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Expression of pro-inflammatory cytokines by flow-sorted alveolar macrophages in severe pneumonia
Abstract:
The aim of the present study was to further characterize the role of alveolar macrophages (AM) in acute human lung inflammation by evaluating their capacity to produce pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8. Patients with severe community-acquired pneumonia (CAP; n=12) and healthy volunteers (n=10) underwent bronchoalveolar lavage (BAL). AM were separated to high purity (>96%) using fluorescence-activated cell sorting. We determined the TNF-alpha, IL-6 and IL-8 cytokine gene expression in AM ex vivo using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, we measured in vitro unstimulated, lipopolysaccharide (LPS)- and LPS/interferon-gamma inducible TNF-alpha, IL-6 and IL-8 cytokine release and evaluated samples of BAL fluids for the same pro-inflammatory cytokines using an enzyme-linked immunosorbent assay (ELISA). We found increased TNF-alpha, IL-6 and IL-8 messenger ribonucleic acid (mRNA) levels in AM from CAP patients that were significantly elevated only for IL-8. When challenged with endotoxin in vitro, AM obtained from CAP patients showed a strongly reduced potential to release TNF-alpha and IL-6 compared to healthy controls, whereas IL-8 secretion did not differ significantly between groups. Moreover, stimulation of AM from CAP patients with LPS plus IFN-gamma augmented TNF-alpha and IL-6 cytokine release to near normal levels. Interestingly, no TNF-alpha protein was measured in BAL samples from CAP patients, whereas IL-6 and IL-8 protein levels were found to be significantly increased. Together, highly purified alveolar macrophages from community-acquired pneumonia patients show relatively low ex vivo tumour necrosis factor-alpha and interleukin-6 but not interleukin-8 messenger ribonucleic acid levels that are associated with a decreased pro-inflammatory cytokine release in vitro which, however, can be restored by concurrent interferon-gamma stimulation.
Insights
Alveolar macrophages in pneumonia patients show reduced pro-inflammatory cytokine release, particularly tumor necrosis factor-alpha and interleukin-6. This impaired cytokine production can be restored with interferon-gamma stimulation, offering insights into lung inflammation.
Area of Science:
- Immunology
- Respiratory Medicine
- Cell Biology
Background:
- Alveolar macrophages (AM) play a critical role in lung inflammation.
- Community-acquired pneumonia (CAP) involves significant inflammatory responses in the lungs.
Purpose of the Study:
- To characterize the pro-inflammatory cytokine production of AM in patients with severe CAP.
- To evaluate the gene expression and protein release of TNF-alpha, IL-6, and IL-8 by AM from CAP patients and healthy controls.
Main Methods:
- Bronchoalveolar lavage (BAL) was performed on CAP patients and healthy volunteers.
- High-purity AM were isolated using fluorescence-activated cell sorting.
- Cytokine gene expression (RT-PCR) and protein release (ELISA) were measured ex vivo and in vitro after stimulation with LPS and/or IFN-gamma.
Main Results:
- AM from CAP patients had elevated IL-8 mRNA but not TNF-alpha or IL-6 mRNA compared to controls.
- In vitro, CAP-derived AM showed reduced TNF-alpha and IL-6 release upon LPS stimulation, while IL-8 release was comparable.
- Interferon-gamma stimulation restored TNF-alpha and IL-6 release in CAP-derived AM.
Conclusions:
- Alveolar macrophages in CAP patients exhibit a diminished capacity for TNF-alpha and IL-6 release, despite increased IL-8 mRNA.
- The impaired cytokine response of AM in CAP can be modulated by interferon-gamma.
- Findings highlight the complex role of AM in acute lung inflammation and potential therapeutic targets.