AUGMENTED TUMOR-NECROSIS-FACTOR RESPONSE TO LIPOPOLYSACCHARIDE AFTER THERMAL-INJURY IS REGULATED POSTTRANSCRIPTIONALLY

Background and Objective: Thermal injury has been shown to enhance mac rophage sensitivity to lipopolysaccharide (LPS), resulting in augmente d tumor necrosis factor alpha (TNF-alpha) production. This study was d esigned to examine whether enhanced TNF-alpha response after thermal i njury and LPS stimulation is regulated at the level of transcription. Design: Tumor necrosis factor alpha release in alveolar macrophages ha rvested from sham- or thermal-injured Wistar rats was determined using an L929 cytotoxicity bioassay on days 1, 3, and 5 following 40% scald burn and incubation for 24 hours with LPS (0 or 10 mu g/mL). Separate groups of rats underwent intraperitoneal injection of LPS (5 mg/kg) 3 days following sham or thermal injury. Lung tissue RNA was isolated a nd probed for TNF-alpha messenger RNA (mRNA), using nuclease protectio n analysis. Finally, pooled alveolar macrophages were harvested 3 days following sham or thermal injury and cultured in the presence or abse nce of LPS (10 mu g/mL) for 4 hours. The RNA from the pooled alveolar macrophages was extracted and probed for TNF-alpha mRNA levels. Result s: Thermal injury alone did not significantly increase alveolar macrop hage TNF-alpha bioactivity, whole-lung TNF-alpha mRNA levels, or poole d alveolar macrophages TNF-alpha mRNA levels when compared with levels in sham-injured rats. However, alveolar macrophages from postburn day 3 (PBD 3) demonstrated increased sensitivity to LPS (10 mu g/mL) comp ared with alveolar macrophages from sham-injured animals undergoing si milar LPS treatment (2365+/-1011 vs 169+/-79 ng/mL; P<.05). Whole-lung mRNA levels in both sham-injured and PBD-3 rats receiving intraperito neal LPS, while elevated approximately 2.5-fold from those of non-LPS treated rats, were not different from each other. Finally, pooled alve olar macrophages from sham-injured and PBD-3 rats cultured in the pres ence of LPS had approximately 1.7-fold and threefold increased TNF-alp ha. mRNA levels, respectively, compared with alveolar macrophages not cultured with LPS. Conclusions: Thermal injury induces priming of alve olar macrophages, resulting in significant increases in macrophage TNF -alpha production after exposure to LPS. The majority of this effect a ppears to be regulated at a posttranscriptional level, since there wer e only moderate increases in TNF-alpha mRNA levels after LPS stimulati on, which did not coincide with large differences in bioactivity.