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Original Article

Tumor Necrosis Factor Induces Resistance of Macrophages to Legionella pneumophila Infection

Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida 33612

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor– (TNF-) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF- restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) /ß increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody–treated cultures corresponded with enhanced levels of TNF- in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF- is important in controlling L. pneumophila replication, and IL-1 can regulate TNF- levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The etiologic agent of Legionnaires' disease is Legionella pneumophila, a facultative intracellular bacterium. This organism invades and infects macrophages and monocytes preferentially (1-3). For example, L. pneumophila readily replicates in thioglycollate-elicited peritoneal macrophages from genetically permissive A/J mice (4). Stimulation of macrophages in vitro with Legionella antigens induces production of inflammatory cytokines such as tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and interleukin-1 (IL-1) (5-9). These cytokines have immunoregulatory effects as well as diverse physiological activities such as fever induction, which is a hallmark of Legionnaires' disease. It has been reported that proinflammatory cytokines, especially IL-1, may serve as a growth factor for a microbe such as Escherichia coli in vitro or in vivo (10, 11). However, studies in this laboratory failed to show that such cytokines, when added to bacteriological medium, enhanced the in vitro growth of Legionella. In the present study we extended investigations in which recombinant acute-phase cytokines added to macrophage cultures from A/J mice were examined to determine their influence on susceptibility of the phagocytes to infection with Legionella. We found that addition of recombinant TNF- induced the ability of the macrophages to provide a suppressive environment for replication of the Legionella. Treatment of the macrophages with recombinant IL-6 had no effect.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals.
Female A/J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and used at 7–10 weeks of age. All mice were housed in animal facilities at this institution and cared for according to National Institutes of Health Guidelines. They were euthanized by CO₂ asphyxiation.

Legionella.
A virulent strain of L. pneumophila serogroup 1 was obtained from a case of fatal Legionellosis at Tampa General Hospital and maintained as a frozen stock at -70°C at two passages (4, 12). The bacteria were cultured on buffered charcoal yeast extract agar (BCYE, Becton Dickinson Microbiology Systems, Cockeysville, MD) and supplemented with L-cysteine (Sigma, St. Louis, MO) for 48 hr at 37°C. The bacteria were harvested and suspended in pyrogen-free saline and adjusted spectrophotometrically to a concentration of 3 x l0⁹ bacteria/ml (4).

Macrophages.
Thioglycollate-elicited peritoneal macrophages were obtained from the mice 4 days after intraperitoneal (ip) injection of 3 ml of thioglycollate broth (Difco Laboratories, Detroit, MI). The cells were collected by peritoneal lavage with 5 ml PBS plus 1% FCS. After washing, the macrophages were adjusted to a concentration of 1 x 10⁶ cells/ml in 10% fetal calf serum RPMI 1640 supplemented with 4 x 10^-5 M 2-mercaptoethanol. The macrophages were adhered to either 96-well (10⁵ cells/well) or 6-well (3 x 10⁶ cells/well) tissue culture plates (Costar, Cambridge, MA) for 2 hr in an atmosphere of 5% CO₂ at 37°C.

Infection.
Nonadherent cells were removed by centrifugation, and the resulting monolayers were incubated overnight (4). The adherent cells were infected for 30 min with the bacteria at a ratio of 10:1 L. pneumophila per macrophage. Nonphagocytized bacteria were removed, and fresh medium was added. At various times, the macrophages were lyzed, and the number of colony forming units (CFU) in the cultures was determined by standard plate technique. For this purpose, the macrophages were lyzed with 100 µl of 0.1% saponin (Sigma), and serial 10-fold dilutions were plated on BCYE agar. After 72–96 hr of incubation at 37°C, CFUs were determined for at least three plates per dilution using an Autocount plate counter (Dynatech Laboratories, Inc., Chantilly, VA).

ELISA.
The level of TNF- activity in culture supernatants was determined by a standard sandwich ELISA technique with hamster anti-TNF- antibody using a Duoset Mouse ELISA development system kit (Genzyme, Cambridge, MA) exactly as described by the manufacturer (5). IL-1 and IL-6 ELISA assays were performed as described previously (12).

