Search

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.

Betaine, taurine, and inositol participate as osmolytes in liver cell volume homeostasis and interfere with cell function. In this study we investigated whether osmolytes are also released from the intact liver independent of osmolarity changes. In the perfused rat liver, phagocytosis of carbon particles led to a four- to fivefold stimulation of taurine efflux into the effluent perfusate above basal release rates. This taurine release was inhibited by 70-80% by the anion exchange inhibitor DIDS or by pretreatment of the rats with gadolinium chloride. Administration of vasopressin, cAMP, extracellular ATP, and glucagon also increased release of betaine and/or taurine, whereas insulin, extracellular UTP, and adenosine were without effect. In isolated liver cells, it was shown that parenchymal cells and sinusoidal endothelial cells, but not Kupffer cells and hepatic stellate cells, release osmolytes upon hormone stimulation. This may be caused by a lack of hormone receptor expression in these cells, because single-cell fluorescence measurements revealed an increase of intracellular calcium concentration in response to vasopressin and glucagon in parenchymal cells and sinusoidal endothelial cells but not in Kupffer cells and hepatic stellate cells. The data show that Kupffer cells release osmolytes during phagocytosis via DIDS-sensitive anion channels. This mechanism may be used to compensate for the increase in cell volume induced by the ingestion of phagocytosable material. The physiological significance of hormone-induced osmolyte release remains to be evaluated.

Background & Aims: CD95 (Apo-1/Fas) ligand suppresses inflammatory responses in immune-privileged organs. In this study, modulation of the hepatic CD95 receptor/ligand system by interferon gamma and cyclosporin A was investigated., Methods: CD95 receptor and ligand expression were measured at the messenger RNA level by using quantitative reverse-transcription polymerase chain reaction and immunocytochemistry in primary cultures of rat Kupffer cells, hepatocytes, and T lymphocytes. Soluble CD95 in culture supernatants was detected by enzyme-linked immunosorbent assay and apoptosis by the TUNEL method., Results: Interferon gamma treatment led to an increase in CD95 ligand messenger RNA levels in Kupffer cells followed by an overexpression of the soluble CD95 receptor. Supernatants derived from 24-hour but not from 48-hour interferon gamma-treated Kupffer cells killed lymphocytes by a CD95-dependent mechanism. Cyclosporin A inhibited CD95 ligand expression in Kupffer cells and lymphocyte killing. In liver parenchymal cells, interferon gamma increased messenger RNA levels of the transmembrane CD95 isoform and sensitivity of these cells toward CD95-mediated apoptosis., Conclusions: The expression pattern of CD95 receptor and ligand in response to interferon gamma points to a coordinated interplay between Kupffer cells, hepatocytes, and T lymphocytes in which Kupffer cells may regulate programmed cell death of T lymphocytes and hepatocytes.

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of alpha-smooth-muscle actin. In parallel with the accumulation of alpha-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13+/-0.01 micromol/h per mg of cell protein within 7-14 days. This value compares with GS activity in liver parenchymal cells of 0.57+/-0.03 micromol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.