Introduction

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Legionella pneumophila (Lp) is a gram-negative, facultative intracellular pathogen and is the etiologic agent of Legionnaires' disease. Once the bacteria enter the respiratory tract and cause pneumonia, most of the invading microorganisms are located within inflammatory phagocytic cells. Alveolar macrophages are especially thought to be the preferential site for growth of Lp in human lung tissue (1). The mechanism by which Legionella infection of the lung is controlled is not yet clear, but the development of cell-mediated immunity (CMI) is essential in host defense to Lp infection. T helper 1 (Th1) cells are essential for the development of CMI and may play a pivotal role in the defense against Lp infection. Interleukin (IL)-12 and the interferon (IFN)- that is induced by IL-12 from Th1 cells play central roles in the development of the protective Th1-type immune response (2). It is known that the Th1 cytokine IFN- can activate macrophages and monocytes to inhibit Lp growth (3, 4). In contrast, other cytokines, including IL-10, facilitate growth of Lp in permissive mononuclear phagocytes, due in part to IL-10-mediated inhibition of tumor necrosis factor (TNF)- secretion and IFN--mediated mononuclear phagocyte activation (5). The induction of such key cytokines by alveolar macrophages is, therefore, considered one of the important immunoregulatory functions of macrophages during the development of T-helper cell phenotypes. However, the induction mechanism of these cytokines by Lp infection in alveolar macrophages is not well understood.

To investigate the in vitro cytokine regulation and induction mechanism of macrophages by Lp infection, previous studies in this laboratory and by others used mainly human monocytes, mouse peritoneal macrophages, and macrophage cell lines that were not derived from the lung (3). However, it has been reported that macrophages obtained from disparate anatomical sites differ functionally and phenotypically (10) and that phenotypically distinct macrophage subpopulations can be identified and isolated from the bronchoalveolar lavage of the human lower respiratory tract (11). The few studies with alveolar macrophages focused only on the growth of Lp in the macrophages (12, 13), and cytokine responses of alveolar macrophages to Lp infection have not been well studied.

MH-S cells are a continuous cell line of murine alveolar macrophages, which were established after transformation of cells obtained by bronchoalveolar lavage from Balb/c mice with simian virus 40 (14). Results of characterization studies of MH-S cells (15) indicated that this cell line may facilitate studies where homogeneous populations of alveolar macrophages are desirable, especially those involved in determining the immunologic responses of alveolar macrophages to Lp infection and their potential role in lung pathology. The necessity of an in vitro alveolar macrophage system to study Lp infection is obvious because the preferential infection site for Lp is the lung and particularly alveolar macrophages, which are the major target cells for Lp infection but functionally and phenotypically different from other tissue macrophages (10, 16, 17). In fact, our previous study showed the immune response of alveolar macrophages to Lp infection is different than that of peritoneal macrophages (18). Thus, establishment of an in vitro alveolar macrophage system is desirable for the successful study of macrophage-Lp interactions. Therefore, in the present study, we attempted to establish an alveolar macrophage cell line model, designated MH-S, for Lp infection and to analyze cytokine responses to Lp infection using this system in comparison with primary alveolar macrophages obtained from mouse alveolar lavage.

	Materials and Methods

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Mice

Inbred female Balb/c mice were used for this study. They were purchased from Jackson Laboratories (Bar Harbor, ME) and were 10 to 12 wk of age when used.

Bacteria

Virulent L. pneumophila M124 (Lp-V), serogroup 1, was obtained from a case of fatal legionellosis (19). Avirulent L. pneumophila (Lp-Av) was prepared by multiple passages of M124 as described previously (20). The Lp-Av showed no lethal activity for experimental animals (20). Both Lp-V and Lp-Av were cultured on buffered charcoal yeast extract (BCYE) medium (Gibco Laboratories, Madison, WI) for 3 d at 37°C. The bacteria were suspended in pyrogen-free saline and the bacterial concentration was determined by spectrophotometry.

Macrophages

Two different types of macrophages were used in this study. The MH-S murine alveolar macrophage cell line was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI 1640 medium containing 10% heat- inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT). The MH-S cells were adhered to 6-well tissue culture plates at a concentration of 5 × 10⁵ cells/ml for 2 h in 5% CO₂ at 37°C. Primary alveolar macrophages were obtained as previously described by repeated bronchoalveolar lavage with phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO) containing 5 mM ethylenediaminetetraacetic acid (18). The collected cells were washed three times with Hanks' balanced salt solution (HBSS) by centrifugation and were resuspended in RPMI 1640 medium containing 10% FCS and antibiotics (50 µg of polymyxin B per ml and 50 µg of vancomycin per ml). The primary alveolar macrophages were adhered to 24-well tissue culture plates at a concentration of 5 × 10⁵ cells/ml for 24 h in 5% CO₂ at 37^oC. All resulting cell monolayers were washed with HBSS, supplied with 10% FCS-RPMI 1640 medium without antibiotics, and then used for experiments.

