Expression and Regulation of Tumor Necrosis Factor in Macrophages from Cystic Fibrosis Patients

Cachexia and anorexia commonly occur in patients with cystic fibrosis (CF), particularly those with severe pulmonary compromise and heavy tracheobronchial colonization with Pseudomonas aeruginosa. Current understanding of the pathophysiology of cachexia attributes much of the anorexia and weight loss to the effects of the cytokine tumor necrosis factor (TNF), which is secreted by endotoxin-stimulated macrophages. It has further been suggested that TNF may play a role in the pathobiochemistry of CF cachexia, secondary to the localized inflammatory response in the lung or wider systemic activation of cells of the monocyte-macrophage series in response to endotoxin. This study investigates TNF production and gene expression by peripheral blood monocyte-derived macrophages from CF patients, compared with normals (NL). The results indicate that although both cell populations responded dose-dependently to lipopolysaccharide (LPS); CF macrophages, upon stimulation with LPS at concentrations of 1 to 1,000 ng/ml, consistently produced substantially higher amounts of TNF than NL macrophages. At the molecular level, Northern blot analysis also revealed that both macrophage populations expressed TNF mRNA in response to LPS in a dose-dependent manner. However, at the same LPS concentrations, CF macrophage TNF mRNA expression was 2- to 4-fold greater than that of NL macrophages. LPS had no effect in either macrophage population on mRNA for CHO-B, a constitutive probe. To investigate differences between NL and CF macrophage TNF regulation, nuclear run-on/half-life studies as well as studies addressing potential differences in LPS membrane interactions and signal transduction were performed. Nuclear run-on assays demonstrated a 180% greater transcription rate in CF, compared with NL macrophages, in the presence of LPS, with half-life studies showing comparable levels of CF and ML TNF mRNA stability. Tolerance studies and measurement of serum factors, including endotoxin, interferon-γ, and LPS binding protein, did not reveal any differences between the two populations to explain the enhanced CF macrophage TNF response to LPS. However, although LPS-induced macrophage TNF production was equally suppressed in both populations by dexamethasone, CF macrophage TNF production was clearly less inhibited than NL macrophage TNF production in the presence of amiloride. Taken together, these results provide evidence that TNF is produced in excess by LPS-stimulated CF peripheral blood monocyte-derived macrophages, compared with NL macrophages similarly derived and stimulated. This may be due in part to an increased rate of TNF gene transcription by CF macrophages, which may be related to a CF macrophage amiloride-sensitive alteration in LPS signal transduction.