Cytokines.
Following infection, the macrophages were treated with varying combinations of recombinant (r) cytokines or anticytokine antibodies for 24 and 48 hr. The concentrations of reagents used were rTNF- (10 ng/ml, Genzyme), rIL-1 /ß (10–50 ng/ml, Genzyme), rIL-6 (20 ng/ml, Pharmagen, San Diego, CA), anti-TNF- (20 µg/ml, Genzyme), anti-IL-1/ß (10–20 µg/ml, Genzyme) or anti-IL-6 (20 µg/ml, Pharmagen). Each of the antibodies was monoclonal. Polyclonal anti-Fc antibody (Pharmagen) that blocked Fc receptor was used at a concentration of 10 ng/ml, either alone or with the anti-IL-1 antibody. At each time point supernatants were removed, and CFUs were determined.

Statistical Analysis.
All experiments were performed at least three times, and results were calculated as the mean ± SEM. Statistical significance was determined by the Student's t test. Values of P < 0.05 were considered significantly different.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thioglycollate-elicited peritoneal macrophages from A/J mice were shown previously to be permissive for growth of virulent L. pneumophila (4). The number of bacteria increased several hundred–folds after 48 hr of culture of the infected macrophages. Approximately 10% of the macrophages were initially infected at a ratio of 10:1 bacteria/macrophage. At 72 or 96 hr of culture, there was no difference in the number of Legionella per culture with this ratio of bacteria per phagocyte as compared with macrophages infected at the higher ratio.

Treatment of the macrophages with recombinant TNF- caused a marked decrease in the ability of the cells to replicate Legionella as determined by CFU analysis of the cultures at 24 and 48 hr following infection (Fig. 1). Treatment of the infected macrophages with monoclonal antibody to TNF- (20 µg/ml) reversed this effect, resulting in a slight but significant increase in replication of the organisms. When the macrophages were treated similarly with recombinant IL-1/ß, there was little change in their ability to replicate the Legionella (Fig. 2). Although there was a slight increase in growth of the bacteria in these treated cultures, this increase was not significant. For these experiments a concentration of 20 ng/ml of recombinant IL-1 /ß was used. In additional experiments with recombinant IL-1/ß, even an excess of 20 ng/ml did not have a detectable effect on the growth of the Legionella in the macrophages. However, addition of monoclonal neutralizing antibodies to IL-1/ß (10 µg/ml) resulted in a marked decrease of bacterial growth in the cell cultures (Fig. 2). This decrease was greater than two log₁₀ inhibition of growth. Addition of monoclonal antibody to an irrelevant antigen (i.e., murine serum IgG) had no effect (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Altered susceptibility of macrophages from A/J mice to L. pneumophila infection after addition of recombinant TNF- or monoclonal antibody to TNF-. Macrophage cultures were infected for 30 min with L. pneumophila and treated immediately after infection with rTNF- or anti-TNF- antibody. Controls consisted of cells in medium alone or treated with an irrelevant protein (i.e., medium plus monoclonal antimurine IgG (10 µg/ml)). The cells were cultured for 24 and 48 hr, washed and lyzed with saponin before determining CFUs. Data represent mean values ± standard deviations for five independent experiments. Significant differences are indicated by (*) P < 0.05.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Modulation of L. pneumophila growth in macrophage cultures treated with recombinant IL-1 or antibody to this cytokine. Macrophages were infected for 30 min with virulent L. pneumophila and treated with rIL-1/ß or anti-IL-1/ß monoclonal antibodies. The macrophages were then cultured for 24 and 48 hr, washed three times, lyzed with saponin, and CFUs were determined. The controls consisted of culture medium alone or culture medium plus murine monoclonal anti-IgG (10 µg/ml). The data represent mean value ± standard deviation for five independent experiments. Significant differences are indicated by (*) P < 0.05.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Effect of blocking Fc receptors prior to treatment of macrophages with anti-IL-1/ß antibodies. Macrophages were infected for 30 min with virulent L. pneumophila and treated with monoclonal anti-IL-1/ß, anti-IL-1/ß plus anti-Fc or anti-Fc antibody alone, incubated for 24 and 48 hr, then washed three times, lyzed with saponin, and CFUs were determined. Data represent mean value ± standard deviation for three independent experiments. Significant differences are indicated by (*) P < 0.05.

