Wolfgang Jelkmann

Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys

The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1 beta, IL-1 alpha, and tumor necrosis factor alpha lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor beta 2 and interferon gamma did not inhibit. Furthermore, IL-1 beta (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.

Inhibition of Erythropoietin Production by Cytokines: Implications for the Anemia Involved in Inflammatory States

In patients with the anemia of chronic diseases, the plasma level of EPO is often low in relation to the blood hemoglobin concentration. Because infectious and inflammatory processes cause activation of cytokine-producing macrophages and lymphocytes, we investigated whether isolated inflammatory cytokines influence the synthesis of EPO in vitro. IL-1 and TNF-alpha were shown to inhibit EPO mRNA levels and EPO formation in the human hepatoma cell cultures HepG2 and Hep3B, and to lower EPO formation in isolated perfused rat kidneys. IFN-alpha and IFN-beta also induced some inhibition of EPO production in HepG2 cultures. IL-3, TGF-beta 2, and IFN-gamma did not inhibit. IL-6 stimulated the production of EPO in Hep3B cells but was ineffective in HepG2 cells and lowered EPO production in isolated perfused rat kidneys. IL-1, TNF-alpha, and possibly other cytokines could contribute to defective EPO production in renal and nonrenal immune responses.

A comparison of the effects of renal artery constriction and anemia on the production of erythropoietin

It is generally assumed that the O2 supply to the kidneys is the major determinant of the synthesis of erythropoietin (Ep). In the present study, the O2 supply of the kidneys of rats was lowered by the reduction of renal blood flow (rbf). Plasma Ep was determined after about 18 h of bilateral application of Goldblatt clips with graded inner diameters. The results were compared to findings in anemic rats, in which the systemic O2 supply was lowered by exchange transfusion of blood with plasma. We found a linear correlation between Ep levels in plasma and the degree of reduction of rbf. However, there was an exponential relationship between Ep levels and the concentration of hemoglobin in blood. In addition, the elevation of plasma Ep was only moderate, when rbf was reduced (maximum 0.07 IU Ep/ml plasma). The increase in Ep concentration was much more pronounced in anemia (up to about 7 IU Ep/ml plasma). From these results it may be concluded that decreasing oxygen supply to the kidney through reduction in renal blood flow (ischemic hypoxia) is less effective in increasing erythropoictin production than reducing the hemoglobin concentration (anemic hypoxia). The possibility must be considered that the increase in renal production of erythropoietin due to anemic hypoxia is triggered by one or more extrarenal signals.

Inhibition of erythropoietin production by phorbol ester is associated with down-regulation of protein kinase C-α isoenzyme in hepatoma cells

The role of protein kinase C (PKC) in the control of erythropoietin (Epo) production was studied using the human hepatoma cell line HepG2. Inhibition of PKC by staurosporine and the selective PKC inhibitor CGP 41251 significantly reduced Epo formation. No inhibition occurred with the inactive staurosporine derivative CGP 42700. Treatment with phorbol 12-myristate 13-acetate (PMA) for 24 h dose-dependently inhibited Epo formation, thus suggesting that down-regulation of PKC might be responsible for this inhibition. Immunoblotting experiments showed that incubation of HepG2 cells with PMA for 24 h resulted in a selective and almost complete down-regulation of PKC-alpha. Thus, PKC-alpha may play a permissive role in Epo synthesis in HepG2 cells.

Inhibition of erythropoietin production by cytokines and chemotherapy may contribute to the anemia in malignant diseases.

The kidneys and the liver are the main sites of synthesis of the hormone erythropoietin. Tissue hypoxia is the primary stimulus of its production. In general, plasma levels of erythropoietin increase exponentially with lowered O2 carrying capacity of the blood. This response is missing in uremic patients. In addition, patients with malignant and inflammatory diseases often exhibit inappropriately low plasma erythropoietin levels1.

Interleukin 1β and phorbol ester induce tumour necrosis factor α production in a hepatic cell line (HepG2)

Tumour necrosis factor α (TNFα) is a pleiotropic cytokine that is produced mainly by monocytes and macrophages. TNFα appears to be responsible for many of the inflammatory and necrotic changes seen in malignant or infectious liver diseases. In addition, blood levels of TNFα have been reported to be elevated in those with hepatic diseases. Although TNFα is synthesized mainly by monocytes and macrophages, its production has recently been found in non-haemopoietic cells as well. Therefore we have used the human liver cell line HepG2 to test for the inducible production of TNFα in hepatic parenchymal cells. No constitutive TNFα gene expression was detected by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). However, treatment with the proinflammatory cytokine interleukin 1β (IL-1β) or phorbol 12-myristate-acetate (PMA) led to a marked increase in TNFα mRNA levels. Maximal TNFα mRNA levels were observed after 3-h incubation periods, decreased thereafter and became undetectable after 12 h. The culture supernatant from cells treated with IL-lβ or PMA contained significant amounts of TNFα protein which was immunologically detectable and biologically active. We believe that our report of inducible TNFα production in this widely available hepatic cell line adds a valuable tool for understanding TNFα gene expression in nonhaematopoietic cells.

Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures

The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogeneous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus non-hypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.

Oxygen sensing by H2O2-generating heme proteins?

Erythropoietin (Epo) production in the kidneys and liver will increase up to 100-fold if the Epo-producing tissue becomes hypoxic because of anemia or a decrease in the arterial PO2 (Jelkmann 1992). Today the only cell culture system in which hypoxia-inducel Epo production can be studied are the hepatoma cell lines HepG2 and Hep3B. In these cells a close correlation exists between increasing grades of hypoxia and Epo messenger RNA (mRNA) levels in the cells (Fandrey and Bunn 1993) and secreted Epo protein in the culture medium supernatant (Wolff et al. 1993).

Interleukin-1 and Tumor Necrosis Factor-α Suppress Hypoxia-Induced Production of Erythropoietin In Vitro

The glycoprotein growth factor erythropoietin (EPO) is primarily produced by the fetal liver and by the kidney in adults. EPO specifically stimulates the proliferation and maturation of erythroid precursor cells, especially colony and burst forming units-erythroid in the bone marrow [17]. Stimuli for EPO synthesis and secretion can be various kinds of lowered O2 supply to a still poorly defined sensor mechanism [2]. Human EPO serum levels increase in response to hypobaric hypoxia [9] or hypoxic hypoxia [14]. Acute hemorrhage in humans results in a transient elevation of EPO production [5, 28, 32]. In chronic anemia there is an inverse relationship between hemoglobin concentration and the serum EPO level [10, 18]. Thus, EPO production would be expected to be greatly stimulated following surgical or accidental blood loss or in septicemia with respiratory failure. However, EPO is inappropriately low for the degree of anemia in patients with chronic renal failure [25], inflammatory [4, 19] and malignant disease [29], or in acute renal allograft rejection [3]. A common feature among these anemia-associated conditions, that is shared with severe trauma, burning, and sepsis [11], is the release of cytokines by activated monocytes [1, 8, 15, 16, 24, 34]. These monokines, namely, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), have been shown to evoke systemic reactions in critically ill patients known as acute-phase response [11].

Control of the Production of Erythropoietin by a Renal Oxygen-Sensor?

The glycoprotein hormone erythropoietin (Epo) is an obligatory growth and differentiation factor for the erythrocytic progenitors. The plasma level of Epo in the human is about 5 to 25 mU/m1 under normoxic conditions. Tissue hypoxia is a major stimulus for erythropoiesis. The plasma level of Epo may increase to 1000 mU/ml or more after the induction of anemia or hypoxic hypoxia (12, 20, 31).