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Comparative effects of lipopolysaccharide on newborn versus adult rat hepatocyte and nonparenchymal cell cocultures.

OBJECTIVE
To examine the effects of development on the response of hepatocytes and nonparenchymal cells to an endotoxin challenge as an in vitro model of organ system dysfunction at differing developmental ages.


DESIGN
In vitro animal cell culture model.


SETTING
University teaching hospital research laboratory.


SUBJECTS
Adult and newborn Sprague-Dawley rats.


INTERVENTIONS
The method of hepatocyte and nonparenchymal cell coculture was utilized and modified to allow evaluation of cells derived from newborns. Cells were isolated from adult rats by standard perfusion technique. Hepatocytes and nonparenchymal cells were isolated from newborn rats by use of identical enzymatic degradation after fine mincing of the organ. Isolated cells were purified by density gradient. The hepatocytes were incubated in standard cell culture plates for 24 hrs before the addition of nonparenchymal cells. Hepatocytes were incubated with similar-age nonparenchymal cells. After an additional 24 hrs, serial log dilutions of lipopolysaccharide were added as a stimulus and the system was cultured an additional 24 hrs. The response of the hepatocytes was assessed by determination of 3H-leucine incorporation in acid-precipitated protein. In addition, the production of tumor necrosis factor, interleukin (IL)-1, and IL-6 by isolated nonparenchymal cells from adult and newborn rats was determined after stimulation with serial log dilutions of lipopolysaccharide by bioassay.


MEASUREMENTS AND MAIN RESULTS
Both adult and newborn hepatocytes cocultivated with nonparenchymal cells demonstrated a comparable and statistically significant dose response to lipopolysaccharide (p < .01). The newborn hepatocytes demonstrated a greater rate of protein synthesis than the adult hepatocytes at all concentrations of lipopolysaccharide. Tumor necrosis factor production by newborn and adult nonparenchymal cells was similar at all lipopolysaccharide doses. IL-1 production demonstrated a positive dose response to lipopolysaccharide in the adult and newborn nonparenchymal cells, with a trend (p = .17) toward greater IL-1 secretion by the adult cells. There were significant differences in IL-6 production by isolated nonparenchymal cells at lipopolysaccharide doses of 0.01, 0.1, and 10 micrograms/mL. While a similar trend was apparent in the cocultured cells, the significance was not apparent, except at the highest lipopolysaccharide dose.


CONCLUSIONS
The dose responses of newborn and adult hepatocytes to nonparenchymal cells stimulated with lipopolysaccharide were similar, although newborn hepatocytes appeared to have an inherently higher rate of protein synthesis compared with adult hepatocytes. Cytokine production was similar in nonparenchymal cells of both ages, although IL-1 production by stimulated newborn nonparenchymal cells appeared to be less than IL-1 production by adult nonparenchymal cells.