Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.     

Cytokine-mediated regulation of transferrin synthesis in mouse macrophages and human T lymphocytes

Transferrin (Tf) plays an important role during immunologic activation by donating iron to activated lymphocytes. Therefore, synthesis by lymphomyeloid cells has been investigated. Mouse macrophages and macrophage cell lines synthesized Tf, with levels being markedly increased by gamma-interferon (gamma-IFN) and, to a lesser extent, by interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF alpha). Tf was also produced by phytohemagglutinin-stimulated human T cells and two T-cell lines and was increased by IL-2. Even after appropriate activation, none was synthesized by human macrophages or monocytic cell lines or by mouse T cells, T-cell lines, or thymus cells. In both species, B-lineage cell lines were negative. Tf was also synthesised by macrophages from congenitally hypotransferrinemic mice and was responsive to gamma-IFN, but levels were lower than those from normal controls. Synthesis by human and murine hepatoma cells was increased by IL-6 but unaffected by IL-1, TNF alpha, or gamma-IFN. Iron decreased synthesis by hepatoma cells but had no effect on the lymphomyeloid cells. Tf mRNA levels paralleled protein synthesis, suggesting that regulation was pre-translational. Thus, Tf synthesis by lymphomyeloid cells is regulated differently from hepatic synthesis, which is consistent with the suggestion that Tf may act in a paracrine (mouse) or autocrine (human) manner on activated lymphocytes.     

Inhibition of iron transport across human intestinal epithelial cells by hepcidin

We investigated the effects of the iron regulatory peptide hepcidin on iron transport by the human intestinal epithelial Caco-2 cell line. Caco-2 cells were exposed to hepcidin for 24 hours prior to the measurement of both iron transport and transporter protein and mRNA expression. Incubation with hepcidin significantly decreased apical iron uptake by Caco-2 cells. This was accompanied by a decrease in both the protein and the mRNA expression of the iron-response element containing variant of the divalent metal transporter (DMT1[+IRE]). In contrast, iron efflux and iron-regulated gene1 (IREG1) expression were unaffected by hepcidin. Hepcidin interacts directly with a model intestinal epithelium. The primary effect of this regulatory peptide is to modulate the apical membrane uptake machinery, thereby controlling the amount of iron absorbed from the diet.     

Cellular expression and regulation of iron transport and storage proteins in genetic haemochromatosis

: Genetic haemochromatosis is a common iron overload disorder of unknown aetiology. To characterize the defect of iron metabolism responsible for this disease, this study localized and semiquantified the mRNA and protein expression of transferrin, transferrin receptor and ferritin in the liver and duodenum of patients with genetic haemochromatosis. Biopsies were obtained from iron-loaded non-cirrhotic patients with genetic haemochromatotic and control patients with normal iron stores. Additional duodenal biopsies were obtained from patients with iron deficiency. Immunohistochemical and in situ hybridization analysis for transferrin, transferrin receptor and ferritin was performed. Hepatic transferrin, transferrin receptor and ferritin protein expression was localized predominantly to hepatocytes and was increased in patients with genetic haemochromatosis when compared with normal controls. Interestingly, hepatic ferritin mRNA expression was not increased in these same patients. In the genetic haemochromatotic duodenum, ferritin mRNA and protein was localized mainly to crypt and villus epithelial cells and the level of expression was decreased compared with normal controls, but similar to iron deficiency. Duodenal transferrin receptor mRNA and protein levels colocalized to epithelial cells of the crypt and villus were similar to normal controls. Early in the course of genetic haemochromatosis and before the onset of hepatic fibrosis, transferrin receptor-mediated iron uptake by hepatocytes contributes to hepatic iron overload. Increased hepatic ferritin expression suggests this is the major iron storage protein. While persisting duodenal transferrin receptor expression may be a normal response to increased body iron stores in patients with genetic haemochromatosis, decreased duodenal ferritin levels suggest that duodenal mucosa is regulated as if the patient were iron deficient.     

Surface membrane characteristics of cells from human acute monocytic leukaemia.

