Molecular mechanisms for changes in hepatic protein synthesis induced by schistosomiasis infection in mice

Mice infected with Schistosoma mansoni and littermate controls were evaluated serially for 12 weeks. Infected mice gained weight at the same rate as controls, but starting with the sixth week their livers became enlarged with granulomas and fibrous tissue, and they developed hypoalbuminemia. To evaluate the regulation of the albumin and type I collagen gene expression, total RNA was isolated from infected and control mice and translated in an mRNA-dependent rabbit reticulocyte lysate system. Protein synthesis was decreased 1.5-3-fold with RNA from infected vs. control liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell-free products showed a reduction in albumin but an increase in type I procollagen synthesis in infected mice. Immunoprecipitation of the cell-free product confirmed that albumin synthesis was reduced in greater proportion than other liver proteins in schistosome-infected mice. Hybridization of RNA from infected liver with cloned mouse albumin cDNA (pmalb-2) demonstrated a reduction in albumin mRNA to 37% of control, while hybridization with a chick type I pro alpha 2 collagen cDNA probe (pCg-45) revealed increased procollagen mRNA in infected liver beginning at 6 weeks postinfection. These results suggest that in murine schistosomiasis a reduction in biologically active albumin mRNA results in decreased albumin synthesis and may be responsible in part for hypoalbuminemia. In addition, increased collagen mRNA is associated with increased collagen synthesis during hepatic fibrosis.     

Fibroblast stimulation in schistosomiasis. IX. Schistosomal egg granulomas from congenitally athymic mice are deficient in production of fibrogenic factors

Schistosomal egg granulomas spontaneously secrete fibrogenic factors, suggesting that there exists a molecular link between granulomatous inflammation and hepatic fibrosis in schistosomiasis. To further assess this possibility, we compared elaboration of fibrogenic factors by egg granulomas isolated from Schistosoma mansoni-infected euthymic mice that develop substantial liver fibrosis, with those elaborated by similarly infected congenitally athymic mice that develop minimal fibrosis. Conditioned medium from cultures of granulomas from euthymic mice stimulated fibroblast proliferation, chemotaxis, and synthesis of collagen, collagenase, tissue inhibitor of metalloproteinases, and hyaluronate, whereas those prepared from cultures of granulomas isolated from athymic mice were relatively or absolutely deficient in such activities. These observations provide a correlation between the presence of fibrosis in vivo and the production of fibrogenic factors and reinforce our hypothesis that granuloma-derived fibrogenic factors play a role in the pathogenesis of liver fibrosis in schistosomiasis. Furthermore, the results of this study suggest a central role of T lymphocytes in the fibrogenic process.     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.     

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.     

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.     

Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5''-methylthioadenosine.

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Variability in the intensity of albumin synthesis in mouse hepatocytes

Using immunoenzyme method followed by the cytophotometrical determination of individual protein content in single cells, a comparative analysis was made of albumin contents in hepatocytes of inbred mouse stocks significantly differing in their albumin contents in the blood. Some reasons of heterogeneity of parenchymal liver cells in respect to albumin synthesis were identified. The content of albumin in hepatocytes was shown to correlate with the cell ploidy. A probable contribution of the tissue level to gene expression control is discussed.     

A contribution to the study of acute schistosomiasis (an experimental trial)

In an attempt to establish an experimental model of acute schistosomiasis, sequential histological changes were investigated in the skin, lung, liver and spleen of mice infected with 30 or 100 cercariae of Schistosoma mansoni according to four sets of experiments: single infection, repeated infections, unisexual infection and infection in mice born from infected mothers. Animals were killed every other day from exposure up to 50 days after infection. Only mild, isolated, focal inflammatory changes were found before the appearance of mature eggs in the liver, even when repeated infections were made. Severe changes of reactive hepatitis and splenitis appeared suddenly when the first mature eggs were deposited, around the 37th to 42nd day after infection. The mature eggs induced lytic and coagulative necrosis of hepatocytes around them which was soon followed by dense infiltration of eosinophils. So, mature egg-induced lesions appeared as the major factors in the pathogenesis of acute schistosomiasis in mice. Mice born from infected mothers were apparently able to rapidly modulate the egg-lesions, forming early fibrotic granulomas. The murine model of acute schistosomiasis appeared adequate for the study of pathology and pathogenesis of acute schistosomiasis.     

Dynamics of fibrosis production and resorption in intestinal schistosomiasis of mice.

