tive Legionella pneumophila-induced lung infec- ... model of replicative L. pneumophila lung infec- ... primarily in alveolar macrophages.19 Previous in vitro.
American Journal of Pathology, Vol. 145, No. 6, December 1994 Copyright C) American Society for Investigative Pathology
Animal Model Replicative Legionella pneumophila Lung Infection in Intratracheally Inoculated A/J Mice A Murine Model of Human Legionnaires' Disease
Joan Brieland,* Pat Freeman,t Robin Kunkel,t Clarence Chrisp,* Mary Hurley,t Joseph Fantone,t and Cary Engleberg,t§ From the Unit for Laboratory Animal Medicine, * and the Departments of Pathology,t Medicine,5 Microbiology and Immunology,t University ofMichigan, Ann Arbor, Michigan
The role of host immune responses in the pathogenesis of Legionnaires' disease is incompletely understood, due in part to the current lack of an animal model that is both susceptible to replicative Legionella pneumophila-induced lung infection and for which species-specific immunological reagents are available. We have developed a model of replicative L. pneumophila lung infection in intratrachealy inoculated A/J mice. L. pneumophila was obtained in the exponential growth phase and inoculated into the trachea of 6- to 8-week-oldfemale A/J mice. Microbiological and histopathological evidence of infection was demonstrated in mice inoculated with 106 colonyforming units. Development of an acute pneumonia that resembled human Legionnaires' disease coincided with exponential growth of the bacteria in the lung 24 to 48 hours after intratracheal inoculation of L. pneumophila This was associated with increased plasma levels of interferon-y at 24 hours after inoculation. After 48 hours, the bacteria were gradualy eliminatedfrom the lung over the next 5 days, corresponding with resolution of the inflammatory response in the lung, thereby mimicking the outcomefrequently seen in the immunocompetent human host. Treatment of animals with anti-interferon-vyantibody enhanced bacterial replication and disease progression, indicating an important role of host immune re-
sponse in resolution of the infection. Because of the availability of murine-specific reagents, this model of replicative L. pneumophila lung infection in A/J mice after intrapulmonary inoculation of L. pneumophilapotentialy provides an important toolfor future studies investigating the role of host immune responses in the pathogenesis of Legionnaires' disease in the immunocompetent
host. (AmiJPathol 1994, 145:1537-1546)
Legionnaires' disease is an important cause of epidemic and sporadic pneumonia in humans. The causative agent, Legionella pneumophila, is a facultative intracellular bacteria of mononuclear phagocytic cells (MPCs).1'2 Humans usually become infected via inhalation of L. pneumophi/a-contaminated aerosols from waterborne environmental sources.3 After inhalation, L. pneumophila multiplies in human MPCs, primarily in alveolar macrophages.19 Previous in vitro and in vivo studies indicate that susceptibility to L. pneumophila pneumonia is determined in large part by permissiveness of host MPCs to growth of the bacteria.6710 12 In the susceptible host, development of cell-mediated immunity in response to L. pneumophila plays a key role in the inhibition of bacterial growth, thereby facilitating resolution of Legionella infections.13,14 Although effector mechanisms of cell-mediated immunity to L. pneumophila in the lung are incompletely understood, in vitro studies indicate Supported by NIH Grants RR-00200, RO1-A126232, R01-A124731, R-29-HL-49136, and R01-HL-44085. Accepted for publication September 1, 1994. Address reprint requests to Dr. Joan K. Brieland, Unit for Laboratory Animal Medicine, 018 ARF, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, Ml 48109-0614.