Infection and Stimulation

The cell monolayers were infected with Lp (infectivity ratio, 10 bacteria per cell) for 30 min, washed to remove nonphagocytized bacteria, and incubated in RPMI 1640 medium containing 10% FCS with no antibiotics. In some experiments, cells were stimulated with 1 µg/ml Escherichia coli lipopolysaccharides (LPS) (Sigma). The cultures were then incubated for up to 48 h at 37°C in 5% CO₂.

Viable Bacteria in Cell Cultures (CFU Assay)

The number of viable bacteria (colony-forming unit [CFU]) in cell lysates was determined by standard plate counts on BCYE medium, as described previously (19). After incubation, the cell monolayers were lysed with 0.1% saponin and the number of viable bacteria in the lysates was determined.

Microscopic Examination

The cell monolayers on a glass coverslip were fixed in methanol, stained with diluted Giemsa solution (Sigma), and examined with a microscope (BH-2; Olympus Co., Tokyo, Japan).

Reverse Transcriptase/Polymerase Chain Reaction

RNA isolation from MH-S cells infected with or without Lp was performed as described previously (18). Total RNA was extracted from cells by either the single-step method with TRI-reagent (Molecular Research Center, Cincinnati, OH) or the microspin technique with RNeasy mini kit (Qiagen, Valencia, CA) in accordance with the manufacturer's manual. Reverse transcription (RT) of total RNA (1 µg) was performed with avian myeloblastosis virus transcriptase in a commercial reaction mixture (Reverse Transcription System; Promega, Madison, WI). The resulting complementary DNA was subjected to polymerase chain reaction (PCR) with primers for ₂-microglobulin (BMG), IL-1, IL-6, IL-10, TNF-, IL-12 p35, and IL-12 p40. The primer sequences for BMG, IL-1, IL-6, IL-10, and TNF- were described previously (18, 21). The sequences of primer for IL-12 p35 were 5'-AAG ACA TCA CAC GGG ACC AAA CCA-3' (sense) and 5'-CGC AGA GTC TCG CCA TTA TGA TTC-3' (antisense). The sequences of primer for IL-12 p40 were 5'-CCA CTC ACA TCT GCT GCT CCA CAA G-3' (sense) and 5'-ACT TCT CAT AGT CCT TTG GTC CAG-3' (antisense). The PCR was performed in a Minicycler (MJ Research, Watertown, MS) for either 25 cycles and 60°C annealing temperature (BMG), 30 cycles and 60°C annealing temperature (IL-1, IL-6, IL-10, TNF-), or 40 cycles and 62°C annealing temperature (IL-12 p35 and IL-12 p40). PCR products were analyzed on ethidium bromide-stained 2% agarose gels, semiquantitated, and normalized to BMG using densitometry readings (Bio-Rad Laboratories, Hercules, CA).

Enzyme-Linked Immunosorbent Assay

The amount of IL-1 and IL-12 p40/p70 in culture supernatants was determined by sandwich enzyme-linked immunosorbent assay (ELISA) using antimouse IL-12 p40 and biotin-labeled anti- IL-12 p40/p70 monoclonal antibodies for IL-12 (PharMingen, San Diego, CA), and antimouse IL-1 and alkaline phosphatase- labeled goat antirabbit immunoglobulin antibodies for IL-1 (Genzyme Diagnostics, Cambridge, MA), respectively, in accordance with the manufacturer's manual.

Statistical Analysis

Statistical analysis was performed with the paired Student's t test.

	Results

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Lp Growth in MH-S Alveolar Macrophages and Primary Alveolar Macrophages