A histological, morphometric and immunocytochemical study of schistosomal periovular granulomas in the liver and intestines of mice revealed that intestinal granulomas are smaller and contain less collagen than those in the liver. After curative treatment intestinal granulomas undergo a relatively more rapid resorption, although the general pattern of collagen degradation apparently does not differ from that observed in the liver. Tendency to form scattered, usually isolated granulomas that are only mildly fibrogenic, coupled with a well-balanced process of resorption appear as the explanation why intestinal fibrosis is not an outstanding feature of schistosomiasis as it is in the liver.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Fibroblast stimulation in schistosomiasis. VII. Egg granulomas secrete factors that stimulate collagen and fibronectin synthesis

Tissue fibrosis in schistosomiasis is largely responsible for the important morbidity that results from infection with the trematode worms, Schistosoma. Neither the migrating larval forms (cercariae) nor the intravascular adult worms appear to incite pathological responses that are important in chronic schistosomiasis. On the other hand, eggs deposited in tissue incite chronic granulomatous inflammatory responses that are the hallmark of infection and precede the onset of adjacent tissue fibrosis. We previously reported that products of the egg granulomas can stimulate a number of relevant responses in fibroblast cultures that in vivo would be expected to promote tissue fibrosis. We report here that the granulomas secrete factors that in vitro can stimulate collagen and fibronectin synthesis in fibroblasts. We determined that activity stimulating collagen synthesis is congruent to 10 Kd (gel filtration) with a pI of congruent to 5.5 (isoelectric focusing); additional activity is also detected in some other fractions (congruent to Kd; pI approximately 7.0). In contrast, the activity stimulating fibronectin synthesis was congruent to 22 Kd with a pI of 5.5. Activity was also present in fractions of 50 Kd with pI of approximately 7.5. Fibroblasts grown in granuloma supernatant-containing medium contained greater steady-state levels of specific mRNA coding for type I procollagen and fibronectin compared with cells cultured in unsupplemented medium. These observations support the hypothesis that biologically active molecules secreted by granuloma cells are instrumental in the initiation of tissue fibrosis in schistosomiasis.     

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells.

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.     

In vivo molecular analysis of lymphokines involved in the murine immune response during Schistosoma mansoni infection. II. Quantification of IL-4 mRNA, IFN-gamma mRNA, and IL-2 mRNA levels in the granulomatous livers, mesenteric lymph nodes, and spleens during the course of modulation.

Parasite egg-induced granulomas are the primary pathogenic lesions in murine schistosomiasis mansoni. This cell-mediated granulomatous response is specific for soluble egg Ag and appears to be mediated predominantly by CD4+ Th2 cells. As infection progresses from the acute to the chronic phase, the cell-mediated anti-soluble egg Ag responses attenuate in a process termed modulation. In this study the hypothesis that modulation is effected by a chronic phase increase in Th2-inhibiting Th1 cell activity was investigated. Northern blot quantification of mRNA specific for the Th2 lymphokine, IL-4, and the Th1 lymphokines, IFN-gamma and IL-2, in the spleens, mesenteric lymph nodes, and granulomatous livers of mice infected for various lengths of time over the course of modulation was performed. Also, the capacity of mitogen- and Ag-stimulated spleen cells to produce message for these lymphokines was compared. Peak tissue levels of both IL-4 mRNA and IFN-gamma mRNA were seen in acutely infected mice, and levels of both messages declined as infection became chronic. Stimulated spleen cells from acutely infected mice also produced higher levels of IL-4 and IFN-gamma mRNA than cells from chronically infected mice. IL-2 mRNA was never detected in any tissue sample but was detected in the stimulated spleen cells, again with acute phase levels higher than chronic phase levels. Hence, this study shows no evidence for increased Th1 cell activity during chronic infection and suggests that modulation may be effected by a generalized suppression of lymphokine synthesis.     

Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D

Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned. Three recombinant plasmids containing type I collagen specific sequences were characterized: p alpha 1R1 is 1600 bp and spans approximately 500 amino acid residues within the triple helical region of alpha 1(I) and p alpha 1R2 is 900 bp in size and covers the entire 3' noncoding and about half of the C-terminal propeptide region of alpha 1(I) collagen mRNA. The third recombinant p alpha 2R2 is 1500 bp and contains alpha 2(I) sequences specific for the entire 3' noncoding and C-terminal propeptide region. Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse greater than human greater than chick. Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed two distinctly different molecular weight patterns characteristic of alpha 1(I) (4.7 and 5.7 kb) and alpha 2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe. Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA. The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria.(ABSTRACT TRUNCATED AT 250 WORDS)