Burn injury sensitizes rat Kupffer cells via mechanisms dependent on gut-derived endotoxin

Abstract

Background

Bacterial translocation occurs after thermal injury in association with intestinal barrier loss. Recently, we found that sensitization of Kupffer cells involved gut-derived endotoxin; therefore, the purpose of this work was to study the mechanisms of sensitization of Kupffer cells in burn injury.

Methods

Rats received a 30% body surface area full-thickness steam burn 24 h before experiments. Serum alanine aminotransferase (ALT) was measured to assess liver damage, and plasma endotoxin in the portal vein were measured. Kupffer cells were isolated 24 h after the burn. Intracellular calcium ([Ca²⁺]_i) in Kupffer cells was measured using a microspectrofluorometer with the fluorescent indicator, fura-2, and tumor necrosis factor (TNF)-α was measured by enzyme-linked immunosorbent assay (ELISA).

Results

Lipopolysaccharide (LPS)-induced mortality was increased by burn treatment. This increase was blocked by gadolinium chloride, a Kupffer-cell toxicant. Accordingly, Kupffer cells were involved in this system. The LPS-induced increase of ALT was upregulated by the burn injury. This increase was blocked by pretreatment with antibiotics. Endotoxin levels were increased to almost 300 pg/ml (normal, <20 pg/ml) in the portal veins of rats that received a burn. This increase was blunted by antibiotics. In Kupffer cells isolated from untreated control rats, [Ca²⁺]_i increased to 82 ± 7 nM after the addition of LPS (100 ng/ml). Levels were elevated twofold over control levels in the cells from rats with burn (174 ± 15 nM). In addition, TNF-α production by Kupffer cells isolated from rats with burn was increased fourfold over the based level. Sterilization of the gut with antibiotics completely blocked all effects of the burn on [Ca²⁺]_i and TNF-α release.

Conclusions

Kupffer cells isolated from rats with burn exhibited sensitization to LPS, involving gut-derived endotoxin. It is concluded that burns sensitize Kupffer cells to LPS via mechanisms that are dependent on gut-derived endotoxin.