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

David M. Steinhorn, Frank B. Cerra

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 μg/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.

Original languageEnglish (US)
Pages (from-to)121-127
Number of pages7
JournalCritical Care Medicine
Volume25
Issue number1
DOIs
StatePublished - Jan 1997
Externally publishedYes

Fingerprint

Coculture Techniques
Lipopolysaccharides
Hepatocytes
Interleukin-1
Interleukin-6
Cell Culture Techniques
Tumor Necrosis Factor-alpha
Hospital Laboratories
Proteins

Keywords

  • cell culture
  • cytokine
  • development
  • hepatocyte
  • Kupffer cells
  • multiple organ dysfunction syndrome

ASJC Scopus subject areas

  • Critical Care and Intensive Care Medicine

Cite this

Comparative effects of lipopolysaccharide on newborn versus adult rat hepatocyte and nonparenchymal cell cocultures. / Steinhorn, David M.; Cerra, Frank B.

In: Critical Care Medicine, Vol. 25, No. 1, 01.1997, p. 121-127.

Research output: Contribution to journalArticle

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AU - Cerra, Frank B.

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N2 - 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 μg/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.

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