotyped by FACS using specific
mAbs for CD3, CD4, and CD8 T cells and NK cells. First, a marked
increase in the number of mononuclear cells from lung tissue of
infected-C57BL/6 mice was noticed, in sharp contrast to that seen with
infected IL-12 p40-/- or control C57BL/6 mice (Fig. 6A). This observation,
taking into account cell populations within both alveolar and
interstitial spaces, consolidated the differences in cellular profiles
seen in BAL. Upon FACS with these cell preparations, marked increases
in the number of CD4, CD8, and NK cells in the lung of C57BL/6 mice
were observed (Fig. 6, B and C). The
increase of CD4 T cell population was most prominent, being at least 10
times higher than the control levels. In contrast, there were markedly
impaired responses to mycobacterial infection of CD4, CD8, or NK cells
in the lung of IL-12 p40-/- mice.
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To investigate the role of lung mononuclear cells in cytokine
responses and the memory immune responses against pulmonary
mycobacterial infection, mononuclear cells isolated by a tissue
dispersion protocol from lungs of C57BL/6 and IL-12
p40-/- mice at days 14 and 27 post-i.t. inoculation
of mycobacteria were cultured with or without a BCG-derived antigen PPD
(purified protein derivative), and the resultant culture supernatants
were examined for IFN- and IL-4 release by ELISA. At day 14
postinfection, lung mononuclear cells from PBS-treated control C57BL/6
or infected-IL-12 p40-/- mice released little IFN-
either spontaneously or upon PPD stimulation (Fig. 7A), but these cells
responded well to the stimulation of a nonspecific mitogen PMA (not
shown). In contrast, lung mononuclear cells from BCG-infected C57BL/6
mice spontaneously released significant amounts of IFN- (247 pg/ml),
and PPD stimulation resulted in a further increase. At day 27, while
the lung mononuclear cells from PBS control or infected IL-12
p40-/- mice released little IFN- without stimulation
or only small amounts of IFN- upon PPD stimulation, cells from
C57BL/6 mice released 598 pg/ml of IFN- spontaneously, which
represented a further increase over the amount released by the cells
purified at day 14 (Fig. 7A), and 4233 pg/ml of
IFN- upon PPD stimulation, which represented a much more pronounced
recall response to Ag challenge than at day 14. In contrast to IFN-,
little type 2 cytokine IL-4 was released by cells either from
IL-12-/- or C57BL/6 mouse lungs (all below 6 pg/ml; Fig. 7B), in keeping with findings from BAL and serum
(Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF- but not type 2 cytokines IL-4 and GM-CSF,
with enhanced circulating levels of type 1 cytokines. This cytokine
response was closely associated with the evolution of a pulmonary
granulomatous response. The mononuclear cell population isolated from
lung tissues contained markedly increased CD4, CD8, and NK cells and
released IFN- upon recall Ag stimulation in vitro. These responses
effectively controlled the local growth and systemic spread of bacilli.
In contrast, IL-12-/- mice demonstrated a complete lack
of IFN- and a severely impaired TNF- response in the lung and
peripheral blood, which, nevertheless, did not lead to an enhanced Th2
response. These mice failed to develop a protective granulomatous
response with an apparent defect in eliciting T cell, particularly CD4
T cell responses in the lung. The mononuclear cells from the lung of
these mice showed little IFN- recall response to Ag restimulation in
vitro. As a result, mycobacteria underwent uncontrolled local
replication and systemic dissemination to extrapulmonary sites.
Our data provide the first evidence that the type 1 cytokines and
tissue granuloma formation responses represent two critical components
in the protective immune-inflammatory response against pulmonary
mycobacterial infection and that IL-12 plays an irreplaceable role in
the initiation of such Th1 responses in the lung. IL-12 has recently
received increasing attention regarding its role in Th1
differentiation, IFN- release, and T cell/NK cell activation (27).
However, recent identification of IFN--inducing factor (IL-18), a
cytokine found to have many functional commonalities and synergistic
activities with IL-12, suggests that IL-12 may not be the only driver
for Th1 responses (5). Indeed, IFN- production was found to be only
partially reduced in IL-12-/- mice during endotoxemia
(22). And Cooper et al. have very recently reported that after i.v.
infection with M. tuberculosis, mRNA expression of both
IFN- and TNF- in the liver of IL-12 -/- mice was
markedly delayed in earlier periods of time but increased to similar
levels seen in wt C57BL/6 mice by day 35, which was associated with a
full recovery of macrophage activation. This alternative mechanism for
the loss of IL-12 function was attributed very likely to IL-18, which
was markedly induced in the liver of both wt and IL-12
-/- mice post-systemic mycobacterial infection (19).
