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Infect Immun, January 1998, p. 65-69, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

J. K. Brieland,1,* D. G. Remick,2 M. L. LeGendre,2 N. C. Engleberg,3,4 and J. C. Fantone2

Unit for Laboratory Animal Medicine1 and Departments of Pathology,2 Internal Medicine,3 and Microbiology and Immunology,4 The University of Michigan Medical School, Ann Arbor, Michigan 48109-0614

Received 12 August 1997/Returned for modification 8 September 1997/Accepted 10 October 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (106 L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-gamma ), tumor necrosis factor alpha (TNF-alpha ), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-alpha activity was significantly lower, while protein levels of IFN-gamma and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-alpha .

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).

Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-gamma ) and tumor necrosis factor alpha (TNF-alpha ) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-gamma limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-gamma in combination with other cytokines including TNF-alpha facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-alpha secretion and IFN-gamma -mediated MPC activation (46).

IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-gamma , TNF-alpha , and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10-12). Using this murine model of Legionnaires' disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice. Female pathogen-free 6- to 8-week-old A/J mice (Jackson Laboratory, Bar Harbor, Maine) were used for all experiments. The animals were house in microisolator cages in Horsefall units and were cared for in accordance with standard guidelines.

Preparation of bacteria. L. pneumophila serogroup 1, strain AA100, a redesignation of a primary clinical isolate from the Wadsworth Veterans Administration Hospital (Wadsworth, Calif.), was provided by Paul Edelstein. For preparation of the intratracheal inoculum, L. pneumophila was quantified on buffered charcoal-yeast extract agar (Becton Dickinson, Cockeysville, Md.) that had been incubated for 48 h and resuspended in phosphate-buffered saline (PBS) at 4 × 107 organisms/ml (10, 25).

Inoculation of A/J mice with L. pneumophila. A/J mice were inoculated intratracheally with L. pneumophila as previously described (10, 49). Briefly, the mice were anesthetized with ketamine (2.5 mg/mouse intraperitoneally) and tethered, and an incision was made through the skin of the ventral neck. The trachea was isolated, and 25 µl of the bacterial suspension (i.e., containing 106 L. pneumophila cells), followed by 10 µl of air, was injected directly into the trachea with a 26-gauge needle. The skin incision was closed with a sterile wound clip.

Recovery of L. pneumophila from infected lung tissue. At specific time points postinoculation (p.i.), the mice were humanely euthanized and the lungs were removed. Lung tissue was finely minced in sterile water (10 ml/lung) and subsequently homogenized (2 min/sample) with a Stomacher (Tekmar, Cincinnati, Ohio) (6, 10). Lung homogenates were subsequently serially diluted in sterile water and cultured on buffered charcoal-yeast extract agar containing polymyxin B, cefamandole, and anisomycin (Becton Dickinson) for 72 h (10, 22). The lower limit of detection of L. pneumophila with this system is 103 CFU per lung.

Collection of lung homogenate supernatant and BALF for cytokine analysis. Lung homogenate supernatant was procured by filtering lung homogenates, prepared as described above, through a 0.45-µm-pore-size filter (Gelman Sciences, Ann Arbor, Mich.) to remove the bacteria. Alternatively, for collection of bronchoalveolar lavage fluid (BALF), the mice were humanely sacrificed and their lungs were lavaged with 1.6 ml of PBS (6). The resultant lavage fluid was subsequently filtered as described above. Filtered lung homogenates and BALF were stored at -20°C until use for cytokine analysis.