View larger version (42K): [in this window] [in a new window]   Figure 4.   Production of TNF- protein following treatment of infected macrophage cultures with anti-IL-1/ß. Macrophages were infected for 30 min with L. pneumophila, treated with monoclonal anti-IL-1/ß antibodies, and incubated for 24–48 hr. Supernatants were removed and assayed for presence of TNF- by ELISA. Values are shown as ng/ml, and data represent mean value ± standard deviation for five independent experiments. Significant differences are indicated by (*) P < 0.05.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Macrophages infected for 30 min with L. pneumophila, treated with rIL-6 or anti-IL-6 antibody, and assayed for bacterial growth at 24 and 48 hr. Values represent mean CFUs ± standard deviation for five independent experiments. Significant differences are indicated by (*) P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies in this laboratory have shown that infection of A/J mice macrophages with L. pneumophila induces secretion of proinflammatory cytokines such as TNF- and IL-1 (5, 12). In the present study we found that addition of recombinant TNF- to L. pneumophila–infected macrophages resulted in a marked resistance of the cells to bacterial growth. The ability of TNF- to control infection in the macrophages was supported by neutralization experiments revealing that treatment of the macrophages with monoclonal anti-TNF- antibody restored susceptibility of the cells to intracellular replication of the bacteria. This suggests that endogenously produced TNF- is able to inhibit the replication of the bacteria in macrophage cultures, since neutralization by antibody to this cytokine resulted in increased growth of the bacteria in the cells.

Treating the macrophage cultures with either rIL-6, anti-IL-6 antibody, or other recombinant cytokines as a control had no significant effect on the replication of the Legionella at 24 or 48 hr or even longer following infection (unpublished data). Although in vitro infection of macrophages by Legionella was previously shown to enhance secretion of soluble IL-6 and also enhance the levels of mRNA to IL-6 in the cells, it was apparent in the present study that addition of exogenous recombinant IL-6 or antibody to this cytokine had no significant effect on susceptibility of the cells to infection with Legionella.

It seems probable that the effect of anti-TNF- antibodies on Legionella growth may be important. Matsiota-Bernard et al. (15) first reported that TNF- stimulates human monocytes to resist infection with L. pneumophila. Skerret and Martin (16) extended these observations using rat alveolar macrophages. In our studies, in all probability the murine peritoneal macrophages in culture were marginally burdened with the organisms by 24 hr after infection, and further intracellular replication was impossible. However, it appeared paradoxical that the infected macrophages produced significant levels of TNF-, yet the cells were still permissive for bacterial growth. Although exogenous TNF- was capable of controlling L. pneumophila in this in vitro infection study, endogenous production may be insufficient to control the excessive growth of the bacteria within the cells. However, at the level of the whole animal the role of increased levels of endogenously produced TNF-, even at low levels, may be important. It is accepted that TNF- may act synergistically with IFN- producing T cells by augmenting the ability of macrophages to inhibit bacterial replication (8). It is possible that TNF-, produced by macrophages, is used by PMNs to extend the effective function of macrophages. The combination of these events may be involved in the control of L. pneumophila infection in vivo. However, the mechanisms by which TNF- results in inhibition of intracellular bacterial multiplication is still unclear. Recent studies have shown that TNF- induced by L. pneumophila may be mediated, at least in part, by endogenous nitric oxide activity (16-18). For example, Summersgil et al. (17) demonstrated that nitric oxide activity correlates with the intracellular level of this cytokine. It is possible that a combination of these factors (i.e., induction of nitric oxide and depletion of intracellular iron) is responsible for the TNF-–associated inhibition of L. pneumophila replication in macrophages. It may also be possible that the capacity of replicating organisms to parasitize new macrophages as the culture proceeds is inhibited by the exogenous cytokine. Anyway, treatment of the L. pneumophila–infected macrophages with recombinant IL-1/ß did not endow these cells with the ability to inhibit L. pneumophila growth.

