Transforming growth factor-beta-induced collagen synthesis by human liver myofibroblasts is inhibited by alpha2-macroglobulin.

BACKGROUND: Transforming growth factor-beta (TGFbeta) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFbeta in different systems has been shown to be influenced by alpha2-macroglobulin (alpha2M), a serum protein with strong protease-scavenging and cytokine-binding properties. AIMS: In the present study, alpha2M derived from normal human plasma has been tested for its ability to modulate the TGFbeta-induced collagen production by human liver fat-storing cells (FSC), which had transformed into alpha-smooth muscle actin-expressing myofibroblasts in culture. METHODS: Alpha2M has been tested after activation with methylamine (alpha2M-Me), an in vitro equivalent of protease activated alpha2M. The binding of 125I-TGFbeta1 to activated forms of alpha2M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of 3H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of alpha2M was studied by means of immunofluorescence. RESULTS: TGFbeta (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFbeta. Alpha2M-Me reduced this TGFbeta-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 microg/ml alpha2M-Me onward. Upon addition of 100 microg/ml alpha2M-Me the effect of TGFbeta was reduced by 60% to 128+/-31% (mean+/-SD) of control values in the absence of TGFbeta. Human liver myofibroblasts endocytosed alpha2M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFbeta-activity by alpha2M-Me may be explained by receptor-mediated clearance of alpha2M-TGFbeta complexes by the cells. CONCLUSIONS: TGFbeta-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated alpha2M. From these results, we hypothesize that alpha2M may have an antifibrogenic effect in vivo by interference with TGFbeta-induced matrix synthesis during liver fibrosis.

Interleukin-6 production by human liver (myo)fibroblasts in culture. Evidence for a regulatory role of LPS, IL-1 beta and TNF alpha.

BACKGROUND/AIMS: Interleukin-6 is a major trigger for the synthesis of acute phase proteins by liver parenchymal cells. Acute phase proteins may contribute to the regulation of liver fibrosis by inhibition of proteases (e.g. collagenase) and by binding of cytokines. Since liver (myo)fibroblasts play an important role in the production of extracellular matrix in fibrotic livers, a study was undertaken into whether these cells are able to synthesize interleukin-6, which would give them the opportunity to contribute to regulation of synthesis of acute phase proteins by neighbouring parenchymal cells. METHODS: In the present study we investigated interleukin-6 production by two cell types obtained from human liver tissue: human fat-storing cells obtained from 5-15% Percoll fractions, which transformed in culture into myofibroblasts co-expressing alpha-smooth muscle actin and vimentin (VA cells) and fibroblasts obtained from 30-40% Percoll fractions which express vimentin only (V vells). Interleukin-6 production was measured in culture media of these cells using an enzyme-linked immunosorbent assay after incubation with lipopolysaccharide, and mediators like interleukin-1 beta, tumor necrosis factor-alpha, transforming growth factor-beta and interferon-gamma, known to be present in elevated concentrations in fibrotic livers. RESULTS: Unstimulated human liver (myo)fibroblasts produced considerable amounts of interleukin-6 (287 ng/mg cellular protein (VA cells), and 54 ng/mg cellular protein (V cells), within 48 h). Biological activity of these high concentrations of interleukin-6 measured in the enzyme-linked immuno-sorbent assay was confirmed in the B9-bioassay for interleukin-6 and by stimulation of alpha 2-macroglobulin production in rat liver parenchymal cell cultures. Lipopolysaccharide, interleukin-1 beta and tumor necrosis factor-alpha were potent stimulators of basal interleukin-6 production by VA and V cells, 1 microgram/ml lipopolysaccharide enhanced basal interleukin-6 production 3-fold within 48 h. 100 U/ml interleukin-1 beta and 1000 U/ml tumor necrosis factor-alpha each stimulated basal interleukin-6 production by VA cells 2-5 fold, whereas V cells were stimulated 10-25 fold. These effects were specific since the stimulation by lipopolysaccharide was completely inhibited by polymyxin B and the enhancing effects of interleukin-1 beta and tumor necrosis factor-alpha were neutralized by specific antibodies. Transforming growth factor-beta and interferon gamma did not influence interleukin-6 synthesis by either cell type in culture. CONCLUSIONS: These results indicate that transformed fat-storing cells (VA cells) and fibroblasts (V cells) may function as a local source of interleukin-6 in the human liver. Since interleukin-6 plays a key role in the regulation of the production of acute phase proteins by liver parenchymal cells, we hypothesize that human liver (myo)fibroblasts may stimulate local production of acute phase proteins in the fibrotic liver, thus contributing to local regulation of inflammatory and fibrogenic reactions.

Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1 beta, IL-4, TGF beta and IFN gamma.

BACKGROUND/AIMS: Different cytokines have been described in fibrotic livers, including interleukin-1, interleukin-4 and interferon gamma, which are capable of regulating collagen production in human skin and lung fibroblasts. METHODS: To investigate possible involvement of interleukin-1, interleukin-4 and interferon gamma in the regulation of collagen production in human liver fibrosis, we studied the effects of these cytokines on collagen synthesis by nonparenchymal human liver cells in vitro. The effects of interleukin-1, interleukin-4 and interferon gamma were compared with the effect of transforming growth factor-beta, a well-known stimulator of collagen synthesis in liver fibrosis. Using a Percoll gradient we isolated two types of fibroblast-like cells from human liver tissue: fat-storing cells, which transformed in culture into myofibroblasts co-expressing vimentin and alpha-smooth muscle actin (VA-cells), and fibroblasts expressing vimentin only (V-cells). Production of collagen was measured in confluent cell cultures by incorporation of 3H-proline into collagenase degradable proteins. RESULTS: The cytokines studied had comparable effects on collagen synthesis in confluent cultures of VA-cells obtained from three different human livers and in confluent cultures of V-cells. Interleukin-1 beta and interleukin-4 enhanced collagen synthesis dose-dependently. 100 U/ml interleukin-1 beta stimulated collagen synthesis up to 174 +/- 25% (mean +/- sd, VA-cells) and 140 +/- 7% (V-cells) of control values. 1000 U/ml interleukin-4 enhanced collagen formation up to 195 +/- 58% (mean +/- sd, VA-cells) and 153 +/- 4% (V-cells) of control values after 48 h. These values were comparable to the stimulatory effects induced by transforming growth factor-beta (235 +/- 33% (mean +/- sd, VA-cells) and 150 +/- 18% of control values (V-cells) after incubation with 10 ng/ml transforming growth factor-beta for 48 h). Interferon gamma reduced both basal (36 +/- 29% (mean +/- sd) of control values in VA-cells, and 59 +/- 9% in V-cells) and transforming growth factor-beta induced collagen synthesis. CONCLUSIONS: These results indicate that in addition to the well-known role of transformed fat-storing cells (VA-cells) in collagen synthesis, fibroblasts (V-cells) may contribute to collagen production in human liver tissue. Moreover, these data demonstrate that in addition to the extensively documented collagen-inducing mediator transforming growth factor-beta, other cytokines present in fibrotic liver tissue like interleukin-1 beta and interleukin-4 may contribute to the enhanced synthesis of collagen, whereas interferon gamma may reduce collagen formation during liver fibrosis in man.

Uptake of methylamine-activated alpha 2-macroglobulin by rat liver.

Serum clearance of alpha 2M-Me or alpha 2M-Tr is rapid and identical. Alpha 2M-Tr is almost exclusively taken up in the liver by the parenchymal cells; the uptake of alpha 2M-Me is equally shared between endothelial and parenchymal cells. Blocking the scavenger receptor on endothelial cells by polyinosinic acid reduces the uptake of alpha 2M-Me to 40% of the control value; under these conditions, alpha 2M-Me is only associated with the parenchymal cells. These results show the following: (1) activation of alpha 2M by methylamine or trypsin is different; (2) the scavenger receptor on endothelial cells functions as a system for the uptake of alpha 2M-Me in addition to the specific alpha 2M receptor on parenchymal cells.

Role of the scavenger receptor in the uptake of methylamine-activated alpha 2-macroglobulin by rat liver.