Cytokine analysis. IFN-gamma , IL-10, and IL-12 protein levels in lung homogenates and/or BALF were measured by commercially available cytokine-specific murine enzyme-linked immunosorbent assay (ELISA) kits (Intertest-gamma , Intertest-IL-10, and Intertest-IL-12X total mouse IL-12, respectively; Genzyme Corp., Cambridge, Mass.) according to the manufacturer's directions. This IL-12 ELISA detects all three forms of IL-12 (i.e., p70 heterodimer, p402 homodimer, and p40 monomer). TNF-alpha activity in lung homogenate was measured by a cytotoxicity assay using the WEHI 164 subclone 13 cell line as previously described (24). Briefly, lung homogenate samples were serially diluted directly into 96-well microtiter plates (Costar, Cambridge, Mass.). The WEHI cells were suspended at 5 × 105 cells per ml in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.5 µg of actinomycin D (Calbiochem, La Jolla, Calif.) per ml and added to the samples. A standard of human recombinant TNF-alpha was run in each assay. Samples were incubated overnight at 37°C, after which 20 µl of dimethyl-azole tetrazolium bromide (MTT) (5 mg/ml; Sigma, St. Louis, Mo.) was added to the wells and allowed to incubate at 37°C for an additional 4 h. Viable cells (i.e., cells not lysed by TNF-alpha ) metabolize the MTT-tetrazolium to produce dark formazan crystals. The crystals were dissolved in isopropanol-HCl, and the plates were read in a microELISA reader (Bio-Tek Instruments, Inc., Winooski, Vt.) at 550 nm. TNF-alpha activity was calculated based on the human recombinant TNF-alpha standard that was run in the same assay.

Interventional studies. Mice were depleted of endogenous IL-12 by intraperitoneal inoculation with rabbit serum containing neutralizing antibody to IL-12 (0.5 ml/mouse) 2 h prior to intratracheal inoculation with L. pneumophila. This antiserum, a generous gift from Steven Kunkel, Department of Pathology, University of Michigan Medical School, Ann Arbor, has previously been shown to be efficacious in neutralizing endogenous intrapulmonary IL-12 in other murine models of lung injury (31). Results of preliminary experiments demonstrated that this concentration of antiserum neutralized >= 95% of endogenous IL-12 activity in lung homogenates of L. pneumophila-infected mice for up to 5 days (data not shown). Alternatively, in selected experiments, mice were inoculated with preimmune rabbit antisera (0.5 ml/mouse) prior to intratracheal inoculation with L. pneumophila. Results of these preliminary experiments demonstrated that neither recovery of L. pneumophila nor cytokine levels in BALF were significantly altered in mice treated with preimmune rabbit serum, compared to similarly infected mice not administered antiserum (data not shown). Consequently, in all subsequent experiments, L. pneumophila-infected mice administered anti-IL-12 serum were compared to similarly infected untreated mice.

Statistical analysis. Student's t test was used to compare differences between treatment groups. For comparison of multiple groups to a single control, analysis of variance with post-hoc Tukey analysis was performed. P < 0.05 was considered significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Endogenous IL-12 facilitates resolution of primary replicative L. pneumophila lung infection. In initial experiments, induction of intrapulmonary IL-12 during replicative L. pneumophila lung infection was assessed. A/J mice were inoculated intratracheally with L. pneumophila (106 organisms per mouse). At 0 to 120 h p.i., the mice were humanely euthanized and the lungs were either lavaged or homogenized. IL-12 activity in BALF and in whole lung homogenates was subsequently quantified by a murine-specific IL-12 ELISA. As shown in Fig. 1, IL-12 was significantly enhanced in BALF and in whole lung homogenates within 24 h p.i. Furthermore, whole lung homogenates from infected mice contained greater than fivefold more IL-12 than did BALF from similarly infected mice.


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  		FIG. 1.   IL-12 activity in BALF and lung homogenates of A/J mice inoculated intratracheally with L. pneumophila. A/J mice were inoculated intratracheally with L. pneumophila as described in Materials and Methods. At specific time points thereafter, the mice were sacrificed and IL-12 was assessed in BALF (A) and whole lung homogenates (B) by ELISA. Results represent the means ± standard errors of the means for three to five animals per time point, *, significantly greater than value for uninfected mice (i.e., 125 pg of IL-12 per ml of BALF; 935 pg of IL-12 per lung) (analysis of variance, P < 0.05).