To determine the extent that the murine alveolar macrophage cell line and the primary alveolar macrophages could support the growth of Lp, both macrophage types were infected with a 10:1 (bacteria:cell) ratio of either Lp-V or Lp-Av bacteria and assayed for CFU at 6, 24, and 48 h after infection. As evident in Figure 1, both MH-S murine alveolar macrophages (Figure 1A) and primary alveolar macrophages (Figure 1B) were permissive for growth of Lp-V, even though these cells were derived from Balb/c mice, which are resistant to Lp infection and their peritoneal macrophages are nonpermissive for growth of Lp (22). The extent of permissiveness to Lp growth in MH-S cells was comparable with primary alveolar macrophages as well as A/J mouse peritoneal macrophages (22), human alveolar macrophages (12), and rat alveolar macrophages (13), with more than 2 logs multiplication within 2 d. The Lp-Av, on the other hand, was able to survive within the cells but incapable of replication. Microscopic analysis of Lp-infected MH-S cells supported the evidence of multiplication of bacteria in the MH-S cells determined by CFU assay (Figure 2). A few bacteria in a cell were observed at the early infection stage, such as 6 h after infection, but obviously multiplied at 24 h after infection as demonstrated by evidence of many bacteria in a cell.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Growth of virulent versus avirulent L. pneumophila in the MH-S murine alveolar macrophage cell line (A) and primary alveolar macrophages (B). Macrophage monolayers were infected with either Lp-V or Lp-Av (infectivity ratio, 10 bacteria per macrophage) for 30 min. The number of viable bacteria (CFU) in macrophages was determined by standard plate count method. Solid circles indicate growth of Lp-V, open circles indicate growth of Lp-Av. The data shown are representative of three experiments. Each data point represents the mean CFU ± SD for triplicate macrophage cultures. *P < 0.05 significantly different from Lp-Av-infected cells.

View larger version (66K): [in this window] [in a new window]   Figure 2.   Microscopic analysis of L. pneumophila replication in MH-S murine alveolar macrophage cells. Macrophage monolayers on the slides were infected with Lp-V and stained with diluted Giemsa solution 6 and 24 h after infection. Phagocytized bacteria (arrows) were observed in MH-S alveolar macrophages at 6 h (A) and 24 h after infection (B) (original magnification: ×1,000).

Cytokine Messenger RNA Expression of Lp-Infected MH-S Alveolar Macrophages and Primary Alveolar Macrophages

The cytokine expression profile of MH-S cells and primary alveolar macrophages in response to infection with either Lp-V or Lp-Av was examined for determination of the immune response of these cells to Lp infection. When the MH-S cells were infected with Lp-V, the expression levels of messenger RNA (mRNA) for IL-1, IL-6, IL-10, and TNF- increased at 6 h after infection (Figure 3), but the expression level of mRNA for IL-12 did not (data not shown). In order to quantify the message expression, the relative expression levels of IL-1, IL-12 p35, and IL-12 p40 messages to the endogenous housekeeping gene BMG were measured in both macrophage types. As a control for induction of cytokines, E. coli LPS was used. As evident in Figures 4 and 5, IL-1 and IL-12 p40 messages tested were significantly induced by LPS stimulation, showing that both macrophage types were satisfactory regarding cytokine responses. Furthermore, the expression level of mRNA for the cytokine responses of MH-S alveolar macrophages to Lp infection appeared similar to that of primary alveolar macrophages. When the cells were infected with Lp-V, the expression levels of mRNA for IL-1 significantly increased at 6 h after infection and decreased after that. In the case of Lp-Av, the expression level of mRNA for IL-1 was slightly increased at 6 h after infection, followed by a decrease in both macrophage types (Figure 4). IL-12 p40 message levels were significantly increased at 24 h after infection with Lp-V but not at 6 h (Figure 5). As a control, LPS stimulation induced a significant increase at this early time point. However, the levels of IL-12 p40 message in Lp-Av-infected cells showed no significant increase in both macrophage types. On the other hand, IL-12 p35 message, which is known to be expressed in various cells constitutively (23), was consistent through the infection (Figure 6).

View larger version (64K): [in this window] [in a new window]   Figure 3.   Cytokine mRNA expression of L. pneumophila-infected MH-S murine alveolar macrophage cells. Macrophage monolayers infected with either Lp-V or Lp-Av, or stimulated with LPS (1 µg/ml). The mRNA expression for cytokines was determined by RT-PCR as described in MATERIALS AND METHODS.

View larger version (16K): [in this window] [in a new window]   Figure 4.   Semiquantitative analysis of mRNA expression for IL-1 in L. pneumophila-infected MH-S murine alveolar macrophages (A) and primary alveolar macrophages (B). The PCR products for IL-1 were semiquantitated and normalized to BMG using densitometry readings. The data are presented as the ratio (mean ± SD) of IL-1 to BMG densities from three independent experiments. Open bars, 6 h after infection; solid bars, 24 h after infection. *P < 0.05 significantly different from the control group at the same time point.

