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We have found that Legionella dumoffii strain Tex-KL (ATCC 33343) invades into and proliferates in the human lung alveolar epithelial-cell line A549 in vitro. The organism associated with the A549 cells at a 10-fold greater magnitude than L. pneumophila Philadelphia-1 during in vitro coculture for 1 h. Thereafter, L. dumoffii Tex-KL invaded the cells at a significantly higher rate (100- to 1,000-fold) than did L. pneumophila Philadelphia-1. After internalization, however, both bacteria proliferated at the same rate. This in vitro finding led us to examine the bacterial localization in lungs in a fatal case of L. dumoffii pneumonia. Double immunostaining revealed the bacteria in surfactant apoprotein A-positive cells (i.e., type II alveolar epithelial cells). Next, we infected guinea pigs intratracheally with L. dumoffii Tex-KL. The animals became sick with a fever from 24 h to 48 h after infection with 104 to 109 cfu of L. dumoffii Tex-KL. The lung tissues were examined through electron microscopy at definite intervals. Many bacteria were found not only inside phagocytic cells in the alveolar space, but also in type I and type II alveolar epithelial cells. These findings strongly suggest that L. dumoffii has an ability to invade into and proliferate in human alveolar epithelial cells, which may explain the rapid and fulminant progress of pneumonia caused by L. dumoffii.
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Bacteria that cause acute lower respiratory infection have a variety of strategies for pathogenesis. For example, Streptococcus pneumoniae (1) and Klebsiella pneumoniae (2) form thick capsules to evade complement killing and phagocytosis. Haemophilus influenzae (3) and S. pneumoniae (4) have lipopolysaccharide (LPS) and pneumolysin, respectively, which are toxic to the cilia of epithelial cells. Mycoplasma pneumoniae adheres to normal mucosa and sticks strongly to epithelial cells (5). Chlamydia psittaci and Chlamydia pneumoniae can enter into and proliferate intracellularly within epithelial cells (6). Legionella pneumophila resists macrophage killing and proliferates in macrophages, which correlate to cause pneumonia (7, 8). Pathogenic mechanisms of Legionella species, however, are not well understood except for those of L. pneumophila (9).
By 1994, 3,524 confirmed cases of Legionella pneumonia had been reported to the surveillance system at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA (10). Among Legionella species that cause human pneumonia, Legionella dumoffii is the fourth most common causative agent (10). The bacteria was identified in water in 1979 by Cordes and colleagues (11) and named in 1980 by Brenner and associates (12). Lewallen and coworkers (13) reported that L. dumoffii can cause pneumonia in humans. Pneumonia caused by this bacterium is rapidly progressive and fulminant, and is often fatal (14, 15). It has been reported that L. dumoffii can grow intracellularly in a human macrophage cell line (16) and in protozoa (17, 18). However, the virulence mechanism in humans is not well characterized. In addition, no reasons have been proposed for the ability of L. dumoffii to cause more serious and rapidly progressive types of pneumonia in humans.
A549 cells were established from a human lung cancer and have characteristics of type II alveolar epithelial cells; for example, the cells secrete surfactant apoprotein (19). While working with in vitro assays for an adherence, invasion, and intracellular growth of Legionella species, we observed that L. dumoffii Tex-KL exhibits highly adherent and invasive behavior toward A549 cells. This observation prompted us to investigate the localization of L. dumoffii in inflammatory sites in the lungs. We have studied the localization of L. dumoffii in human lung specimens from an autopsied patient who died of acute pneumonia caused by this organism. We also infected guinea pigs intratracheally with L. dumoffii Tex-KL to examine sites of bacterial proliferation in the lungs. We observed L. dumoffii Tex-KL not only inside alveolar macrophages (AM), but also inside type I and type II alveolar epithelial cells.
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Bacteria and Culture
L. dumoffii Tex-KL (ATCC 33343) and L. pneumophila Philadelphia-1 (serogroup 1, ATCC 33152; American Type Culture Collection, Rockville, MD) were donated by the CDC. These organisms
were grown on buffered charcoal-yeast extract with 
-ketoglutarate
(BCYE) agar plates, which contained a Legionella agar base (Difco
Laboratories, Detroit, MI) supplemented with (per liter of deionized
water) 0.4 g of L-cysteine and 0.25 g of ferric pyrophosphate. The pH was adjusted to 6.9 with 5 M KOH after autoclaving. The bacteria were stored in tryptic soy broth (Difco) supplemented with 30% (vol/
vol) glycerol at 
80° C.
Cell-Culture Methods
The cell line A549 (JCRB0076) was donated by the Health Science Research Resources Bank, Osaka, Japan. The cells were established from a human alveolar epithelial carcinoma, and have characteristics of well-differentiated type II pneumocytes. The cells were maintained in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 5% fetal calf serum (FCS; GIBCO Laboratories, Grand Island, NY). For experiments, the A549 cells were seeded into 24-well flat-bottom tissue culture plates (Falcon No. 3047; Becton Dickinson Labware, Oxnard, CA) and Lab-Tek chamber slides (Nunc, Inc., Naperville, IL) at a concentration of 5 × 105/ml, incubated for 18 h at 37° C in a humid atmosphere containing 5% CO2 in air, and used for bacterial adhesion and invasion assays.
Adhesion and Invasion Assay
The bacteria were harvested from BCYE agar plates that had been
incubated for 1 d and were suspended in culture medium at a concentration of approximately 2 × 107 to 2 × 109 cfu/ml. Of this suspension,
0.5 ml was inoculated onto A549 cells in each well. After cocultivation
for 1 h at 37° C in a CO2 incubator, each well was washed twice with
PBS to remove nonadherent bacteria. At this time, for performance
of an adhesion assay, 1 ml sterile distilled water was added to the wells
for each bacterial concentration and adherent cells were scraped from
the bottom of each well with a rubber policeman. The cell suspension
was transferred to a test tube and sonicated with a Bransonic Model
32 sonicator (150 W; Branson Cleaning Equipment Company, Shelton, CT) for 20 s. After brief vortexing and a serial 10-fold dilution of
the suspension, the number of bacteria that associated with the cells
was determined by plating on BCYE-agar plates. For an invasiveness assay, 0.5 ml of the culture medium, supplemented with 100 µg/
ml of gentamicin (Schering-Plough Co., Ltd., Osaka, Japan), was added
to each remaining well and incubated for 1 h at 37° C to kill any extracellular bacteria. The supernatant fluids were discarded and the cells were washed twice with PBS to remove residual gentamicin. After 0, 1, or 2 d of incubation with 0.5 ml of culture medium with no antibiotics, 0.5 ml of sterile distilled water was added to each well and the number of bacteria that invaded into and proliferated within the cells was
determined as described earlier. It was confirmed that L. dumoffii
Tex-KL and L. pneumophila Philadelphia-1 could not proliferate in
the cell-culture medium. Cultures in Lab-Tek chamber slides were used
for both Gimenez staining and electron-microscopic examination.
Transmission Electron Microscopy of Infected A549 Cells
At appropriate intervals after bacterial infection, the monolayers of A549 cells on Lab-Tek chamber slides were washed with PBS and fixed with a solution of 2.5% glutaraldehyde for 30 min at room temperature. The monolayers were washed twice with 0.15 M cacodylate buffer and fixed with a solution of 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer for 1 h at 4° C. The fixed monolayers were dehydrated in a graded series of 30% to 100% ethanol at room temperature and embedded in Epon (Nissin EM Co., Ltd., Tokyo, Japan) on the slide glasses. The embedded monolayers were removed from the slide glasses after a brief heating, and were cut into fine, small pieces. These pieces containing A549 cells were affixed to the top of the solidified Epon. Thin sections were then cut off, stained with uranyl acetate and lead citrate, and examined with a JEM-1200EX electron microscope (JEOL, Tokyo, Japan).
