Dominant negative down-regulation of endotoxin-induced tumor necrosis factor alpha production by Lps(d)/Ran.

We recently showed that adenoviral transfer and expression of the Lps(d)/Ran gene isolated from endotoxin-resistant C3H/HeJ mice could protect endotoxin-sensitive mice from endotoxic shock. Elevation of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), is thought to be essential for the development of septic shock. To investigate the extent to which Lps(d)/Ran affects TNF-alpha production, we transduced primary macrophages from endotoxin-sensitive and -resistant mice with adenoviral vectors expressing the wild-type and the mutant Lps/Ran cDNAs and other control genes, and compared the amount of TNF-alpha produced by these various transduced macrophages. Successful transfer and expression of Lps(d)/Ran cDNA in endotoxin-sensitive C3H/HeOuJ macrophages reduced TNF-alpha production upon lipopolysaccharide (LPS) stimulation, as compared with macrophages transduced with vectors expressing the wild-type Lps(n)/Ran cDNA, the green fluorescent protein gene, or the lacZ gene. On the other hand, successful transfer and expression of the wild-type Lps(n)/Ran cDNA in primary macrophages from endotoxin-resistant C3H/HeJ mice failed to induce TNF-alpha production to any significant extent unless a very high LPS concentration was used. Given our previous demonstration that Lps(n)/Ran functions effectively in restoring LPS responsiveness in B cells from C3H/HeJ mice, we conclude that Lps/Ran is involved in a CD14-independent signal transduction pathway. This dominant negative down-regulation by Lps(d)/Ran on TNF-alpha production by macrophages and probably other innate immune responses may be key to the development of an effective gene therapy for endotoxic or septic shock.