ABSTRACT
Great progress has been made in understanding the development of non-alcoholic fatty liver disease (NAFLD) but less is known about the mechanisms underlying the progress from steatosis to steatohepatitis (NASH). Our aim was to evaluate if the amount and type of storage of fat in hepatocytes is of importance for hepatocyte injury. We also wanted to show if not only the innate immunity but also the adaptive immunity is involved in NASH. Thirty-one patients with NASH or borderline NASH and 18 non-NASH patients were investigated. Liver biopsies were scored for NASH according to Kleiner et al. Paraffin-embedded liver biopsies were stained with antibodies against CD3, TLR4, CD68, Cleaved Caspase-3, ICAM1, Foxp3 and ApopTag by immunohistochemistry. Serum soluble ICAM-1 (sICAM-1) were analysed by ELISA. The volume density of fat was 59% in the NASH patients and microvesicular fat, increased in high NAS score patients. ICAM-1 positive hepatocytes were seen in NASH patients and were localized in areas with microvesicular fat. Non-NASH biopsies were negative for ICAM-1 positive hepatocytes. The sICAM-1 were significantly higher in NASH-patients (339.8 ± 34.07) than in non-NASH patients (229.5 ± 12.14), p = 0.0015. Patients with NAS score over four had higher area of CD68 positive cells p = 0.0011 and Foxp3 positive cells (p = 0.024) than non-NASH patients. In liver tissue with NASH, hepatocytes with microvesicular steatosis seem to be expressing more inflammatory markers, and in this liver tissue an increased number of CD68 cells and regulatory T-cells (Tregs, e.g. Foxp3+ cells) were seen, indicating an involvement of, both the innate and the adaptive immunity.
Subject(s)
Fats/analysis , Hepatocytes/chemistry , Intercellular Adhesion Molecule-1/analysis , Intercellular Adhesion Molecule-1/blood , T-Lymphocytes, Regulatory/immunology , Adaptive Immunity , Adolescent , Adult , Aged , Antibodies, Monoclonal , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Apoptosis , Cytokines/biosynthesis , Cytokines/immunology , Enzyme-Linked Immunosorbent Assay , Fatty Liver/immunology , Fatty Liver/metabolism , Fatty Liver/pathology , Female , Forkhead Transcription Factors/analysis , Hepatocytes/ultrastructure , Humans , Immunity, Innate , Liver/immunology , Liver/pathology , Macrophages/immunology , Male , Middle Aged , Non-alcoholic Fatty Liver DiseaseABSTRACT
BACKGROUND/AIMS: To study the effect of iron and proinflammatory cytokines on the expression of HAMP and other iron regulatory genes in primary rat hepatocytes. METHODS: Primary hepatocytes from rats fed a control or iron-enriched diet were plated on extracellular matrix and incubated with inflammatory stimuli in the presence or absence of serum. Cells were also incubated with desferrioxamine or ferric ammonium citrate. mRNA levels were determined by Real-Time PCR. RESULTS: Hepatocytes from control rats increased their HAMP expression during culturing, whereas the opposite was seen in hepatocytes from carbonyl-iron loaded animals. In the presence of serum, tumor necrosis factor-alpha, lipopolysaccharide and interleukin-6 increased HAMP expression in hepatocytes from both control and iron-loaded rats. Under serum-free conditions only tumor necrosis factor-alpha increased HAMP mRNA levels. Desferrioxamine and ferric ammonium citrate decreased HAMP gene expression. Tumor necrosis factor-alpha significantly increased mRNA levels of TfR2 and decreased those of DMT1 and IREG1. CONCLUSIONS: HAMP expression differs in cultured as compared with freshly isolated hepatocytes, and decreases in iron-loaded hepatocytes in serum free-media, suggesting that additional serum factors influence HAMP expression. Tumor necrosis factor-alpha regulates the mRNA levels of HAMP, IREG1, DMT1 and TfR2 in cultured hepatocytes from both iron-loaded and control animals.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Gene Expression/drug effects , Hepatocytes/metabolism , Interleukin-6/pharmacology , Iron Overload/metabolism , RNA, Messenger/genetics , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antimicrobial Cationic Peptides/metabolism , Cells, Cultured , Disease Models, Animal , Hepatocytes/drug effects , Hepatocytes/pathology , Hepcidins , In Vitro Techniques , Iron Overload/pathology , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND/AIMS: Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver. METHODS: Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells). RESULTS: The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes. CONCLUSIONS: The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism.
