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Binding of Legionella pneumophila to macrophages increases cellular cytokine mRNA
Y Yamamoto1, S Okubo, T W Klein
1Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa 33612.
Abstract:
Infection of macrophages with Legionella pneumophila induces formation of interleukin 1 beta (IL-1 beta), but the molecular basis of this is not understood. Binding of bacteria to macrophage surfaces is the first step in an infection process. Therefore, we examined whether this step was sufficient to increase the cellular level of mRNAs for IL-1 beta and other cytokines. To assess the effect of binding of L. pneumophila on the steady-state levels of cytokine mRNAs, cultures of thioglycolate-elicited macrophages from L. pneumophila-susceptible A/J mice were treated with cytochalasin D and infected with L. pneumophila and the total RNA was extracted for analysis by reverse transcription-PCR with primers for IL-1 alpha, IL-1 beta, IL-6, tumor necrosis factor alpha, granulocyte macrophage colony-stimulating factor, and beta interferon (IFN-beta). L. pneumophila treatment increased the cellular steady-state mRNA levels of all cytokines except IFN-beta. To determine the specificity of this effect, macrophage cultures were treated with cytochalasin D and either bacterial lipopolysaccharide, bovine serum albumin-sensitized latex, Salmonella typhimurium, or Escherichia coli. Lipopolysaccharide treatment increased all mRNAs, bovine serum albumin-sensitized latex had no significant effect, and treatment with S. typhimurium or E. coli increased all mRNAs except that of IFN-beta. These results suggested that the binding of gram-negative bacteria to the macrophage surface was sufficient to induce a unique pattern of cytokine mRNAs. Additional studies that examined the characteristics of the bacterial ligands involved indicated involvement of both heat-labile and heat-stable surface ligands.
Insights
Bacterial binding to macrophages triggers increased cytokine mRNA, except for interferon-beta. This suggests gram-negative bacteria surface ligands initiate a specific cytokine response during infection.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- Interleukin 1 beta (IL-1 beta) production by macrophages upon Legionella pneumophila infection is known, but the underlying molecular mechanisms remain unclear.
- Bacterial surface binding is the initial step in macrophage infection, potentially initiating downstream signaling pathways.
- Understanding early molecular events is crucial for deciphering host-pathogen interactions and immune responses.
Purpose of the Study:
- To investigate if bacterial binding alone is sufficient to elevate cytokine mRNA levels in macrophages.
- To identify the specific cytokines affected by bacterial surface interaction.
- To explore the role of bacterial surface ligands in inducing cytokine mRNA expression.
Main Methods:
- Thioglycolate-elicited macrophages from susceptible A/J mice were treated with cytochalasin D to facilitate bacterial binding.
- Macrophages were infected with Legionella pneumophila or other bacterial stimuli (lipopolysaccharide, Salmonella typhimurium, Escherichia coli) or non-bacterial particles (BSA-sensitized latex).
- Total RNA was extracted and analyzed using reverse transcription-PCR (RT-PCR) to quantify mRNA levels for various cytokines, including IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF, and IFN-beta.
Main Results:
- Infection with Legionella pneumophila significantly increased cellular steady-state mRNA levels for all tested cytokines except interferon-beta (IFN-beta).
- Treatment with bacterial lipopolysaccharide, Salmonella typhimurium, and Escherichia coli also induced increased mRNA levels for most cytokines, excluding IFN-beta.
- Non-bacterial stimuli like bovine serum albumin-sensitized latex did not significantly affect cytokine mRNA levels, indicating specificity.
- Further analysis suggested the involvement of both heat-labile and heat-stable surface ligands on gram-negative bacteria in this response.
Conclusions:
- The binding of gram-negative bacteria to the macrophage surface is sufficient to induce a distinct pattern of cytokine mRNA expression.
- This bacterial binding event initiates an early immune signaling cascade, influencing the production of key inflammatory mediators.
- Bacterial surface components, including both heat-labile and heat-stable factors, play a critical role in modulating macrophage cytokine responses during the initial stages of infection.