Contrasting with these important findings, we found that the generation
of type 1 cytokines, particularly IFN-, and Th1 tissue responses was
fully dependent upon the presence of IL-12 in the lung during the
entire course of pulmonary mycobacterial infection (up to 71 days),
thus suggesting a lack of alternative mechanisms in the lung, unlike at
other tissue sites and establishing an unique role for IL-12 in the
immune response against pulmonary mycobacterial infections. In further
support of this notion, we have recently found that, while lung
mononuclear cells from airway-infected IL-12-/- mice fail
to release IFN- in response to PPD recall stimulation in vitro, such
response is not impaired in spleen-derived mononuclear cells from the
same mice. It should be pointed out, however, that, although
immunogenetically and structurally very similar to M.
tuberculosis, the virulence of M. bovis organisms used
in our study has been attenuated. This fact may explain why the
immune-competent host (C57BL/6 mice) could more readily control lung
infection caused by such mycobacteria as compared with infection by
virulent mycobacteria (21, 23) and why it did not lead to mortality in
IL-12-/- mice during the course of our study (71 days).
Nevertheless, our findings suggest a potential molecular basis to
explain why certain strains of mycobacteria are more virulent in the
lung than in other organs (20), why the previously deemed
bcg-resistant strains of mice based upon observations in the
spleen or liver after systemic infection become susceptible during
local pulmonary infection (21), and why tissue granulomas elicited via
i.v. delivery of BCG resolve much earlier (28) than granulomas elicited
via the airway, as shown in our study. In our current model, the
effective control, although not a complete clearance in the lung by day
71, of mycobacterial infection in C57BL/6 mice was likely through
inhibition of intracellular mycobacterial replication and enhanced
intracellular mycobactericidal activities as a result of macrophage
activation by IFN- and TNF-. Indeed, we observed the
fragmentation of bacilli in lung or spleen macrophages of C57BL/6 mice
but not of IL-12-/- mice.
In our current study, we have also demonstrated that, despite the
normal development and dissemination to peripheral tissues of immune
cells in IL-12-/- mice (22), these mice had an impaired
capability to expand and accumulate CD4, CD8, and NK cells in the lung
during pulmonary mycobacterial infection. This finding, together with
the fact that one of the most prominently increased immune cell types
is CD4 T cells in the lung of immune-competent C57BL/6 mice, highlights
the importance of CD4 T cells in the initiation of immune cascade of
events in the lung. The differentiation of these CD4 T cells, likely
Th1 cells, is known to be critically dependent upon IL-12 (27). Hence,
the lack of IL-12, and the subsequent lack of CD4 Th1 as well as NK
cells, most likely accounted for a complete lack of IFN- release
from these cells in the lung of IL-12-/- mice, whereas a
severely impaired TNF- response was likely a result of the lack of
CD4 cells and macrophage activation mediated by IFN-. However, at
this point, we cannot rule out that IL-12 may also directly activate
lung macrophages to release TNF-. In this regard, Taylor et al. have
recently demonstrated an IL-12-induced, IFN--independent TNF-
expression in a model of visceral leishmaniasis (29), and IL-12 has
been shown to directly activate murine macrophages in vitro (30).
Currently, we are investigating whether activated lung macrophages in
our model can release Th1-type cytokines and whether IL-12 itself has a
direct activating effect on these cells. CD4 cells have been shown to
be required for protection against systemic infection by M.
bovis BCG in CD4-/- mice (31). Our findings also
suggest the involvement of both CD8 and NK cells in immune protection
during pulmonary mycobacterial infection, supporting the previous
findings in models of M. tuberculosis or Mycobacterium
avium infection using mice deficient in CD8 or NK cells (32, 33).
