Augmented tumor necrosis factor response to lipopolysaccharide after thermal injury is regulated posttranscriptionally
J. P. Minei, J. G. Williams, S. J. Hill, K. McIntyre and P. E. Bankey
Department 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.