View larger version (16K): [in this window] [in a new window]   Figure 5.   Semiquantitative analysis of mRNA expression for IL-12 p40 in L. pneumophila-infected MH-S murine alveolar macrophages (A) and primary alveolar macrophages (B). The PCR products for IL-12 p40 were semiquantitated and normalized to BMG using densitometry readings. The data are presented as the ratio (mean ± SD) of IL-12 p40 to BMG densities from three independent experiments. Open bars, 6 h after infection; solid bars 24 h after infection. *P < 0.05 significantly different from the control group at the same time point.

View larger version (22K): [in this window] [in a new window]   Figure 6.   Semiquantitative analysis of mRNA expression for IL-12 p35 in L. pneumophila-infected MH-S murine alveolar macrophages (A) and primary alveolar macrophages (B). The PCR products for IL-12 p35 were semiquantitated and normalized to BMG using densitometry readings. The data are presented as the ratio (mean ± SD) of IL-12 p35 to BMG densities from three independent experiments. Open bars, 6 h after infection; solid bars, 24 h after infection.

IL-12 p40/p70 and IL-1 Protein Production in Lp-Infected MH-S Alveolar Macrophages and Primary Alveolar Macrophages

At the protein level, both Lp-V and Lp-Av infection resulted in only minimum production of IL-12 p40/p70 protein at 24 h after infection in both macrophage types (Figures 7A and 7B). However, the IL-1 protein levels in the culture supernatants were increased in Lp-V-infected macrophages at 24 h after infection in both macrophage types (Figures 7C and 7D). In contrast, both IL-12 p40/p70 and IL-1 protein production in both macrophage types stimulated with LPS were significantly increased at 24 h after stimulation.

View larger version (22K): [in this window] [in a new window]   Figure 7.   IL-12 p40/p70 (A and B) and IL-1 (C and D) protein production by MH-S murine alveolar macrophage cells (A and C) and primary alveolar macrophages (B and D). The production of IL-12 p40/p70 and IL-1 protein was measured in the culture supernatants by ELISA at 24 h after infection. Results are expressed as means ± SD for three independent experiments. *P < 0.05 significantly different from the control group at the same time point.

	Discussion

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The results obtained in this study support the usefulness of MH-S cells for in vitro study of Lp infection. The extent of Lp growth in MH-S cells was comparable with primary alveolar macrophages as well as human alveolar macrophages (12) and rat alveolar macrophages (13). Our previous studies showed that peritoneal macrophages from Balb/c mice were nonpermissive for growth of Lp and Balb/c mice were resistant to Lp when infected intraperitoneally (22). The permissiveness of MH-S cells and Balb/c primary alveolar macrophages for Lp, therefore, may indicate that alveolar macrophages could be more susceptible than peritoneal macrophages in regard to Lp infection.

The cytokine response of MH-S alveolar macrophages to Lp infection was also comparable with primary alveolar macrophages as well as the findings of some previous reports that showed the induction of cytokines, including IL-1, IL-6, and TNF-, in either peritoneal macrophages or macrophage-like cell lines, which were not derived from the lung, by Lp (6, 24). Therefore, the cytokine response of MH-S alveolar macrophages to Lp infection appears to be similar to previously reported in vitro responses.

The study of IL-12 induction in MH-S alveolar macrophages by virulent versus avirulent Lp infection revealed a differential response of macrophages to this bacterial infection. That is, the messages for IL-12 p40, which is inducible by microbial stimulation whereas IL-12 p35 is synthesized constitutively (23), were slowly but significantly induced by Lp-V. However, IL-12 protein secretion was not significantly induced by Lp infection even 24 h after infection. Lp-Av infection did not induce either message or protein secretion of IL-12. In contrast, IL-1 protein secretion was readily induced by Lp-V but not by Lp-Av. These results indicate that Lp-V infection can induce the message for IL-12 p40 in macrophages, but production of IL-12 protein may be inhibited for some reason in Lp-V- infected macrophages by an unknown mechanism. This inhibition was specific for IL-12 because both IL-1 message and protein secretion as well as other cytokine messages, such as IL-6, IL-10, and TNF-, in Lp-V-infected macrophages were readily induced by infection. Although the mechanism of inhibition was not clear, current studies suggest there is an active inhibition mechanism for IL-12 production after Lp infection because LPS-induced IL-12 production could be selectively inhibited by Lp infection (unpublished observation). The weaker or minimum induction of cytokines, including IL-12 in Lp-Av-infected cells, may be due to the lack of growth of avirulent bacteria in the macrophages.

Thus, the MH-S alveolar macrophage cell line can be a useful in vitro model system to study Lp infection. Furthermore, the study indicates that virulent Lp infection of alveolar macrophages induces minimum production of IL-12, which is critical for the development of the Th1 phenotype even though other cytokines are markedly induced.