Double Immunostaining of L. dumoffii and Surfactant Apoprotein A
Formalin-fixed, paraffin-embedded lung tissue of an autopsied human who had died of pneumonia caused by L. dumoffii (15) was provided by Y. Kobashi of the Tenri Hospital, Nara, Japan. The patient had been an 84-yr-old female. She had been given melphalan and steroid therapy to treat multiple myeloma. She contracted L. dumoffii pneumonia while in the hospital.
Tissue sections were deparafinized with xylene and a serial alcohol solution, and were then incubated in 10% H2O2 solution to block endogenous peroxidase. After 5 min in the H2O2 solution the sections were washed three times with PBS. The sections were incubated in "blocking solution" (LSAB kit; DACO, Kyoto, Japan) for 5 min to block nonspecific reactions, and were then incubated with monoclonal anti-surfactant-protein-A antibody (DACO) at room temperature for 30 min. After washing three times with PBS, the sections were incubated with biotinylated antimouse IgG and streptavidin-peroxidase (DACO) according to the manufacturer's protocol, and were then incubated with diaminobenzidine (DAB) solution for chromogenic reactions. After washing with PBS, the sections were incubated in Blocking Reagent II (10% goat normal-serum; Nichirei Co., Tokyo, Japan) to block nonspecific reactions. The sections were incubated with anti-L. dumoffii antiserum (Denka Seiken Co., Ltd., Tokyo, Japan) for 30 min. After washing three times with PBS, the sections were incubated with biotinylated antirabbit IgG (Nichirei) for 10 min. After again washing three times with PBS, the sections were incubated with alkaline phosphatase-labeled streptavidin (Nichirei) for 10 min. Following three further washings with PBS, the sections were incubated with Fast blue solution (Nichirei) for 10 min for chromogenic reactions.
Animals
Female guinea pigs of outbred Hartley strain, weighing 500 g to 600 g, were purchased from Shizuoka Experimental Animals (Hamamatsu, Japan). The experimental protocol was approved by the Institutional Animal Care Committee of our university.
Intratracheal Inoculation of L. dumoffii Tex-KL
After anesthetizing the guinea pigs with an intraperitoneal injection of pentobarbital sodium (Abbott Laboratories, North Chicago, IL), their tracheas were exposed. L. dumoffii Tex-KL, at approximately 104 to 109 cfu in 0.3 ml PBS, was injected intratracheally and the skin was repaired.
Transmission Electron Microscopy of Lungs of Infected Guinea Pigs
At appropriate intervals after bacterial infection, the guinea pigs were humanely killed and the lungs of infected guinea pigs were removed. Pieces of hashed lungs were suspended in PBS with 2% glutaraldehyde for fixation. After 2 h at room temperature, the pieces of lung were washed twice with PBS and then fixed with a solution of 1% osmium tetroxide. After 2 h at room temperature the pieces were dehydrated in a graded series of 30% to 100% ethanol at room temperature and embedded in Epon. Thin sections were stained with lead citrate and uranyl acetate, and were examined with a JEOL JEM-1200EX electron microscope.
Statistical Calculation
The same experiments were repeated at least three times. The results were analyzed for significance through analysis of variance (ANOVA) and by the two-tailed, unpaired t test. Differences were considered significant at p < 0.05.
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Association, Invasion by, and Intracellular Growth of L. dumoffii Tex-KL in A549 Cells
We examined the interaction between L. dumoffii Tex-KL and A549 cells. Figure 1A shows the numbers of bacteria associated with A549 cells (ordinate) immediately after in vitro phagocytosis for 1 h. When 1 × 107 cfu per well of L. dumoffii Tex-KL or L. pneumophila Philadelphia-1 were added, the numbers of bacteria associated with A549 cells were approximately 1 × 105 or 1 × 104 cfu per well, respectively. With greater initial inocula (108 and 109 organisms); however, there was no remarkable difference in the numbers of L. dumoffii Tex-KL and L. pneumophila Philadelphia-1 associated with A549 cells.
