CD14 IS NOT INVOLVED IN RHODOBACTER-SPHAEROIDES DIPHOSPHORYL LIPID A INHIBITION OF TUMOR-NECROSIS-FACTOR-ALPHA AND NITRIC-OXIDE INDUCTION BY TAXOL IN MURINE MACROPHAGES

Taxol, a microtubule stabilizer with anticancer activity, mimics the a ctions of lipopolysaccharide (LPS) on murine macrophages in vitro. Rec ently, it was shown that taxol-induced macrophage activation was inhib ited by the LPS antagonist Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA). To investigate the mechanisms of taxol-induced macrophage activation, the present study focused on the interaction of LPS, RsDPL A, and taxol in the activation of and binding to macrophages. Taxol al one induced murine C3H/He macrophages to secrete tumor necrosis factor alpha (TNF) and to produce nitric oxide (NO) with kinetics similar to that of LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, in con trast, did not yield any detectable TNF and NO production in response to LPS or taxol. RsDPLA inhibited taxol-induced TNF and NO production from C3H/He macrophages in a dose-dependent manner. The inhibition by RsDPLA was specific for LPS and taxol in that RsDPLA did not inhibit h eat-killed Listeria monocytogenes- or zymosan-induced TNF production. Polymyxin B blocked the inhibitory effect of RsDPLA on taxol-induced T NF production. The inhibitory activity of RsDPLA appeared to be revers ible since macrophages still responded to taxol in inducing TNF produc tion after the RsDPLA was washed out with phosphate-buffered saline pr ior to the addition of taxol. Taxol-induced TNF production was not inh ibited by colchicine, vinblastine, or 10-deacetylbaccatine III. A muta nt cell line, J7.DEF3, defective in expression of a CD14 antigen, resp onded equally well to taxol by producing TNF as did the parent J774.1 cells. This suggested that the activation of macrophages by taxol does not require CD14. Taxol-induced TNF production by the mutant cells wa s also inhibited by RsDPLA. I-125-labeled LPS and H-3-labeled taxol wa s reported to bind to J774.1 cells predominantly via CD14 and microtub ules, respectively. The binding of I-125-labeled LPS to J7.DEF3 cells was about 30 to 40% of that to J774.1 cells, The binding of I-125-LPS to J774.1 cells was inhibited by unlabeled LPS and RsDPLA but not by t axol. On the other hand, H-3-labeled taxol bound to both J774.1 cells and J7.DEF3 cells in similar time- and dose-dependent manners, The bin ding of [H-3] taxol to these cells was inhibited by taxol but not by L PS or RsDPLA. Although the binding studies failed to examine cross com petition for binding to macrophages, a possible explanation of these r esults is that LPS, RsDPLA, and taxol share the same molecule(s) on mu rine macrophages for their functional receptor(s), which is neither CD 14 nor tubulin.