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Increased expression of functionally active membrane-associated tumor necrosis factor in acute respiratory distress
L Armstrong1, D R Thickett, S J Christie
1Lung Research Group, University of Bristol Department of Hospital Medicine, Division of Medicine, Bristol, United Kingdom. Lynne.Armstrong@bristol.ac.uk
Abstract:
Membrane-associated tumor necrosis factor (mTNF) has recently been shown to induce inflammatory cellular responses previously attributed to the soluble form. The present study measures for the first time the expression and function of mTNF on the surface of alveolar macrophages (AMs) to determine whether it is associated with the development of acute respiratory distress syndrome (ARDS). TNF expression was determined by flow cytometry, and the function of mTNF on the surface of AMs was determined by an in vitro cytotoxicity assay. Tumor necrosis factor (TNF)-alpha bioactivity was measured by bioassay. Soluble TNF receptor (TNFR) protein and messenger RNA (mRNA) expression were measured by enzyme-linked immunosorbent assay and reverse transcriptase/polymerase chain reaction, respectively. Increased detection of mTNF was observed on the surface of AMs derived from subjects with ARDS (mean percentage increase in fluorescence 22.30 +/- 3.50% for subjects with ARDS compared with 7.09 +/- 1.70% for At Risk subjects [P < 0.003]). mTNF cytotoxicity in the bioassay positively correlated with the mTNF expression determined by flow cytometry (r(2) = 0.97). Although there was increased mTNF expression and cytotoxic function in ARDS, there was no significant increase in soluble TNF expression in the bronchoalveolar lavage fluid or the AM supernatants. Lower levels of CD120b-soluble TNFR were detected in the AM supernatants derived from subjects with ARDS compared with At Risk (mean 0.264 +/- 0.058 versus 0.593 +/- 0.143 ng/ml, respectively [P < 0.05]). By contrast, there was increased CD120b mRNA expression in AMs derived from subjects with ARDS (P < 0.03), suggesting that increased surface expression of this receptor may be important in mediating the signal of mTNF. These data demonstrate for the first time the presence of functionally active mTNF on the surface of AMs in ARDS and highlight a potential mechanism for TNF-mediated lung injury.
Insights
Membrane-associated tumor necrosis factor (mTNF) on alveolar macrophages is elevated in acute respiratory distress syndrome (ARDS). This functionally active mTNF may drive lung injury, independent of soluble TNF.
Area of Science:
- Pulmonary Medicine
- Immunology
- Cell Biology
Background:
- Membrane-associated tumor necrosis factor (mTNF) plays a role in inflammatory responses.
- The specific role of mTNF on alveolar macrophages (AMs) in acute respiratory distress syndrome (ARDS) remains unclear.
Purpose of the Study:
- To investigate the expression and function of mTNF on AMs in ARDS patients.
- To determine the association between mTNF on AMs and ARDS development.
Main Methods:
- Flow cytometry to measure mTNF expression on AMs.
- In vitro cytotoxicity assays to assess mTNF function.
- Bioassays for TNF-alpha bioactivity.
- ELISA and RT-PCR for soluble TNF receptor (TNFR) expression.
Main Results:
- Significantly increased mTNF expression on AMs from ARDS subjects compared to At Risk subjects.
- Positive correlation between mTNF expression and cytotoxic function.
- No significant increase in soluble TNF in bronchoalveolar lavage fluid or AM supernatants.
- Decreased soluble TNFR protein but increased TNFR mRNA in ARDS subjects' AMs.
Conclusions:
- Functionally active mTNF is present on AMs in ARDS.
- mTNF on AMs may contribute to TNF-mediated lung injury in ARDS.
- Increased surface TNFR expression might mediate mTNF signaling in ARDS.