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Next, we compared the invasive activity of L. dumoffii Tex-KL for A549 cells with that of L. pneumophila Philadelphia-1, using gentamicin to kill extracellular bacteria (Figure 1B). The numbers of intracellular L. dumoffii Tex-KL were significantly (p < 0.05) higher than that of L. pneumophila Philadelphia-1. Approximately 103, 104, and 104 cfu per well of intracellular L. dumoffii Tex-KL were observed at initial inocula of 107, 108, and 109 cfu, respectively. L. pneumophila Philadelphia-1 organisms were not detected when we inoculated 107 cfu.
We compared the intracellular growth of L. dumoffii Tex-KL with that of L. pneumophila Philadelphia-1 by continuing cultures. At 1 and 2 d after infection, the cfu per well of both bacteria growing in A549 cells increased approximately 500 and 1,500 times, respectively (data not shown). Despite the same input multiplicity of infection, microcolonies of L. dumoffii Tex-KL, which were visualized by Gimenez staining, were observed intracellularly in many of the A549 cells at 1 d after in vitro phagocytosis, whereas L. pneumophila Philadelphia-1 was observed only in a part of the cells (Figure 2). There were no significant differences between the two organisms in terms of the fold increase in intracellular bacterial growth after 1 and 2 d of infection (data not shown).
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Intracellular Localization of L. dumoffii Tex-KL in A549 Cells
To observe the intracellular localization of L. dumoffii Tex-KL in A549 cells, electron microscopic examinations were performed. At the time immediately after in vitro phagocytosis, L. dumoffii Tex-KL were observed both at the cell surface (Figure 3A) and intracellularly in A549 cells. No significant ruffling of the cell membrane nor coiling phagocytosis (20) were observed in the present study. Intracellular bacteria were localized in membrane-bordered vacuoles, and mitochondria were observed in the vicinity of such L. dumoffii Tex-KL-containing vacuoles (Figure 3B). Many researchers have observed L. pneumophila organisms within membrane-bordered vacuoles, on the membranes of which ribosomes were present in a line, giving these bodies the name of ribosome-lined phagosomes (21). No significant arrangement of ribosomes on the phagosome membrane was seen in the present study. From 6 h after in vitro phagocytosis, rough endoplasmic reticula (RER) and many mitochondria were present near the vacuoles containing L. dumoffii Tex-KL (Figure 3C). Subsequently, at 12 h of infection, the organism proliferated in the vacuoles, and RER were observed studding the membranes of the vacuoles (Figure 3D and E). This construction began to disappear at the late stage of infection (24 h; data not shown).
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Immunohistochemical Staining of Autopsied Human Lung
Double immunostaining was done to examine the localization of L. dumoffii in the lung in the fatal case of human pneumonia. To visualize the type II alveolar epithelial cells, we stained the cells brown with DAB by using an anti-surfactant-protein-A antibody as a primary antibody, which was a significant marker of type II cells. L. dumoffii were stained blue by the Fast blue reaction with anti-L. dumoffii antiserum. Most of the macrophages that localized in alveolar spaces showed no reaction to DAB. L. dumoffii appeared to be present both in foamy macrophages in the alveolar spaces and in type II alveolar epithelial cells, which were stained in a mosaic of blue and brown, lining the vague alveolar wall (Figure 4).
