Augmented tumor necrosis factor response to lipopolysaccharide after thermal injury is regulated posttranscriptionally.
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.


METHODS
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.
Department of Surgery, University of Texas, Southwestern Medical Center, Dallas.
J P
JP
Minei
J G
JG
Williams
S J
SJ
Hill
K
K
McIntyre
P E
PE
Bankey
D000818:Animals; D002056:Burns / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D016676:Macrophages, Alveolar / Q000276:immunology; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha