Suppression of Cox-2 and TNF-alpha mRNA in endotoxin tolerance: Effect of cycloheximide, antinomycin D, and oakadaic acid

Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce de creased levels of inflammatory mediators such as eicosanoids and tumor necr osis factor alpha (TNF alpha) in response to LPS. We hypothesized that tran scriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cel ls were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1 mug/mL) a nd subsequently challenged with LPS (10 mug/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox )-2 and TNF alpha mRNA was suppressed. Cycloheximide (10 muM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNF alpha mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant ce lls but destabilized TNFa: mRNA expression in LPS-tolerant cells. The obser vation that in naive cells Cox-2 and TNF alpha mRNA levels subside at 3 to 4 h after LPS (10 mug/mL or 1 mug/mL) suggested that LPS tolerance may occu r earlier. Therefore, in subsequent experiments, the effect of LPS pretreat ment for only 3.5 h was examined. This abbreviated tolerance regimen dimini shed secondary LPS-induced Cox-e mRNA expression but had a lesser effect on TNF alpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNF alpha mRNA expression in this group. Also, the serine/threonine phosph atase inhibitor okadaic acid augmented Cox-2 and TNFa mRNA expression in th e LPS-tolerant cells. Although LPS-induced TNF alpha production in LPS-tole rant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNF alpha in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of pro teins that alter expression of certain genes. Expression of Cox-e mRNA appe ars to be particularly sensitive to down-regulation and, to a lesser extent , TNF alpha mRNA. However, this seems to vary depending on the LPS pretreat ment regimen. The ability of a phosphatase inhibitor to induce TNF alpha an d expression of Cox-2 and TNF alpha mRNA in LPS tolerance suggests that the re may be alterations in phosphorylation status of signaling pathways, tran scriptional mechanisms, or post-transcriptional mRNA stability.