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Intratracheal Injection of L. dumoffii Tex-KL to Guinea Pigs, and Electron Microscopy
Because guinea pigs are a suitable animal model of human legionellosis, we injected L. dumoffii Tex-KL intratracheally into guinea pigs. An electron-microscopic examination of the lungs was done to observe the localization of bacteria. Although the guinea pigs became sick with a fever within 48 h of infection with at least 104 cfu L. dumoffii Tex-KL, we used models with an inoculum of 109 cfu for the electron-microscopic examination. After 3 h of infection, L. dumoffii Tex-KL were observed inside many kinds of cells in the alveolar spaces. The bacteria were observed mainly in phagocytic cells, but also in alveolar epithelial cells. One of the cells that contained bacteria was found lining the alveolar wall and projecting hemispherically into the alveolar space. The cells contained lamellar bodies in their cytoplasm, had many microvilli at their surfaces, and were considered as type II alveolar epithelial cells (Figure 5A). Another type of cell that contained bacteria had few cytoplasmic organelles and was located in the surface of the interalveolar septum. These were considered type I alveolar epithelial cells (Figure 5B). At 24 h after intratracheal injection, great numbers of L. dumoffii Tex-KL were observed in the lungs of guinea pigs, and many alveolar cells had been destroyed. The structure of alveoli also became unclear. As in the early stage of infection, the bacteria were observed not only in phagocytic cells but also in other types of alveolar cells. Some of these cells, which were considered type II (Figure 5C) and type I (Figure 5D) alveolar epithelial cells, had large vesicles in which proliferating bacteria were observed.
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There are now known to be 42 species of the genus Legionella. Many species have been isolated from environmental sources, but 21 of them have been isolated from humans, suggesting their pathogenicity for humans. Although biochemical and morphologic characteristics are similar among Legionella species, they show a variety of modes of interaction with human monocytes (8) and macrophages (22), and with macrophages of experimental animals (23, 24), protozoans (18), and established cell lines (25).
In this study, we found that L. dumoffii internalize and proliferate in alveolar epithelial cells of humans and guinea pigs. This was shown by experimental infection by live L. dumoffii Tex-KL in guinea pigs, and by positively double-immunostained specimens from an autopsied patient who had died of acute L. dumoffii pneumonia. As far as we know, this is the first report that a species of Legionella can internalize and proliferate in alveolar epithelial cells in vivo.
When we inoculated 107 cfu of L. dumoffii Tex-KL in vitro, the bacteria associated with A549 cells in 10-fold greater numbers than did L. pneumophila Philadelphia-1 (Figure 1A). In cases of higher initial inocula (108 and 109 organisms), there were no significant differences in the cell-association rate of the two strains of bacteria. This could have been because the numbers of associated bacteria were near maximum in the system. L. dumoffii Tex-KL also invaded A549 cells at a significantly higher rate (100- to 1,000-fold) than did L. pneumophila Philadelphia-1 (Figure 1B). The results suggested that L. dumoffii Tex-KL had greater adherence and invasive activity for A549 cells than did L. pneumophila Philadelphia-1. After internalization into A549 cells, L. dumoffii Tex-KL proliferated very rapidly, with a doubling time of approximately 3 h (data not shown). However, there were no significant differences between L. dumoffii Tex-KL and L. pneumophila Philadelphia-1 in intracellular growth rates. The precise biochemical analysis of the entry mechanism is now under investigation, but L. dumoffii Tex-KL entered Vero cells through receptor-mediated endocytosis (26).
In A549 cells, most of the L. dumoffii Tex-KL organisms proliferated within membrane-bounded vacuoles. In the same areas, RER were observed to be wrapped around the vacuoles (Figure 3). These findings resembled the ultrastructural features of protozoan cells infected by virulent strains of L. pneumophila (27). A similar structural appearance is found when Brucella abortus proliferates intracellularly in trophoblasts (28) and Vero cells (29). Swanson and Isberg (30) showed that the vacuoles in which L. pneumophila replication occurs resemble nascent autophagosomes in ultrastructural studies of infected macrophages, in which the luminal protein, BiP, of the endoplasmic reticulum (ER) was labeled by immunoperoxidase cytochemistry. They also showed that association of L. pneumophila with ER increased, and that bacterial growth was enhanced by stimulating host autophagy. They suggested that L. pneumophila exploits the autophagic machinery of macrophages to establish an intracellular niche favorable for replication. Abu Kwaik (27) demonstrated that a mutant strain of L. pneumophila that failed to replicate within host cells was defective in the recruitment of organelles, including ribosomes and RER. The functional roles of the interactions between the bacteria and host organelles are still not clear. The recruitment of organelles may be necessary for the intracellular replication of L. dumoffii Tex-KL in A549 cells.
