Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration.

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.

Overexpression of prothymosin alpha, concomitant with c-myc, during rat hepatic carcinogenesis.

By using a subtraction-enhanced display technique, we identified a cDNA clone representing alpha-prothymosin, from rat hepatocellular carcinoma (HCC). alpha-Prothymosin has been reported to be involved in cell proliferation and regulated by c-myc gene in vitro. In the present study, we investigated the gene expression pattern of alpha-prothymosin and analyzed its correlation with c-myc during rat hepatic carcinogenesis and liver regeneration. Hepatic alpha-prothymosin mRNA levels, concomitant with c-myc, were increased at the early stage of hepatic carcinogenesis (6 weeks), and remained nearly 10 fold higher as the tumor progressed. In comparison, alpha-prothymosin mRNA levels were only slightly and moderately increased at early (3-6 hr) and at later stage (24-30 hr) of liver regeneration after 70% partial hepatectomy. In situ hybridization revealed that overexpressed alpha-prothymosin mRNA was restricted to the tumor nodules and to tumor cells invading blood vessels. These data provide evidence that overexpression of alpha-prothymosin, concomitant with c-myc, is related to rat hepatic carcinogenesis.

Hepatic amino acid and protein metabolism in non-anorectic, moderately cachectic tumor-bearing rats.

BACKGROUND/AIMS: Cancer cachexia is characterized by loss of lean body mass. Under this condition peripheral proteins are broken down and transferred to visceral organs and the tumor. The liver is the principal organ in the regulation of protein and amino acid metabolism, but liver amino acid kinetics in cancer are unclear. Therefore, we examined the effects of increasing tumor loads on hepatic protein turnover and amino acid handling. METHODS: A MCA-induced sarcoma was implanted subcutaneously in Lewis rats (200-225 g). Rats were studied when the tumor was 5-15% or 15-30% of body weight. Control rats were sham implanted. Under anesthesia, a primed constant infusion of para-aminohippuric acid and L-[3, 4-3H]-valine was given to calculate hepatic substrate fluxes and protein turnover. Serum alpha 2-macroglobulin concentration was measured to determine the acute phase response. RESULTS: Carcass weight decreased approximately 10% in large-tumor-bearing rats (p < 0.001). Liver wet weight increased from 5.5 +/- 0.1 (g) to 5.9 +/- 0.2 in the small-tumor-bearing group and 7.3 +/- 0.3 (p < 0.001) in the large-tumor-bearing group, with minimal changes in water content. Serum alpha 2-macroglobulin concentration, essential and gluconeogenic amino acid uptake by the liver increased in large-tumor-bearing animals. This contrasted with reduced liver ammonia uptake and unchanged urea production in tumor-bearing rats. In the small-tumor-bearing group liver protein synthesis increased, whereas protein breakdown remained unchanged. In the large-tumor-bearing group protein synthesis also increased, but protein breakdown decreased to zero. CONCLUSIONS: The study shows that in tumor-bearing rats, liver uptake of essential and gluconeogenic amino acids increases without significant increases in urea or glucose production. Synthesis of both structural and export proteins, e.g. acute phase proteins, increases suggesting that the liver becomes a more efficient nitrogen-sparing and active protein-synthesizing organ during the growth of a malignant tumor.

Blockade of the alpha 2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein on rat liver parenchymal cells by the 39-kDa receptor-associated protein leaves the interaction of beta-migrating very-low-density lipoprotein with the lipoprotein remnant receptor unaffected.

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and beta-migrating very-low-density lipoprotein (beta-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated alpha 2-macroglobulin and beta-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated alpha 2-macroglobulin (alpha 2M-T) was cleared rapidly by the liver (maximal uptake of 80.8 +/- 1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)/kg rat reduced the liver uptake to 62.2 +/- 2.3%, 59.3 +/- 1.1%, or 2.9 +/- 0.1% of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of 125I-beta-VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I-alpha 2M-T was 98% inhibited by GST-RAP with an IC50 of 0.3 microgram/ml (4.2 nM), whereas the binding of 125I-beta-VLDL and 125I-beta-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 micrograms/ml (700 nM). Also, the cell association and degradation of alpha 2M-T was blocked by RAP, while the association and degradation of beta-VLDL and beta-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of alpha 2M-T lasted for 1-2 h of incubation at 37 degrees C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (the alpha 2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of beta-VLDL by rat liver parenchymal cells is not mediated by the alpha 2Mr/LRP. The properties of binding of beta-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the alpha 2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.