We have previously demonstrated that A/J mice inoculated intratracheally with virulent L. pneumophila (106 bacteria per mouse) develop replicative L. pneumophila lung infections, with logarithmic growth of the bacteria within the first 48 h p.i. followed by gradual clearance of the bacteria from the lung at >= 72 h P.I. (10). Because induction of IL-12 activity in the lung of L. pneumophila-infected A/J mice (i.e., at >= 24 h p.i. [Fig. 1]) precedes clearance of the bacteria from the lung (i.e., at >= 72 h p.i.), the potential role of endogenous IL-12 in resistance to primary replicative L. pneumophila lung infection was evaluated. Mice were depleted of endogenous IL-12 by administration of rabbit IL-12 antiserum as described in Materials and Methods. At 24, 72, and 120 h p.i., the mice were humanely euthanized, the lungs were excised and homogenized, and L. pneumophila CFU were quantified in lung homogenates. As shown in Table 1, while there was no significant difference in recovery of L. pneumophila from the lung of mice treated with anti-IL-12 antiserum and similarly infected immunocompetent mice within the first 72 h p.i., significantly more bacteria were recovered in lung homogenates from animals depleted of endogenous IL-12 and sacrificed at 5 days p.i. (compared to similarly infected immunocompetent mice [Student's t test, P <0.05]). Furthermore, 10-fold more bacteria were recovered in lung homogenates of L. pneumophila-infected mice treated with IL-12 antiserum at 5 days p.i. compared to the inoculating dose of bacteria (i.e., 106 L. pneumophila per mouse), suggesting that A/J mice depleted of endogenous IL-12 activity develop persistent replicative intrapulmonary L. pneumophila infection. Taken together, these results demonstrate that IL-12 is induced in the lung in response to L. pneumophila and plays a key role in innate immunity to the bacteria.

                              
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  		TABLE 1.   Effect of IL-12 neutralizing antibody on intrapulmonary replication of L. pneumophilaa

Immunomodulatory activity of endogenous IL-12. Previous in vitro and in vivo studies have demonstrated that growth of L. pneumophila in permissive MPCs is modulated by cytokines including IFN-gamma , TNF-alpha , and IL-10 (10, 12, 14, 15, 46). Because IL-12 has previous been shown to modulate the production of these cytokines (44), in subsequent experiments the effect of endogenous IL-12 on intrapulmonary levels of IFN-gamma , TNF-alpha , and IL-10 during replicative L. pneumophila lung infection was assessed. A/J mice were administered anti-IL-12 serum and inoculated intratracheally with L. pneumophila (106 bacteria per mouse) as described in Materials and Methods. At 24, 72, and 120 h p.i., the mice were humanely euthanized, the lungs were excised, and filtered lung homogenates were obtained. TNF-alpha , IFN-gamma , and IL-10 were subsequently quantified in filtered lung homogenates obtained from anti-IL-12-treated and untreated L. pneumophila-infected mice by cytokine-specific bioassay or ELISA.

In agreement with our previous studies (10, 12), both TNF-alpha and IFN-gamma were significantly induced in the lung of immunocompetent L. pneumophila-infected mice within 24 h p.i. compared to uninfected mice (<10 pg of TNF and 170 pg of IFN-gamma per lung of uninfected mice) (Fig. 2). In contrast, IL-10 was not significantly induced in the lung of L. pneumophila-infected immunocompetent mice at any time point studied compared to uninfected mice (330 pg of IL-10 per lung of uninfected mice). Administration of anti-IL-12 serum to A/J mice prior to intratracheal inoculation with L. pneumophila resulted in a significant decrease in intrapulmonary TNF-alpha activity within 24 h p.i. compared to similarly infected immunocompetent mice (Student's t test, P <0.05). In contrast, there was no significant difference between intrapulmonary levels of IFN-gamma or IL-10 in L. pneumophila-infected mice depleted of endogenous IL-12 compared to similarly infected immunocompetent mice at any time point studied. We have previously demonstrated that TNF-alpha plays a key role in elimination of L. pneumophila from the lung in this murine model system (12). Together, these results suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is likely mediated at least in part by TNF-alpha .


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  		FIG. 2.   Effect of IL-12 on endogenous cytokine activity in the lung during replicative L. pneumophila lung infection. A/J mice were administered IL-12 antiserum as described in Materials and Methods prior to intratracheal inoculation of L. pneumophila. At specific time points p.i., the mice were sacrificed. The lungs were excised, homogenized, and filtered. Levels of TNF-alpha , IFN-gamma , and IL-10 were quantified in filtered lung homogenates of L. pneumophila-infected mice depleted of endogenous IL-12 by ELISA or by bioassay and compared to those of similarly infected immunocompetent (i.e., untreated) mice. Results represent the means ± standard errors of the means for five mice per treatment group. black-square, untreated mice; , anti-IL-12-treated mice. *, significantly less than value for untreated mice (Student's t test, P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, the role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection was assessed in vivo, using a murine model of Legionnaires' disease in A/J mice inoculated intratracheally with virulent bacteria. We demonstrate that IL-12 is induced in the lung during replicative L. pneumophila lung infection and that neutralization of endogenous IL-12 by administration of IL-12 antiserum resulted in impaired ability of A/J mice to resolve a primary replicative L. pneumophila lung infection. These results identify a key role of endogenous IL-12 in innate immunity to L. pneumophila pulmonary infection.