1537
1538
Brieland et al
AJP December 1994, Vol. 145, No. 6
that lymphokine-activated MPCs inhibit the intracellular growth of the bacteria.8 1,13,15-18 Specifically, interferon-y (IFN-y) downregulates cell surface transferrin receptors and decreases intracellular ferritin in human MPCs in vitro, resulting in a net decrease in intracellular iron,19'20 thereby limiting growth of L. pneumophila. IFN-y-activated natural killer cells also have enhanced cytolytic activity and lyse Legionellainfected human monocytes in vitro, suggesting that these cells may also limit the course of L. pneumophila infections in vivo.21 Much of our current knowledge regarding the role of host immune responses in the pathogenesis of L. pneumophila pneumonia has been gleaned from the use of animal models of Legionnaires' disease. Animal models provide a means of testing theories of the pathogenesis of L. pneumophila infections as well as the potential value of therapeutic interventions in a whole animal system.22 Although a variety of species can be experimentally infected with L. pneumophila, the guinea pig is extremely susceptible and is widely used as an animal model for experimental legionellosis.10'14,16,23-25 After intrapulmonary inoculation of virulent bacteria, (ie, via intratracheal (IT) inoculation or aerosolization), guinea pigs develop a fatal pneumonia that closely resembles severe cases of Legionnaires' disease in human patients.10'25-27 The high susceptibility of guinea pigs to replicative fulminant L. pneumophila infection is due both to the permissiveness of guinea pig MPCs to growth of the bacteria and the relative unresponsiveness of guinea pig lymphocytes to L. pneumophila.11 Although the guinea pig has proved a valuable model for evaluation of bacterial virulence and efficacy of antibiotic therapies, studies investigating the role of the host immune response in the development of L. pneumophila lung infections with this species are severely limited by the current lack of commercially available guinea-pig-specific reagents. Because of the availability of murine-specific immunological reagents, development of a murine model of replicative L. pneumophila lung infection would greatly facilitate studies investigating interactions of Legionella with host defenses and/or the pathogenesis of L. pneumophila pneumonia. In addition, mice are relatively inexpensive to acquire and maintain, making them an attractive alternative to existing animal models of Legionnaires' disease. However, previous studies indicate that most inbred strains of mice are highly resistant to L. pneumophila infection. Specifically, peritoneal and/or alveolar macrophages from BDF1, DBA/2, C3H/HeN, C57BL/6 and BALB/c mice are not permissive to growth of the bacteria in vitro and/or in ViVo.6'28'29 In contrast, peri-
toneal macrophages from mice of the A/J strain support growth of L. pneumophila both in vitro and in viVo.28,30 Similarly, alveolar macrophages from A/J mice have recently been shown to support growth of the bacteria in vitro.31 We hypothesized that alveolar MPCs from A/J mice would also support the growth L. pneumophila in vivo, thereby providing a murine model of replicative L. pneumophila lung infection. Results of this study describe the pathogenesis of the replicative L. pneumophila lung infection induced in A/J mice by IT inoculation of virulent bacteria. Because of the availability of species-specific reagents, it is anticipated that this murine model of legionellosis will provide an important tool for future in vivo studies investigating the role of host immune responses in the pathogenesis of L. pneumophila lung infection in the immunocompetent host.
Materials and Methods Animal Care Female pathogen-free 6- to 8-week-old A/J mice (Jackson Laboratory, Bar Harbor, ME) were used for all experiments. The animals were housed in microisolator cages in Horsefall units and were cared for in accordance with standard guidelines.
Bacterial Inoculum L. pneumophila strain AA100, a redesignation of a primary clinical isolate from the Wadsworth Veterans Administration Hospital, Wadsworth, CA, was provided by Dr. Paul Edelstein. Bacteria were passed three times on buffered charcoal-yeast extract (BCYE) agar.32 For preparation of the IT inoculum, L. pneumophila was quantitated from BCYE agar plates that had been incubated for 48 hours and resuspended in phosphate-buffered saline (PBS) at the desired concentration as previously described.33
Animal Inoculation A/J mice were inoculated IT with L. pneumophila by using previously described methodology.34 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 pi of the bacterial suspension (1 02 to 108 L. pneumophila), followed by 10 p1 of air, was injected directly into the trachea with a 27gauge needle. The skin incision was closed with a
L.
pneumophila Lung Infection in A/J Mice
1539
AJP December 1994, Vol. 145, No. 6
sterile wound clip. Control mice were similarly inoculated with PBS (25 pl/mouse).