Naturally produced inhibitors of IL-1 may influence the effects of the cytokine on macrophage susceptibility to infection by L. pneumophila. These include IL-1 type 2 receptor (decoy), soluble forms of IL-1 receptor, and IL-1 receptor antagonist (IL-1ra). For example, IL-1 receptors are capable of being shed in response to stimuli that may include microbial infection (19). Such soluble forms of receptors are also capable of binding IL-1 and IL-1ß, thus possibly inhibiting biological activity. It is known that IL-1ra is produced at the macrophage level and may antagonize potential beneficial and protective effects that IL-1 might display. This effect might be dependent on the system being studied and could result in apparent ineffectiveness of IL-1/ß antibodies. Thus, it is possible that IL-1/ß may appear beneficial in controlling virulent L. pneumophila infection, but the potential beneficial effects could be antagonized by the presence of specific inhibitors. Although IL-1 has been reported to facilitate the growth of E. coli in vitro (11), such effect was not seen with L. pneumophila. In contrast, treatment of macrophages with neutralizing antibodies to IL-1/ß resulted in inhibition of replication of the L. pneumophila in the macrophages.

In attempts to understand the mechanisms involved in this effect, TNF- levels were measured in the supernatants of cells treated either with recombinant IL-1 or anti-IL-1/ß. The TNF- levels increased within 24 hr after treatment of the macrophages with antibody to IL-1. Addition of recombinant IL-1 (10 µg/ml) to the macrophages, either at 24 or 48 hr of culture, had no significant effect (unpublished data). However, it was not clear whether the increased TNF- levels after treatment of the macrophages with the antibody to IL-1/ß reflected an increased production or enhanced release of the cytokine. Measurement of the release of IL-1, IL-1 receptors, and/or IL-1ra would address directly how TNF- and IL-1 interact in this system. Such experiments are planned. Although it is not clear how the increased TNF- levels occurred, TNF- and IL-1, as well as other proinflammatory cytokines, are known to act as part of a tightly regulated network. It is conceivable that if one arm of the cytokine response is compromised, as in the case of neutralization of IL-1, enhancement of a cytokine can result in overlapping activity profiles, insuring that infection can be effectively controlled. Nevertheless, the results of these studies with cytokine treatment of macrophages infected with L. pneumophila support the view that proinflammatory cytokines such as TNF- and IL-1 are important factors in infection with an opportunistic organism like L. pneumophila. Even though it is clear that TNF- acts directly on macrophages to control infection by this bacterium, the effects of other cytokines could be due to other effector mechanisms.

It was surprising to find that IL-6 or antibody to this cytokine had no discernible effect on the ability of the macrophages to be infected with Legionella. Other studies in this laboratory had shown that murine macrophages stimulated with Legionella antigens evinced enhanced production of IL-6 (12). This interleukin is considered an important proinflammatory cytokine involved in immunoregulation of T cells and other aspects of cellular immunity. The effect of exogenous addition of this cytokine on susceptibility of macrophages, as occurred with TNF- or antibody to IL-1, shows that the influence of cytokines on susceptibility of macrophages to this intracellular pathogen is more complex than appeared evident from initial studies. The defense response to an opportunistic bacterium such as L. pneumophila likely involves complex interactions among many molecules, including proinflammatory cytokines. Thus, further studies are warranted to determine the mechanisms involved.

	Footnotes

 
This work was supported by Grant AI 6618 from the National Institute of Allergy and Infectious Diseases.

¹ To whom requests for reprints should be addressed at the University of South Florida College of Medicine, Box 10, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. E-mail: hfriedma{at}com1.med.usf.edu

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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