Localization of alpha 2-macroglobulin protein and messenger RNA in rat liver fibrosis: evidence for the synthesis of alpha 2-macroglobulin within Schistosoma mansoni egg granulomas.

alpha 2-Macroglobulin (alpha 2M) in the rat is a strong-reacting acute-phase protein with potent protease-inhibiting and cytokine-binding properties. Production of alpha 2M is ascribed mainly to liver parenchymal cells. In the present study, we investigated, by means of immunohistochemistry and in situ hybridization, whether fibrosis in the rat liver induced by Schistosoma mansoni eggs leads to local production of alpha 2M. alpha 2M protein and messenger RNA (mRNA) in the unaffected liver tissue, as well as serum values of alpha 2M, were comparable in control rats and egg-injected rats, at 1, 3, and 8 weeks after injection of the eggs. alpha 2M was homogeneously distributed across the liver lobule. In contrast, at the sites of the granulomas, a strong increase in alpha 2M was observed. alpha 2M mRNA was expressed by granuloma cells, but not by the surrounding liver parenchymal cells. Within the granulomas, alpha 2M protein was present in numerous spindle-shaped cells and was diffusely distributed in the extra-cellular matrix. Using double-staining techniques, a subpopulation of the alpha 2M-positive cells in the granulomas appeared to be desmin-positive, suggesting a myofibroblast origin. In addition, parenchymal cells directly surrounding the granulomas contained alpha 2M protein in approximately 50% of the granulomas 1 week after injection of the eggs. In situ hybridization on consecutive sections revealed that these parenchymal cells showed only background activity of alpha 2M mRNA, suggesting uptake of alpha 2M-protein by these parenchymal cells and previous activation of alpha 2M by proteases within the granuloma. The significance of the present study is that alpha 2M is produced locally at sites of inflammation and liver fibrosis, without measurable increase of serum levels of alpha 2M. Unexpectedly, alpha 2M present at the sites of the granulomas is not produced by the liver parenchymal cells, but rather by granuloma cells.

alpha2-Macroglobulin is mainly produced by cancer cells and not by hepatocytes in rats with colon carcinoma metastases in liver.

Localization and production of alpha2-macroglobulin (alpha2M), a multifunctional binding protein with protease and cytokine scavenging properties, was studied in situ in rat livers containing experimentally induced colon carcinoma metastases by means of immunocytochemistry and in situ hybridization methods. The study was performed to investigate whether alpha2M production by hepatocytes plays a role in the defense against the growth of metastases on the basis of its protease inhibiting capacity. It was found that colon cancer cells in all developmental stages of the metastases contained large amounts of messenger RNA (mRNA) of alpha2M but hardly any alpha2M protein. Cancer cells in culture contained large amounts of both mRNA and protein of alpha2M. In contrast, stromal cells and liver cells did not show positivity for alpha2M mRNA above background levels. The exception was a few layers of hepatocytes around the latest stage of metastases. Hepatocytes contained both alpha2M mRNA and protein only when Kupffer cells were present, indicating that alpha2M mRNA production was induced via Kupffer cells. On the other hand, alpha2M protein was found in high amounts in the sinusoids and stroma of all metastases, irrespective of their developmental stage. Increased levels of alpha2M could not be detected in serum in all but one rat tested (n=8). It is concluded that production of alpha2M by hepatocytes occurs only around the latest developmental stage of metastases and that alpha2M does not play a significant role in the defense against metastatic cancer growth in rat liver. In contrast, cancer cells produce and secrete large amounts of alpha2M, which seems to be linked with their tumorigenicity. We suggest that this alpha2M captures cytokines rather than proteases by complex formation. These complexes were observed using immunocytochemical staining for alpha2M protein indicating that it was captured by either stromal cells, sinusoidal cells, or hepatocytes that are in direct contact with cancer cells, Therefore, changes in serum levels of alpha2M were limited, indicating that these levels do not reflect local production and effects of alpha2M.

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.