The clinical and laboratory features of 4 patients with acute monocytic leukaemia are described. Surface membrane receptor studies of these patients have shown increasing strength of receptor expression occurs with increasing morphological maturity of the monocyte. In addition, the study shows that expression of membrane receptors can vary from patient to patient and also with time and treatment in an individual patient. A further finding has been the loss of positivity with specific anti-monocyte serum when the disease becomes refractory to therapy.     

Immune regulation of human colonic electrolyte transport in vitro.

The role of lamina propria cells in regulating human colonic ion transport was investigated in vitro. Normal human colonic mucosae were mounted in Ussing chambers, and short circuit current changes (delta SCC) were monitored in response to immune cell activation. Anti-human immunoglobulin E (anti-IgE) and formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used to stimulate mast cells and phagocytes respectively. Anti-IgE (100 micrograms/ml) and fMLP (100 microM) evoked rapid onset, inward delta SCC (mean (SEM) max delta SCC 19.3 (2.8) and 29.4 (4.7) microA/0.63 cm2 respectively). A pharmacological approach was used to identify the charge carrying ion species and to characterise mediators involved in the SCC response. Responses to each secretagogue were significantly attenuated by bumetanide, indicating that the delta SCC was at least partly due to electrogenic chloride secretion. Piroxicam reduced the delta SCC to mast cell and phagocyte activation by 91.1 (3.4)% and 48.2 (25.2)% respectively, implicating eicosanoids as mediators of the responses. Mepyramine (100 microM) reduced the SCC responses to anti-IgE by 79.6 (12.0)% but did not significantly alter delta SCC responses to fMLP. Desensitisation to repeated anti-IgE or fMLP stimulation, and cross desensitisation between each of the stimuli, were features of immune cell activation. In summary, we have shown that activation of immune cells can stimulate electrogenic chloride secretion. Such events in vivo will result in gradient driven secretory diarrhoea, which may occur as a protective response to enteric-dwelling parasites, or as a feature of local bowel inflammation.     

Cytokine-mediated regulation of ovarian function: interleukin-1 inhibits gonadotropin-induced androgen biosynthesis

Resident ovarian macrophages have long been recognized as potential in situ regulators of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, interleukin-1 (IL-1) has recently been shown to exert profound regulatory effects at the level of the ovarian granulosa cell. In this report, we examine the possibility that the adjacent theca-interstitial (androgen-producing) cell may also be a site of IL-1 reception and action. The basal accumulation of androsterone, the major androgenic steroid synthesized by whole ovarian dispersates from immature rats, in the presence of insulin (1 microgram/ml), increased 8- to 9-fold after treatment with human CG (1 ng/ml). Although IL-1 alpha or IL-1 beta (10 ng/ml) by themselves were without effect on basal androsterone accumulation, both cytokines (IL-1 beta greater than IL-1 alpha) inhibited human CG hormonal action (in the presence of insulin) in a dose-dependent manner, the maximal inhibitory effect being 75%. Similar results were obtained when using highly purified theca-interstitial cells derived from the same animal model suggesting that IL-1-attenuated androgen biosynthesis is due, at least in part, to IL-1 acting directly at the level of the theca-interstitial cells. The IL-1 effect proved relatively specific since all other known interleukins (IL-1, IL-3, IL-4, IL-5, and IL-6) were without effect. Moreover, IL-1 beta action was effectively immunoneutralized when concurrently applied with anti-IL-1 beta (but not nonimmune) sera. Significantly, the antigonadotropic action of IL-1 could not be accounted for by a decrease in the viable cell mass. Tracer studies with radiolabeled steroid substrates suggested that IL-1-attenuated ovarian androsterone accumulation is due, if only in part, to inhibition of transformations catalyzed by (theca-interstitial) 17 alpha-hydroxylase/17:20 lyase, stimulation of theca-interstitial (or granulosa 20 alpha-hydroxysteroid dehydrogenase-mediated conversions, or both. Taken together, these findings indicate that relatively low concentrations of IL-1, possibly originating from somatic ovarian cells or resident ovarian macrophages, are capable of exerting an inhibitory effect upon gonadotropin-supported androgen production. As such, these and previous observations suggest that intraovarian IL-1 may play a dual regulatory role in the developing ovarian follicle by targeting both the granulosa and theca-interstitial cells as its sites of action.     

Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells

Resveratrol, a natural product present in wine, has recently been shown to inhibit the growth of a number of cancer cell lines in vitro. In the current study, we have demonstrated that resveratrol inhibits the growth of THP-1 human monocytic leukaemia cells in a dose-dependent manner with a median effective dose of 12 microM. It did not induce differentiation of THP-1 cells and had no toxic effect on THP-1 cells that had been induced to differentiate into monocytes/macrophages by phorbol myristate acetate. A significant fraction of resveratrol-treated cells underwent apoptosis as judged by flow cytometric analysis of DNA content, DNA fragmentation and caspase-specific cleavage of poly(ADP-ribosyl) polymerase. Resveratrol treatment had no effect on the expression of Fas receptor or Fas ligand (FasL) in THP-1 cells, nor did it induce clustering of Fas receptors. In addition, THP-1 cells were resistant to activating anti-Fas antibody, and neutralizing anti-Fas and/or anti-FasL antibodies had no protective effect against resveratrol-induced inhibition of THP-1 cell growth. The effect of resveratrol on THP-1 cells was reversible after its removal from the culture medium. These results suggest that (1) resveratrol inhibits the growth of THP-1 cells, at least in part, by inducing apoptosis, (2) resveratrol-induced apoptosis of THP-1 cells is independent of the Fas/FasL signalling pathway and (3) resveratrol does not induce differentation of THP-1 cells and has no toxic effect on differentiated THP-1 cells. Thus, resveratrol may be a potential chemotherapeutic agent for the control of acute monocytic leukaemia.     

Bacterial lipopolysaccharide induces phosphatidylcholine breakdown in human leukaemia monocytic U937 cells.

We investigated the effect of bacterial lipopolysaccharide (LPS) on phospholipid (PL) turnover in human monocytic leukaemia U937 cells. Cells were pre-labelled with [3H]choline, [14C]ethanolamine and [3H]inositol for 24 h. By monitoring the radiolabel association with cellular PL, the data indicated that LPS (10 micrograms/ml) drastically altered the catabolism of choline-containing PL; it induced their breakdown by 50% within 20 min. The reutilization of choline or its phosphates for PL synthesis was also suggested as a result of regaining radiolabel in the next 40 min. Choline-containing PL then underwent a second degradation after 60 min; 50% decline in radiolabel was detected at 120 min. In contrast, LPS did not induce the breakdown of phosphatidylethanolamine and phosphatidylinositol through phospholipase C/phospholipase D (PLC/PLD). No significant redistribution of the radiolabel in PL was detected in any cases during chasing. The data clearly indicate that LPS stimulates phosphatidylcholine breakdown, implying that the liberation of phosphatidic acid or diacylglycerol via PLC/PLD reaction may be relevant to the initiation of LPS-induced monocytic activation.     

Conventional transport assays underestimate sugar transport rates in human red cells

The time course of protein-mediated 3-O-methylglucose uptake by human red cells and by red cell ghosts containing or lacking 4 mM MgATP was measured at ice temperature and sub-saturating sugar levels by conventional sampling procedures and at 20 degrees C by use of a quench-flow apparatus. The temporal resolution of the quench-flow apparatus (as fast as 5-ms sample times) was confirmed by analysis of alkaline hydrolysis of dinitrophenolacetate. Red cell sugar uptake at 4 degrees C is consistent with two processes [fast (tau = 120 s) and slow (tau = 1100 s)] that occur in series. Intracellular ATP increases the size and the rate of equilibration of the fast compartment and slows the rate of filling of the slow compartment. Red cell ghost volume and protein content are unaffected by lysis/resealing in the presence of ATP. Uptake at 20 degrees C is also consistent with two processes [fast (tau = 10 ms) and slow (tau = 15 s)] that occur in series. ATP increases the size of both compartments and the rate of filling of the small compartment at 20 degrees C. Preliminary estimates indicate that the sugar uptake capacity of human red cells at 20 degrees C is underestimated by as much as 8-fold by measuring sugar uptake over 2 s vs. 26 ms. We discuss the implications of multiphasic sugar uptake in the context of models for protein-mediated sugar transport.