Recently, Bermudez and Goodman (31) reported that Mycobacterium tuberculosis could proliferate in A549 cells in vitro, and suggested that there was another route to tubercular infection through alveolar epithelial cells. Mody and colleagues (32) reported that L. pneumophila can invade and replicate within rat alveolar epithelial cells in vitro. It has been generally accepted that inhaled bacteria are trapped by AM, and that bacteria proliferate intracellularly in the macrophages when they can disturb the cells' killing mechanisms (7). This process is considered a major pathogenetic route to pneumonia in humans. The finding that L. dumoffii Tex-KL invaded and proliferated in A549 cells implies that alveolar epithelial cells may constitute an additional site for the organism's intracellular growth. We therefore performed immunologic staining of the autopsied lung of a human with pneumonia caused by L. dumoffii, to observe localization of the bacteria (Figure 4). The bacteria were observed not only in alveolar phagocytic cells, but also in type II alveolar epithelial cells that were surfactant-apoprotein-A-positive. This result suggested that L. dumoffii were able to enter alveolar epithelial cells of the human lung in vivo.
It is nearly impossible to examine specimens biopsied in the early stage of human pneumonia. We therefore injected L. dumoffii Tex-KL intratracheally into guinea pigs and performed an electron-microscopic examination to examine the localization of the organism at an early stage of infection (Figure 5). Morphologic differences between type I and type II alveolar epithelial cells were clear. Type I alveolar epithelial cells are thin and flat, except for the part containing the nucleus, and the cells have poor cytoplasm and few organelles. The cells cover most of the alveolar surface. Type II alveolar epithelial cells line the alveolar wall and project hemispherically into the alveolar space. Type II cells also have many microvilli at their surfaces. Although the cells contain many organelles, lamellar bodies are considered significant structures in distinguishing type II cells morphologically from other cells in alveolar spaces. L. dumoffii Tex-KL were observed not only inside alveolar macrophages, but also inside type I and type II alveolar epithelial cells. It has been suggested that although type I alveolar epithelial cells have a weak phagocytic activity that is less than 5% that of the AM, type II alveolar epithelial cells have no phagocytic activity (33). Because L. dumoffii Tex-KL was observed in type II alveolar epithelial cells, we suggest that these bacteria have the ability to invade "nonprofessional" phagocytes, and/or to induce phagocytosis by type II cells.
As far as we know, neither Legionella spp. nor most of the bacterial pathogens responsible for human pneumonia have been shown morphologically to invade alveolar epithelial cells in vivo, although bacterial invasive activity into epithelial cells in vitro (5, 6, 31, 32) and in specimens obtained from other organs (34) has been shown. Therefore, the present study is the first to show that Legionella bacteria can invade alveolar epithelial cells in vivo. We used guinea pigs for infection, and the results suggest that some strains of L. dumoffii may be able to adhere to and invade alveolar epithelial cells at an early stage of infection in humans, and then to produce pneumonia.
Even now, no reason has been given to explain why L. dumoffii can cause more serious and rapidly progressive pneumonia in humans than can other Legionella spp., although most cases of L. dumoffii infection occur in immunocompromised patients. Our present results strongly suggest that, besides the ability to grow in AM, L. dumoffii has an additional mode of pathogenesis in being able to invade and grow inside alveolar epithelial cells. Because the bacteria inside epithelial cells can evade the bactericidal activity of phagocytes, and can destroy the epithelial cells, this may lead to more serious and rapidly progressive pneumonia.
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Correspondence and requests for reprints should be addressed to Koji Maruta, Department of Microbiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. E-mail: k-malta{at}wombat.med.uoeh-u.ac.jp
(Received in original form October 31, 1997 and in revised form January 27, 1998).
Acknowledgments: Supported by Grant-in-Aid for Scientific Research 097701192 from the Ministry of Education, Science, Sports and Culture, Japan.
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References |
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