Susceptibility to Plasmodium berghei infection in rats is modulated by the acute phase response.

Brown Norway (BN) and Sprague Dawley (SD) rats are known to differ in their susceptibility to infection with sporozoites of Plasmodium berghei, as measured by the density of liver schizonts. Because of the known inhibitory effect of non-specific immunomodulators on schizont development, we compared some aspects of the acute phase response in these two rat strains. LPS induced IL-6 production was measured in supernatants of spleen cells and peritoneal macrophages of both strains. SD rats, which are the least susceptible to P. berghei sporozoites, showed significantly higher IL-6 production by macrophages from both sources. When LPS was administered in vivo, SD rats also had a significantly higher IL-6 response. Hepatocytes from both strains were cultured in the presence of IL-6. After three days of culture, alpha 2-Macroglobulin concentrations in the supernatants of SD hepatocytes were much higher than those from BN rats. Kupffer cell depletion in both BN and SD rats was correlated with a significant increase in liver schizont density, but did not abrogate the difference in susceptibility. From these results we conclude that the higher cytokine production capacity of SD rats compared to BN rats, may contribute to the difference in susceptibility to P. berghei sporozoites between these strains, but that other yet unknown factors are also involved.

Different zonal distribution of the asialoglycoprotein receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein and the lipoprotein-remnant receptor of rat liver parenchymal cells.

Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

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.

Effects of monocytic products, recombinant interleukin-1, and recombinant interleukin-6 on glycosylation of alpha 1-acid glycoprotein: studies with primary human hepatocyte cultures and rats.

Changes in the carbohydrate moieties of acute-phase glycoproteins (APGPs) often accompany the increase in their secretion by the liver during inflammation. In this study, we investigated whether factors known to regulate APGP gene expression are also involved in the altered glycosylation. For this purpose, the glycosylation pattern of alpha 1-acid glycoprotein (AGP) as secreted by human hepatocytes, cultured in the presence and absence of dexamethasone and monokines, was studied by crossed affino- (concanavalin A) immunoelectrophoresis (CAIE). The monokines rIL-1 and rIL-6, in the presence of dexamethasone, both stimulated AGP secretion and caused a change in glycosylation towards an increased Con A reactivity, including the appearance of two strongly reactive forms (D and E) normally not present. Dexamethasone alone did not influence either process. When tested in vivo in rats, rIL-6 also induced an increased presence of Con A-reactive forms of AGP in serum. In conclusion, the changes in secretion and glycosylation of AGP as seen during inflammation seem to be mediated by the same factor(s).

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 effects of alpha M-foetoprotein, an acute phase protein, and BaSO4-induced injury on IgE-mediated, systemic anaphylaxis in the rat.

A recently developed method for inducing fatal, IgE-mediated, bronchial and cardiovascular anaphylaxis in the rat was used to compare the effects of exogenously administered, purified alpha M-foetoprotein (alpha M FP) and BaSO4 pretreatment (as mean to induce an acute phase reaction with increased alpha M FP serum levels) with regard to mortality, bronchoconstriction and cardiovascular events. The BaSO4 pretreatment protected the rats almost completely against mortality, whereas exogenously administered alpha M FP offered no protection at all. With respect to the antigen-induced bronchoconstriction alpha M FP greatly inhibited the increase of the pulmonary resistance (RI), whereas the BaSO4 pretreatment suppressed either the dynamic lung compliance (Cdyn) or RI considerably. The cardiovascular events were only influenced by the BaSO4 pretreatment demonstrating a small but highly significant reduction of the initial fall in blood pressure together with a remarkable recovery within almost I h in the majority (91%) of the animals. Both exogenously administered alpha M FP and BaSO4 pretreatment increased the alpha M FP serum levels from a normal value of 59 +/- 4 micrograms/ml (n = 22), to 2732 +/- 252 micrograms/ml (n = 9) and 855 +/- 200 micrograms/ml (n = 22), respectively. From these data we conclude that the antianaphylactic activity of alpha M FP is limited to bronchoprotection of the more central parts of the lungs, whereas BaSO4 pretreatment covers a much broader antianaphylactic profile. This implies that BaSO4 pretreatment does not only induce alpha M FP but also other endogenous antianaphylactic factors.