José V. Castell

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

The three monokines interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL‐6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C‐reactive protein, serum amyloid A, fibrinogen, α1‐antitrypsin, α1‐antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL‐1β as well as TNFα, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C‐reactive protein. These data suggest that IL‐6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.

Recombinant human interleukin‐6 (IL‐6/BSF‐2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

Recombinant human IL‐6 (rhIL‐6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL‐6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C‐reactive protein, haptoglobin, α1‐antichymotrypsin and fibrinogen were strongly induced (26‐, 23‐, 8.6‐, 4.6‐ and 3.8‐fold increases, respectively). Moderate increases were found for α1‐antitrypsin (2.7‐fold) and α1‐acid glycoprotein (2.7‐fold). RhIL‐6 had no effect on α1‐macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, α1‐antichymotrypsin, α1‐antitrypsin and α1‐acid glycoprotein, dexamethasone enhanced the action of rhIL‐6. We conclude that rhIL‐6 controls the acute phase response in human liver cells.

In vitro Investigation of the Molecular Mechanisms of Hepatotoxicity

Iatrogenic Hepatitis: Intrinsic and Idiosyncratic Toxicity. Substances capable of producing liver damage and, more specifically, hepatocyte damage are known as hepatotoxins. They are classified (Zimmerman and Ishak, 1995, Castell et al., 1992) according to whether they exert their effects in all individuals, in a dose-dependent and hence predictable manner (intrinsic hepatotoxins), or in certain individuals, occasionally after several contacts, in a non-dose dependent and therefore unpredictable way (idiosyncratic hepatotoxins). These substances can act directly on cells (active hepatotoxins), or become toxic after biotransformation (latent hepatotoxins). Idiosyncratic hepatotoxicity is the consequence, either of an unusual metabolism of the drug by susceptible individuals which produce too large amounts of toxic metabolites (metabolic idiosincrasy), or is due to an immune-mediated attack to sensitised hepatocytes (drug hypersensitivity).

6 – Isolation, Culture and Use of Human Hepatocytes in Drug Research

The major contributions of in vitro systems, hepatocytes and slices, for studying hepatotoxicity include: the ranking of a series of compounds, the evaluation and comparison of species susceptibility, and investigation toward understanding the mechanism of the toxicity. A continuous goal for these systems is to demonstrate that the data obtained are predictive and can be used reliably in extrapolating from animals to humans for safety assessment. Further validation of the systems for risk assessment will increase the use of data obtained within companies and by regulatory authorities. Within companies the in vitro data could aid in the planning of investigational in vivo studies and the data could be used in combination with computer modeling to assess risk. In the future, in vitro data may change the type and number of in vivo studies performed, resulting in a further reduction in the total number of animals used. A major impact of the hepatocyte and slice in vitro systems is in clearly defining species susceptibility, including human, to the side-effects or toxicity of the compound in question. This information could contribute to defining the safety margin or establishing the risk for humans. Furthermore, understanding the mechanism of toxicity may lead to the development of a compound with a wider safety margin or may result in a means of counteracting the effect in vivo.

Isolation and culture of human hepatocytes

Small tissue samples obtained in the course of liver surgery (i.e. for trauma, tumour resections, cholecystectomy etc.) or from tissue resected for pathological examination are available on a regular basis, and constitute a reliable source of viable human hepatocytes. The enzymatic perfusion of liver through the blood vessels is the procedure of choice to obtain human hepatocytes for culturing. Another key factor is the use of appropriate enzymes to weaken cell-cell junctions and liberate cells with minimal membrane damage [1,2]. On the basis of these considerations, a two-step microperfusion technique for isolating hepatocytes has been successfully applied to very small samples of human liver [3].

Proliferative response and metabolic effects of growth factors in human hepatocytes

Liver regeneration is the outcome of several pathologies, including not only partial hepatectomy but also liver transplantation, viral or toxic hepatitis, cirrhosis, hepatocellular carcinoma, and fulminant hepatic failure [1,2]. Thus, understanding the nature and mechanisms of action of the factors involved in the control of liver cell proliferation is important for establishing the basis for improved therapies for patients with chronic or acute hepatic injury. Human hepatocytes in primary culture retain most of their basic metabolic hepatic functions if adequate chemically-defined culture media and substrata are used. Indeed, adult human hepatocytes can be stimulated to synthesize DNA when they are cultured at very low cell density, under defined culture conditions (supplementation of culture medium with certain nutrients and hormones) and in the presence of specific hepatotrophic growth factors [3–6]. Therefore, primary cultured human hepatocytes have become a useful tool for investigating human liver cell metabolic functionality, drug biotransformation processes, gene expression and the proliferative response to growth factors.