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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)-α 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 (r2 = 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.