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Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages
J T Summersgill1, L A Powell, B L Buster
1Department of Medicine, University of Louisville School of Medicine, Kentucky 40292.
Abstract:
The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.
Insights
Nitric oxide (NO) radicals are crucial for killing the bacterium Legionella pneumophila (Lp) in macrophages. Inhibiting NO production allows Lp to survive, highlighting NO's essential role in bacterial defense.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Intracellular pathogens like Legionella pneumophila (Lp) pose significant health challenges.
- Macrophages play a critical role in the innate immune response against bacterial infections.
- Nitric oxide (NO) is a signaling molecule with known antimicrobial properties.
Purpose of the Study:
- To investigate the role of nitric oxide (NO) radicals in the killing of Legionella pneumophila (Lp) within infected macrophages.
- To determine the impact of NO production inhibition on Lp survival in different cell types.
- To explore the relationship between NO production, intracellular iron levels, and bacterial killing.
Main Methods:
- Murine (RAW 264.7) and human (HL-60) macrophage cell lines were infected with Lp.
- Cells were treated with gamma-interferon (IFN) to induce antimicrobial responses.
- Nitric oxide (NO) production was inhibited using N-monomethyl-arginine (NGMMA).
- Bacterial viability was assessed by colony-forming units (CFU) over 24 hours.
- NO production and intracellular iron levels were quantified.
Main Results:
- IFN-treated RAW 264.7 cells showed a significant decrease in viable Lp (3.8 to 0.7 log CFU/ml) after 24h.
- In the presence of NGMMA (NO inhibitor), Lp survival increased in IFN-treated RAW 264.7 cells (persisted at 2.2 log CFU/ml).
- NO production was significantly higher in IFN-treated RAW 264.7 cells without NGMMA (58.6 microM) compared to those with NGMMA (5.65 microM).
- Sodium nitroprusside (NO donor) killed Lp in a dose-dependent manner.
- IFN-treated RAW 264.7 cells exhibited decreased intracellular iron levels compared to IFN+NGMMA-treated cells.
- IFN-treated HL-60 cells displayed bacteriostasis, with no significant NO production detected.
Conclusions:
- Nitric oxide (NO) radicals are critical for the bactericidal activity against Legionella pneumophila in IFN-treated RAW 264.7 macrophages.
- Inhibition of NO production significantly enhances Lp survival in macrophages.
- Intracellular iron depletion may be linked to NO-mediated bacterial killing.
- The absence of detectable NO production in IFN-treated HL-60 cells limits the immune response to bacteriostasis rather than bactericidal activity.