Augmented Tumor Necrosis Factor Response to Lipopolysaccharide After Thermal Injury Is Regulated Posttranscriptionally

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Abstract
Background and Objective: Thermal injury has been shown to enhance macrophage sensitivity to lipopolysaccharide (LPS), resulting in augmented tumor necrosis factor α (TNF-α) production. This study was designed to examine whether enhanced TNF-α response after thermal injury and LPS stimulation is regulated at the level of transcription. Design: Tumor necrosis factor α release in alveolar macrophages harvested 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 μg/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 and probed for TNF-α messenger RNA (mRNA), using nuclease protection analysis. Finally, pooled alveolar macrophages were harvested 3 days following sham or thermal injury and cultured in the presence or absence of LPS (10 μg/mL) for 4 hours. The RNA from the pooled alveolar macrophages was extracted and probed for TNF-α mRNA levels. Results: Thermal injury alone did not significantly increase alveolar macrophage TNF-α bioactivity, whole-lung TNF-α mRNA levels, or pooled alveolar macrophages TNF-α 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 μg/mL) compared with alveolar macrophages from sham-injured animals undergoing similar LPS treatment (2365±1011) vs 169±79 ng/mL; P<.05). Whole-lung mRNA levels in both sham-injured and PBD-3 rats receiving intraperitoneal LPS, while elevated approximately 2.5-fold from those of non-LPS treated rats, were not different from each other. Finally, pooled alveolar macrophages from sham-injured and PBD-3 rats cultured in the presence of LPS had approximately 1.7-fold and threefold increased TNF-α mRNA levels, respectively, compared with alveolar macrophages not cultured with LPS. Conclusions: Thermal injury induces priming of alveolar macrophages, resulting in significant increases in macrophage TNF-α production after exposure to LPS. The majority of this effect appears to be regulated at a post-transcriptional level, since there were only moderate increases in TNF-α mRNA levels after LPS stimulation, which did not coincide with large differences in bioactivity. (Arch Surg. 1994;129:1198-1203)