The traditional two-step EGTA/collagenase method is widely used in studying nitric oxide (NO) production in hepatocytes. The present study first revealed that hepatocytes isolated by this method spontaneously express an iNOS mRNA. Thereafter, based on this novel finding, we characterized the expression and regulation of the gene in primary cultured hepatocytes. Using Northern blot analysis, the iNOS mRNA was observed 4 h after isolation, reached peak at 8 h, and declined to an undetectable level after 24 h. iNOS gene expression was shown to be serum-independent and not due to lipopolysaccharide contamination. Time-course analysis of the effects of actinomycin D demonstrated that the increase in iNOS transcripts is the result of an accompanying great increase in iNOS gene transcription and lower iNOS mRNA stability; also blockage by cycloheximide suggests that it is dependent on de novo protein synthesis. Inhibition by pyrrolidine dithiocarbamate, a NF-kappaB/c-rel inhibitor, further implies the involvement of NF-kappaB/c-rel. To clarify reason(s) for the induction, hepatocytes were isolated with the collagenase buffer perfusion step omitted. As a consequence, iNOS mRNA was undetectable in the hepatocytes. These findings show that the traditional hepatocyte-isolation culture does indeed transiently express a serum-independent but de novo protein synthesis-dependent iNOS mRNA due to collagenase (type IV) buffer perfusion.
cell line (GRX cell line), obtained from mouse liver infected A novel mouse Ito (fat-storing) cell line (A640-IS) was eswith Schistosoma, 1,2 rat fat-storing cell lines obtained from tablished by transformation with a temperature-sensitive munormal and CCl 4 -treated rat liver, 3 and a human cell line tant of simian virus 40 (SV40) and the relationships between obtained from a hepatic mesenchymal tumor. 4 However, as the expression of SV40 large T antigen and the growth, differfar as we know, no Ito cell line obtained from normal mouse entiation, and functions of A640-IS cells were investigated.liver has been established. Moreover, in general, native cell A640-IS cells expressed large T antigen when cultured at functions are often lost after cell immortalization. To resolve 33ЊC. At this temperature, the cells grew actively, assumed a these problems, establishment of cell lines with both profibroblastic shape, and showed few Ito cell characteristics. In longed growth potential and persistent native cell functions contrast, when large T-antigen expression was inhibited by is required. culture at 39ЊC, the cells did not grow but differentiated intoThe large T antigen of simian virus 40 (SV40), an oncoIto cells as assessed by both morphological and functional genic virus, plays an important role in the immortalization characteristics. Expression of the transcription factor SCL of cells. 5,6 Use of a temperature-sensitive mutant of SV40 (stem cell leukemia), which plays a role in the development (tsA640) allows the expression of the large T antigen to be and differentiation of blood cells, was observed at both 33ЊC controlled by changes in temperature. [7][8][9] In this study, a and 39ЊC, although expression was greater at 33ЊC. Therefore, altering the temperature. This is a very useful feature of the temperature-sensitive mutant of SV40 because it allows the The Ito cell (fat-storing cell, lipocyte, and perisinusoidal growth and differentiation of these cells to be regulated. In stellate cell) is one of the perisinusoidal cells in the liver addition, this novel Ito cell line retains the characteristics of and has many unique characteristics, including the ability the parent cells, in contrast to ordinary primary Ito cell culto synthesize extracellular matrix (ECM) proteins and store tures in which the original Ito cell nature is lost with time. vitamin A. However, little is known about the differentiation and functions of Ito cells at present. In general, a homoge- MATERIALS AND METHODSneous cell group is necessary for the examination of cells. However, in ordinary primary culture, Ito cells form a heteroEstablishment of Cell Lines. Sinusoidal cell fractions were obtained using collagenase and pronase-E digestion according to the method geneous cell group. To the best of our knowledge, only four of Knook et al. and Tsutsumi et al. 10,11 with minor modifications.Ito cell lines originating from pathological liver tissues have Care of the animals followed the appropriate guidelines. Under been established to date. The...
Chemokines are a superfamily of structurally related chemoattractant cytokines. JE (monocyte chemoattractant protein-1) and IP-10 (interferon-inducible protein-10) have been detected in the diseased liver. However the in vitro expression is unclear. In this report, we revealed that JE, KC (melanoma growth-stimulating activity gene), and IP-10 mRNAs are not expressed in the normal liver but spontaneously and time-dependently expressed in the primary hepatocytes. The serum-independent gene expression of both JE and KC lasted over 72 h, but that of IP-10 became undetectable 24 h after isolation with collagenase perfusion method. The induction of the genes' expression was not due to LPS contamination but nevertheless was associated with isolation procedure. Actinomycin D blocked their expression. The increase of their transcripts resulted from greater increase in gene transcription and lower mRNA stability. Consistent with c-jun, their mRNA expressions were simultaneously superinduced by cycloheximide (1 microg/ml), suggesting that de novo protein synthesis is involved their transcriptions. Inhibition by pyrrolidine dithiocarbamate (PDTC), a NF-kappaB/c-rel inhibitor, and EMSA imply that NF-kappaB/c-rel is important in their expressions. Of particular interest is that dexamethasone upregulated the spontaneous expression of KC, but suppressed that of JE and IP-10. LPS upregulated the mRNA levels of JE and KC but did not affect that of IP-10. IFN-gamma induced the expression of IP-10; however unlike in macrophages, it did not selectively inhibit that of JE and KC. Our data demonstrated the existence and differential gene expression of JE, KC, and IP-10 in primary cultured hepatocytes, and these are considered to be a reflex of the alteration of hepatocyte cellular physiology during and after isolation.
During infection with plerocercoids of Spirometra erinacei, organisms in the peritoneal cavity of infected animals have many bound inflammatory leukocytes yet survive apparently unharmed. Coculture of IFN gamma and LPS stimulated mouse peritoneal macrophages with live plerocercoids suppressed the mRNA expression of the inducible isoform of nitric oxide synthase (iNOS) and JE, the murine homologue of monocyte chemotactic protein-1 (MCP-1). Excretory/secretory (ES) products from plerocercoids also suppressed the induced iNOS and JE mRNA and reduced nitrite production of macrophages in a dose dependent manner. The suppression of inducible mRNA levels in macrophages cultured for 24 h with ES products varied with the nature of the stimuli; IFN gamma/ LPS-induced iNOS mRNA levels were effected less than were iNOS mRNA levels induced by IFN gamma/IL-2 or IFN gamma/ TNF alpha. Similar findings were obtained when nitrite production was measured. Thus modulation of LPS and cytokine inducible mRNA levels appear to be the primary target of ES products. We speculate that a major physiological role for this inhibitory activity in ES products might be the down regulation of pro-inflammatory gene expression.
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