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The Journal of Immunology, 1998, 160: 6101-6111.
Copyright © 1998 by The American Association of Immunologists

Lack of Both Types 1 and 2 Cytokines, Tissue Inflammatory Responses, and Immune Protection During Pulmonary Infection by Mycobacterium bovis Bacille Calmette-Guérin in IL-12-Deficient Mice1

Julia Wakeham*, Jun Wang*, Jeanne Magram{ddagger}, Kenneth Croitoru*, Robin Harkness{dagger}, Pamela Dunn{dagger}, Anna Zganiacz* and Zhou Xing2,*

* Immunology and Infection Program, Department of Pathology, McMaster University, Hamilton, Ontario, Canada; {dagger} Pasteur Mérieux Connaught, North York, Ontario, Canada; and {ddagger} Hoffmann-La Roche, Inc., Nutley, NJ 07110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12-/-) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-{gamma}, and TNF-{alpha}, 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-{gamma} and TNF-{alpha}, 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary mycobacterial infections, particularly tuberculosis, have become a leading global health threat (1, 2). Incomplete understanding of the nature of protective immune-inflammatory responses and contributing soluble molecules has hampered the development of effective vaccines and therapies. The cell-mediated immune response is an important aspect of host resistance to mycobacterial infection and is believed to be tightly regulated by a balance between the type 1 cytokines including IL-12, IFN-{gamma}, TNF-{alpha}, 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-{gamma} release from Th1 and NK cells. IFN-{gamma} plays a role in anti-mycobacterial immune responses likely via activating macrophages. TNF-{alpha} 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-{gamma}, TNF-{alpha}, 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-{gamma}/TNF-{alpha} 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-{gamma}, and TNF-{alpha}, 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

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-{gamma}, TNF-{alpha}, 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cellular profiles in the BAL

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. 1Go, 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. 1Go, A, B, and C).



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  		FIGURE 1. Cellular profiles in BAL fluids during pulmonary mycobacterial infection. BAL fluids were obtained from C57BL/6 or IL-12-/- mouse lungs at various times post-airway infection and the number of alveolar macrophages/monocytes (AM) (A), lymphocytes (LC) (B), or neutrophils (PMN) (C) was determined on cytospins. Results are expressed as means ± SEM from four mice per time point.

 
Histopathology of lung tissues

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. 2Goa). 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. 2Goc). From day 57 onward, inflammation started to resolve (Fig. 2Goe), and, by day 71, only loose inflammatory accumulation in the peribronchial and perivascular areas with foamy macrophages in alveolar spaces was seen (Fig. 2Gog). There were no overt signs of tissue fibrotic responses.



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  		FIGURE 2. Lung tissue immune-inflammatory responses during pulmonary mycobacterial infection. Morphology was assessed on multiple sections from lung lobes obtained from mice at various times post-airway infection. Day 14: granuloma formation in C57BL/6 lung (a) and little granuloma formation in IL-12-/- lung (b); day 27: intense granulomatous responses in peribronchial and perivascular areas with abundant epithelioid cells (E) in C57BL/6 lung (c) and limited atypical lymphocytic (L) granuloma formation in some perivascular areas with little inflammatory responses in most areas in IL-12-/- lung (d); day 57: resolving granulomatous responses with abundant foamy macrophages (arrows) in C57BL/6 lung (e) and minimal atypical lymphocytic granuloma formation with little inflammatory responses in the majority of lung areas in IL-12-/- lung (f); day 71: further resolving inflammatory responses with a small number of foamy macrophages (arrow) in C57BL/6 lung (g) and limited inflammatory responses with the majority of lung areas free of inflammation (h). a and b, x220; c–h, x440.

 
In contrast, the lungs of IL-12 p40-/- mice from the same time points exhibited minimum granulomatous responses. Some small-sized atypical granulomas were primarily seen in the perivascular areas, which contained lymphocytes and small numbers of macrophages and neutrophils, and noticeably lacked macrophage-derived epithelioid cells (Fig. 2Go, 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. 2Go, 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. 3Go, 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. 3Go, c and d).



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  		FIGURE 3. Mycobacterial burden in lung and spleen tissue during pulmonary mycobacterial infection. Tissue sections (day 57) were stained with Ziehl-Neelson stain and counterstained with hematoxylin. Mycobacterial bacilli were stained red/pink. Few bacilli in the lung or spleen of C57BL/6 mice (a, c) but many seen in macrophages in the lung or spleen of IL-12-/- mice (b, d; arrows). a, c, d, x885; b, x1800.

 
Mycobacterial burden in tissue assessed by colony enumeration assay

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. 4GoA). 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. 4GoB). 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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  		FIGURE 4. Mycobacterial colony enumeration in lung (A) and spleen (B). Lungs and spleens were taken from C57BL/6 or IL-12-/- mice at days 27, 43, and 71 post-airway infection and homogenized. Tissue homogenates were plated onto Middlebrook agar and colonies counted at days 11 and 14. Results are expressed as means ± SEM from five mice per time per mouse strain.