Flynn et al. have reported that the lack of IFN- led to the
formation of a defective necrotic type of granuloma following i.v.
infection with M. tuberculosis in IFN--/-
mice (13). In this regard, recent human studies have linked the IFN-
receptor deficiency to disseminated tuberculous infection in children
immunized with M. bovis BCG (34, 35). Abrogation of TNF-
with a mAb also led to the decreased number and size of granulomas and
increased number of bacilli in the liver after i.v. infection with live
M. bovis BCG (16). Our observations point to the importance
of all of these type 1 cytokines, with IL-12 being a required initiator
in the successful development of protective types of pulmonary
granulomatous inflammation. Indeed, we observed closely correlated
kinetics of augmented IL-12 protein levels, together with IFN- and
TNF- in the lung of immune-competent mice with the development of an
intense granulomatous response and the subsequent inhibition of local
growth and systemic spread of bacilli. The fact that there was
relatively little cytokine or tissue response in the initial 2 wks
post-airways infection indicates the nature of a delayed type
hypersensitivity and the dependence of such tissue responses on the
evolution of specific cellular immune responses (36). Moreover, the
lack of recall immune responses in these mice as shown by Ag
stimulation assay suggests that, in addition to severely impaired
primary immune responses in the lung, the secondary memory immune
responses were also impaired. Thus, the type 1 cytokines hold the key
to the initiation of tissue inflammatory response whereas the tissue
inflammatory response serves as an end check point determining the fate
of infection. It would be of interest to carry out similar studies in
the future using IFN--/- mice to compare the relative
role of IL-12 and IFN- in host responses against pulmonary
mycobacterial infection.
We have also revealed the cytokine profile in the peripheral blood in the course of pulmonary mycobacterial infection. While the role of relatively low but significant circulating levels of type 1 cytokines in immune-competent mice remains speculative at this point, it is likely that these cytokines operate as an integral part of host immune-inflammatory responses to local lung mycobacterial infection. These circulating cytokines may activate the peripheral blood monocytes before their influx into tissue and, more importantly, restrict the growth of mycobacteria by stimulating immune responses at extrapulmonary tissue sites. Our data, that a small number of bacilli detected in the spleen at earlier times was eradicated at later times in immune-competent mice, support this notion. Since the levels of these circulating cytokines were much lower than those in the lung but with similar kinetics, they likely originated primarily from the lung.
Ag-stimulated immune cells from IL-12-/- mice were shown
to release more IL-4 in vitro (22), and a recent study by Mattner et
al. demonstrated a polarized type 2 cytokine response in
IL-12-/- mice postinfection with Leishmaniamajor (37). In contrast, we observed a lack of type 2 cytokine
responses in the lung and peripheral blood throughout the entire course
of pulmonary mycobacterial infection in IL-12-/- mice.
This was further confirmed by the lack of IL-4 release, as opposed to
IFN-, by Ag-stimulated lung mononuclear cells in vitro. Our findings
support those by Cooper et al. that IL-4 mRNA was not increased in the
liver of IL-12-/- mice post-systemic infection (19). The
lack of GM-CSF in C57BL/6 that we observed suggests that this cytokine
is not significantly involved in antimycobacterial activities in the
lung despite its well known effects on macrophage activation (38, 39).
Together, these suggest that the Th2 phenotype is not always controlled
by a balance with the Th1 phenotype, at least in the case of
mycobacterial infection. The lack of type 2 cytokine responses in both
immune-competent and IL-12-/- mice that we have observed
cannot be attributed merely to the C57BL/6 genetic background of these
mice since we have recently found that C57BL/6 mice, like BALB/c mice,
are fully capable of Th2 allergic airways responses, including IL-4
release and airways eosinophilia.
Taken together, our study has demonstrated that type 1 cytokines IL-12,
IFN-, and TNF- and tissue granulomatous inflammatory responses
are required for controlling pulmonary mycobacterial infection by
M. bovis BCG, and that IL-12 represents an imperative
element upstream of this cacade of events. Our findings provide the
rationale for IL-12 and other type 1 cytokine-based therapy,
complementary to chemotherapy, for immune-compromised patients with
pulmonary mycobacterial infections (38).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zhou Xing, Rm. 4H17, Health Sciences Center, 1200 Main Street West, Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5. E-mail address:
3 Abbreviations used in this paper: BCG: bacille Calmette-Guérin; GM-CSF: granulocyte-macrophage CSF; BAL, bronchoalveolar lavage; wt, wild type; PPD, purified protein derivative; i.t., intratracheal.
Received for publication October 20, 1997. Accepted for publication February 19, 1998.
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