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Augmented tumor necrosis factor response to lipopolysaccharide after thermal injury is regulated
J P Minei1, J G Williams, S J Hill
1Department of Surgery, University of Texas, Southwestern Medical Center, Dallas.
Background And Objective:
Thermal injury has been shown to enhance macrophage sensitivity to lipopolysaccharide (LPS), resulting in augmented tumor necrosis factor alpha (TNF-alpha) production. This study was designed to examine whether enhanced TNF-alpha response after thermal injury and LPS stimulation is regulated at the level of transcription.
Design:
Tumor necrosis factor alpha 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 micrograms/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-alpha 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 micrograms/mL) for 4 hours. The RNA from the pooled alveolar macrophages was extracted and probed for TNF-alpha mRNA levels.
Results:
Thermal injury alone did not significantly increase alveolar macrophage TNF-alpha bioactivity, whole-lung TNF-alpha mRNA levels, or pooled 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 micrograms/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-alpha 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-alpha production after exposure to LPS. The majority of this effect appears to be regulated at a posttranscriptional level, since there were only moderate increases in TNF-alpha mRNA levels after LPS stimulation, which did not coincide with large differences in bioactivity.