 
Type 1 and type 2 cytokine profiles in the lung and peripheral blood

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-{gamma}, and TNF-{alpha}, 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. 5GoA). The kinetics of IFN-{gamma} 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. 5GoC). Similarly, the level of TNF-{alpha} 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. 5GoE). In contrast, both IL -12 and IFN-{gamma} were not detected in the lung of IL-12 p40-/- mice (Fig. 5Go, A and C), and the amounts of TNF-{alpha} detected were also minimal in these mice (Fig. 5GoE). The level of IFN-{gamma} or TNF-{alpha} 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 IGo). GM-CSF was present in minimal amounts in BAL, and no difference was found in both C57BL/6 and IL-12 p40-/- mice (Table IGo).



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  		FIGURE 5. Type 1 cytokine profiles in BAL and peripheral blood during pulmonary mycobacterial infection. BAL and serum samples were obtained from C57BL/6 or IL-12-/- mice at various times post-airway infection and measured for IL-12 (A, B), IFN-{gamma} (C, D) and TNF-{alpha} (E, F) by specific ELISA. Results are expressed as means ± SEM from four mice per time point per mouse strain.

 

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  		Table I. IL-4 and GM-CSF content in BAL and sera during pulmonary mycobacterial infection1

 
Systemic circulating levels of above cytokines were examined in sera collected at various time points. As shown in Figure 5Go, 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-{gamma} was measured in C57BL/6 mice whereas there was no IL-12 or IFN-{gamma} detected at any time in IL-12 p40-/- mice. The amounts of TNF-{alpha}, GM-CSF, and IL-4 were very small in sera from both C57BL/6 and IL-12 p40-/- mice (Table IGo).

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. 6GoA). 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. 6Go, 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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  		FIGURE 6. Subtypes of immune cells in the lung during pulmonary mycobacterial infection. Total lung mononuclear cells were obtained and pooled from lungs of three PBS-treated control C57BL/6, two infected C57BL/6, or three infected IL-12-/- mice at day 27, and immunostained for CD3, CD4, CD8, and NK cells and analyzed by FACS. A, Recovery of total lung mononuclear cells per lung from control, infected C57BL/6 or IL-12-/- mice. B, Dot-plot demonstration of percentages of immune subsets by FACS and gating was set in the lymphocyte-rich region. C, Absolute numbers of different subsets of immune cells calculated based upon FACS and total lung cell recovery.

 
In vitro Ag-stimulated cytokine response by lung mononuclear cells

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-{gamma} 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-{gamma} either spontaneously or upon PPD stimulation (Fig. 7GoA), 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-{gamma} (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-{gamma} without stimulation or only small amounts of IFN-{gamma} upon PPD stimulation, cells from C57BL/6 mice released 598 pg/ml of IFN-{gamma} spontaneously, which represented a further increase over the amount released by the cells purified at day 14 (Fig. 7GoA), and 4233 pg/ml of IFN-{gamma} upon PPD stimulation, which represented a much more pronounced recall response to Ag challenge than at day 14. In contrast to IFN-{gamma}, 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. 7GoB), in keeping with findings from BAL and serum (Table IGo).



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  		FIGURE 7. Ag-stimulated cytokine recall response by lung mononuclear cells in vitro. Total lung mononuclear cells were obtained and pooled from lungs of two C57BL/6 or three IL-12-/- mice at days 14 and 27 post-airway infection and cultured with or without 10 µg/ml PPD for 48 h. Collected culture supernatants were assayed for IFN-{gamma} (A) and IL-4 (B) by ELISA. Results are expressed as means ± SEM from triplicate determinations per condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that, following airways inoculation of live M. bovis BCG, immune-competent wt mice were able to mount an active response in the lung of type 1 cytokines IL-12, IFN-{gamma}, and TNF-{alpha} 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-{gamma} 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-{gamma} and a severely impaired TNF-{alpha} 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-{gamma} 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-{gamma} release, and T cell/NK cell activation (27). However, recent identification of IFN-{gamma}-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-{gamma} 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-{gamma} and TNF-{alpha} 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-{gamma}, 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-{gamma} 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-{gamma} and TNF-{alpha}. 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-{gamma} release from these cells in the lung of IL-12-/- mice, whereas a severely impaired TNF-{alpha} response was likely a result of the lack of CD4 cells and macrophage activation mediated by IFN-{gamma}. However, at this point, we cannot rule out that IL-12 may also directly activate lung macrophages to release TNF-{alpha}. In this regard, Taylor et al. have recently demonstrated an IL-12-induced, IFN-{gamma}-independent TNF-{alpha} 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-{gamma} led to the formation of a defective necrotic type of granuloma following i.v. infection with M. tuberculosis in IFN-{gamma}-/- mice (13). In this regard, recent human studies have linked the IFN-{gamma} receptor deficiency to disseminated tuberculous infection in children immunized with M. bovis BCG (34, 35). Abrogation of TNF-{alpha} 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-{gamma} and TNF-{alpha} 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-{gamma}-/- mice to compare the relative role of IL-12 and IFN-{gamma} 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-{gamma}, 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-{gamma}, and TNF-{alpha} 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).


   Acknowledgments
 
We thank Darlene Steele-Norwood for her invaluable technical assistance, Sara DeSilvio for her secretarial assistance, and Dr. Jack Gauldie for his constant support of this work.


   Footnotes
 
1 This work is supported by grants from The Ontario Thoracic Society and The Medical Research Council of Canada. Z. Xing is a scholar of the Medical Research Council of Canada. Back

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: Back

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. Back

Received for publication October 20, 1997. Accepted for publication February 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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