Alpha 2-Macroglobulin (alpha 2M) requires activation by small nucleophiles (e.g. methylamine; giving alpha 2M-Me) or proteolytic enzymes (e.g. trypsin; giving alpha 2M-Tr) in order to be rapidly removed from the circulation by the liver. Separation of rat liver cells into parenchymal, endothelial and Kupffer cells at 10 min after injection indicates that liver uptake of alpha 2M-Me is shared between parenchymal and endothelial cells, with relative contributions of 51.3% and 48.3% respectively of total liver-associated radioactivity. In contrast, alpha 2M-Tr is almost exclusively taken up by the parenchymal cells (90.1% of liver-associated radioactivity). A preinjection of 5 mg of poly(inosinic acid) decreased liver uptake of alpha 2M-Me to 39.9% of the control value, while it had no effect on liver uptake of alpha 2M-Tr. It appears that poly(inosinic acid) specifically reduces the uptake of alpha 2M-Me in vivo by endothelial cells, leaving uptake by parenchymal cells unaffected. In vitro studies with isolated liver cells indicate that the association of alpha 2M-Me with endothelial cells is 21-fold higher per mg of cell protein than with parenchymal cells. The capacity of endothelial cells to degrade alpha 2M-Me appears to be 46 times higher than that of parenchymal cells. Competition studies show that poly(inosinic acid) or acetylated low-density lipoprotein effectively competes with the association of alpha 2M-Me with endothelial and Kupffer cells, but association with parenchymal cells is unaffected. It is suggested that activation of alpha 2M by methylamine induces a charge distribution on the protein which triggers specific uptake by the scavenger receptor on endothelial cells. It is concluded that the uptake of alpha 2M-Me by the scavenger receptor might function as an additional system for the uptake of activated alpha 2M.

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.

Recognition of chylomicron remnants and beta-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver alpha 2-macroglobulin-recognition site.

The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta-VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor-related protein) which shows properties that are distinct from those of the remnant receptor.

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone.

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.

The microheterogeneity of human liver and serum ferritins measured on minute amounts of ferritin in crude samples.

A method is presented to measure the microheterogeneity of ferritin in micrograms amounts, without purifying the samples extensively. Ferritin-containing samples such as serum and homogenized liver-biopsy specimens were mixed with Sephadex G-75 and ampholines. Isoelectric focussing was performed and the pH gradient in the Sephadex was measured. The Sephadex was divided into predetermined pH ranges and the ferritin eluted from these fractions. Ferritin concentration was measured by an enzyme-linked immunosorbent assay. The method proved to be reproducible. The isoferritin profiles of different human serum and liver tissue samples were quite variable. In most cases serum ferritins focussed between approximately pH 5.5 and pH 4.9 and liver ferritins between approximately pH 5.8 and pH 5.3. We examined whether there was a similarity in the isoferritin patterns of serum and liver of distinct patients. We also studied liver tissue and serum from a patient with haemochromatosis and from a child with iron overload of unknown origin. In the serum of our patients the isoferritin pattern had shifted to lower pI when compared with that found in liver tissue. Only in the case of a patient with transfusion iron overload were basic isoferritins measured in the serum. In this case no liver biopsy specimen was available for comparison.

Influence of liver isoferritin profile on quantitative measurement of total ferritin protein in human liver, with emphasis on iron overload.

Ferritin was isolated from normal human liver and from iron-loaded human liver by gel chromatography and by ultracentrifugation. From each of these ferritin batches several isoferritin fractions were isolated by preparative isoelectric focusing. It was our aim to have at our disposal isoferritin fractions with relatively large pI ranges but distinctly different isoferritin profiles on analytical isoelectric focusing. These fractions were compared for their immunoreactivity. The total protein in the isoferritin fractions was measured by the nitrogen content. The immunoreactivity of the isoferritin fractions was measured as the ratio of the reaction in immunoassays to the nitrogen content of these fractions. We used radial immunodiffusion and enzyme-linked immunoassay to measure immunoreactivity. The immunoreactivity did not change obviously with the isoferritin composition of the isolated fractions. It is concluded that pathological changes in the isoferritin composition that might occur in liver ferritin during iron overload does not significantly influence the quantitative measurement of liver ferritin protein by immunological methods.

On the iron content of human serum ferritin, especially in acute viral hepaptitis and iron overload.

A new method has been developed to measure ferritin iron in human serum. The ferritin is bound to antibodies to ferritin, which are coupled to Sepharose 4-B and separated from the serum by centrifugation. The iron is liberated from the bound ferritin and measured colorimetrically. The detection limit of this method proved to be approx. 15 ng Fe/ml serum. The serum ferritin iron concentration has been compared with the serum ferritin protein concentration. During liver disease with liver cell leakage the mean iron content of serum ferritin proved to be less than the mean iron content in the liver ferritin. The mean iron content in the liver ferritin was 16%, range 7%--32% (n = 8). The mean iron content in serum ferritin from the same patients was 5%, range 0%--14%. Also in two cases of haemosiderosis the serum ferritin iron content was low. It is suggested that ferritin loses part of its iron on passage from the tissue cells to the blood. In some cases of severe liver cell (or other cell) leakage the mean iron content in serum ferritin might be high, because then more or less complete ferritin molecules enter the blood.

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis.

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.