In subsequent experiments, immunomodulatory effects of endogenous IL-12 on intrapulmonary IFN-gamma , TNF-alpha , and IL-10 during replicative L. pneumophila lung infection were assessed. This is of particular interest, as IFN-gamma , TNF-alpha , and IL-10 have previously been shown to regulate growth of the bacteria in permissive MPCs in vivo and/or in vitro (10, 12, 46). Results of these studies demonstrated that enhanced growth of L. pneumophila in mice depleted of endogenous IL-12 was positively correlated with a significant reduction in TNF-alpha activity in the lung within 24 h p.i.; however, intrapulmonary levels of neither IFN-gamma nor IL-10 were significantly altered by this therapy (Fig. 2). TNF-alpha has previously been shown to play a key role in resolution of replicative L. pneumophila infection, as it enhances polymorphonuclear leukocyte bacteriocidal activity (7), is directly toxic for L. pneumophila (42), and in combination with other cytokines, including IFN-gamma , induces MPC production of reactive nitrogen intermediates, including nitric oxide, which limit L. pneumophila growth and viability (12). Together, these results suggest that regulation of intrapulmonary L. pneumophila replication by endogenous IL-12 is likely mediated, at least in part, by TNF-alpha .

Previous studies have demonstrated that IL-12 is a potent inducer of IFN-gamma (41). Furthermore, IL-12-mediated inhibition of other pathogenic microbes, including T. gondii (30), Histoplasma capsulatum (1), and Listeria monocytogenes (54, 55), has been shown to occur through an IFN-gamma -mediated mechanism. Therefore, we were somewhat surprised that neutralization of endogenous IL-12 in L. pneumophila-infected A/J mice did not significantly alter intrapulmonary levels in IFN-gamma . However, our results demonstrating IFN-gamma -independent effects of IL-12 on host immune responses to L. pneumophila concur with those of a recent in vitro study by Bermundez et al., who showed that IL-12-induced NK cell-mediated mycobactericidal activity in human MPCs is mediated by a TNF-alpha -dependent, IFN-gamma -independent mechanism (3). Together, these studies suggest that the role of endogenous IL-12 in cytokine networking and host resistance to pathogenic microbes differs with respect to different intracellular pathogens.

While our studies have focused on elucidating immunomodulatory effects of IL-12 on the expression of cytokines which mediate resistance to replicative L. pneumophila lung infection, it is likely that endogenous IL-12 also contributes to innate immunity to L. pneumophila by cytokine-independent mechanisms. Specifically, costimulation of NK cells and/or T cells with IL-12 and other cytokines, including IL-15 or TNF-alpha , has been shown to enhance NK cell and T-cell cytotoxicity (2, 13, 16-18, 38, 53). The potential role of cytotoxic T cells and/or NK cells in resistance to primary replicative L. pneumophila lung infection remains to be thoroughly explored.

In summary, using a murine model of Legionnaires' disease in A/J mice, we have demonstrated that endogenous IL-12 plays a key role in innate immunity to legionellosis, likely in part by its ability to modulate intrapulmonary activity of other cytokines, including TNF-alpha . Further studies, which will identify the potential synergy between intrapulmonary IL-12 and other cytokines such as IL-15 in innate immunity to replicative L. pneumophila lung infection are warranted and will likely provide a rational approach to immunotherapy for treatment of Legionnaires' disease.

    ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants RR00200 and R-29-HL-49136.

    FOOTNOTES

* Corresponding author. Mailing address: University of Michigan Medical School, 018 Animal Research Facility, 1301 Catherine Road, Ann Arbor, MI 48109-0614. Phone: (313) 764-0277. Fax: (313) 936-3235. E-mail: jbrie{at}umich.edu.

Editor:  J. R. McGhee

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Infect Immun, January 1998, p. 65-69, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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