Recovery of L. pneumophila from Infected Mice At specific time points after IT inoculation of L. pneumophila (0, 24, 48, 72, 120, and 168 hours), the mice were humanely sacrificed and the lungs were removed. Lung tissue was finely minced in 10 ml of PBS and subsequently homogenized (2 minutes/sample) with a Stomacher (Teckmar, Cincinnati, OH) as previously described.29 The tissue homogenates were serially diluted and cultured on BCYE agar containing polymyxin B, cefamandole, and anisomycin (BCYE + PAC, Becton Dickinson, Cockeysville, MD) for 72 hours and colony-forming units (cfu) were determined.13 In selected experiments, extrapulmonary dissemination of IT inoculated L. pneumophila was also evaluated. In these experiments, spleens from L. pneumophila-inoculated mice were processed as described above, and cfu in spleen homogenates were determined by culture of spleen homogenate on BCYE + PAC agar plates as described above.
Pathology The inflammatory response in the lung in A/J mice in response to IT L. pneumophila was assessed by light microscopy. Mice were inoculated IT with L. pneumophila and were humanely sacrificed at specific time points (24, 48, 72, 120, and 168 hours) thereafter. The lungs were subsequently excised, inflated, and fixed in 10% buffered formalin. Fixed lungs were sectioned, embedded in paraffin, and hematoxylineosin-stained slides of lung tissue were prepared.
Visualization of L. pneumophila in Lung Tissue L. pneumophila was visualized in lung tissue of infected mice by transmission electron microscopy and by direct immunofluorescence. Briefly, mice were inoculated IT with L. pneumophila. At 24 and 48 hours post-inoculation (PI), the mice were sacrificed and the lungs were excised. The lungs were subsequently inflated with 4% glutaraldehyde in 0.1 mol/L cacodylate, pH 7.3, minced, and fixed at 4 C overnight. The samples were post-fixed in 2% osmium tetroxide for 1 hour at room temperature, dehydrated with graded alcohols to propylene oxide, infiltrated with graded Epon to propylene oxide mixtures, and finally embedded in pure Epon. Sections (1 p thick) were cut and
stained with toluidine blue, and areas of interest were identified. Thin sections were cut on an AO ultracut, ultramicrotome and stained with uranyl acetate and lead citrate before examination on a Philips 400T electron microscope. For immunofluorescence, lung sections were depariffinized and subsequently stained with rabbit antiserum to Legionella that had been conjugated to fluorescein isothiocyanate (FITC) (generously provided by the Michigan Department of Public Health). FITC-labeled lung sections were then examined with a Zeiss epifluorescence microscope.
Statistical Analysis The Student's t-test or the Mann Whitney test was used to compare differences between treatment groups. P < 0.05 was considered significant.
Results Establishment of a Survival IT inoculum of L. pneumophila in A/J Mice Initial experiments were conducted to identify a survival IT dose of L. pneumophila in A/J mice. Female A/J mice were inoculated IT with L. pneumophila strain AA100 (102 to 108 organisms per 25 pi PBS). The mice were observed three times daily for 9 days for mortality. Results of these experiments are shown in Table 1. As shown in Table 1, mice inoculated with >106 bacteria IT died acutely (ie, within 48 hours) after bacterial inoculation. Histopathology of lungs of A/J mice inoculated with >106 bacteria IT indicated an acute hemorrhagic process with the lack of leukocyte recruitment into the lung. Although the mechanism for the acute lethal effect of L. pneumophila in A/J mice inoculated IT with relatively large concentrations of bacteria is incompletely understood, results of previous studies with other mouse strains suggest that it Table 1.
Establisbment of a Survival ITInoculum of L. pneumophila in A/J Mice
Dose of Bacteria
Live/Total
% Mortality
108
3/3 2/3 0/3 0/3 0/3 0/3 0/3
100 66 0 0 0 0 0
107 106 105 104 103 102
A/J mice were inoculated IT with L. pneumophila ( 102 to 108 bacteria per mouse) as described in Materials and Methods. Mice were observed three times daily for 9 days for clinical signs of illness/mortality.
1540
Brieland et al
AJP December 1994, Vol. 145, No. 6
is independent of bacterial growth and may be mediated by the elaboration of bacterial toxin.35"36 Additional results of these experiments established a survival threshold of
