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Biomedical subjects

J H Brock

Publications and source records attributed to J H Brock.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regulation of cellular iron metabolism by erythropoietin: activation of iron-regulatory protein and upregulation of transferrin receptor expression in erythroid cells.

Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

Animals↗
PubMed1996

Iron transport across Caco-2 cell monolayers. Effect of transferrin, lactoferrin and nitric oxide.

Differentiated Caco-2 colon carcinoma cell monolayers grown in bicameral chambers have been used as an in vitro model to study the effect of different carrier molecules on mucosal iron transport. Transfer of iron across the monolayers in the apical-to-basolateral direction was greater from ferric lactoferrin than from iron citrate, while very little transport occurred from Fe-transferrin. However, a greater proportion of iron was retained by the cells when Fe-citrate was the donor. Caco-2 cells expressed transferrin receptors (n = 1.3 x 10(5) /cell; Ka = 2 x 10(8) l/mol), but binding of lactoferrin, though substantial in quantity, had an affinity too low to measure. When monolayers were incubated with 125I-labelled lactoferrin or transferrin some 125I-activity was transported, but almost all was TCA-soluble, suggesting that degradation products rather than intact protein were being transported. Addition of 10 microM S-nitroso-N-acetyl-D,L-penicillamine (SNAP), which produces nitric oxide (NO) in solution, caused a significant increase in iron transport from ferric citrate, but not from Fe-lactoferrin or Fe-transferrin. It is concluded that in this in vitro system lactoferrin but not transferrin enhances mucosal iron transport, and that NO may play a regulatory role in iron absorption.

Biological Transport↗
PubMed1996

Longitudinal evaluation of peritoneal macrophage function and activation during CAPD: maturity, cytokine synthesis and arachidonic acid metabolism.

The release of cytokines and prostaglandins (PG) by peritoneal macrophages (PM luminal diameter of) may influence the cytokine network controlling peritoneal inflammation and in the long-term the function of the peritoneum as a dialysis membrane. In the present study, an evaluation of the long-term effects of peritoneal dialysis on the release of cytokines and prostaglandins, and the expression of surface markers of cellular maturation on blood and mononuclear cells has been performed in patients during their first year on CAPD. Spontaneous release of tumour necrosis factor alpha (TNF alpha) and interleukins 6 (IL-6) by PM luminal diameter of, after 4 or 24 hours in culture, increased significantly with time on CAPD, while there was a small but significant decrease in release of prostaglandin E2 (PGE2). Production of TNF alpha and IL-6 was enhanced following incubation of the cells with lipopolysaccharide (LPS), but the effect of LPS was proportionally greater on blood monocytes than on PM luminal diameter of. There was a significant increase in the concentrations of PGE2 and 6-keto-prostaglandin F1 alpha in overnight dwell peritoneal dialysis effluent with time on CAPD. The levels of TNF alpha and IL-6 in uninfected PDE were below the detection limit of the immunoassay over the whole time period studied. Expression of CD15, which correlates with immaturity, by PM luminal diameter of and blood monocytes increased with time on CAPD, while expression of CD11c, a marker of maturation, decreased on blood monocytes, but did not change significantly on PM luminal diameter of. There was also a slight increase in expression of transferrin receptor in both PM luminal diameter of and monocytes, but this did not reach statistical significance. These findings suggest that peritoneal macrophages and blood monocytes isolated from CAPD patients over a one year period become increasingly immature with time, and this is accompanied by a significant modulation of their ability to secrete inflammatory cytokines. Dysregulation of macrophage function may have important consequences with respect to inflammatory processes and the long-term function of the peritoneal membrane in CAPD patients.

Adolescent↗
PubMed1995

Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells.

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

Ferritins↗
PubMed1995

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.

Animals↗
PubMed1994

Interaction of lactoferrin with mononuclear and colon carcinoma cells.

Lactoferrin is known to bind to macrophages/monocytes and intestinal mucosal cells, but the nature and function of these interactions is not clear. We have therefore examined the interaction of lactoferrin in vitro with the promonocytic cell line U937 and with differentiated human colon carcinoma cells. U937 cells bound more lactoferrin than transferrin, although most of the lactoferrin binding was non-specific. Uptake of iron from transferrin was rapid, but uptake from lactoferrin was slow, and may have been due to prior transfer of iron to transferrin in the culture medium as a result of labilisation of iron from membrane-bound lactoferrin. Unlike transferrin, lactoferrin was not internalised by U937 cells. Lactoferrin significantly reduced uptake of non-transferrin-bound iron by the cells, but had no effect on uptake of transferrin-bound iron. Transport of lactoferrin-bound iron across monolayer cultures of differentiated Caco-2 cells in bicameral chambers was similar to that of ferric citrate, while transport of transferrin-bound iron was lower. Lactoferrin and transferrin themselves were not transported, although some proteolytically degraded material did cross the monolayer. Thus lactoferrin, unlike transferrin, is not an important iron donor to monocytic cells, but may instead serve to regulate iron uptake from other sources. It does not seem to enhance iron transport across mucosal cells.

Biological Transport↗
PubMed1993

Binding of lactoferrin and transferrin to the human promonocytic cell line U937. Effect on iron uptake and release.

We have compared the ability of lactoferrin and transferrin to interact with and donate iron to the monocytic cell line U937. About 10 times more lactoferrin was bound than transferrin, but most lactoferrin bound nonspecifically, and the degree of specific binding was similar for both proteins (2-3 x 10(6) sites/cell). The binding affinity for lactoferrin (83 nM) was about 4-fold lower than for transferrin (21 nM). Lactoferrin did not inhibit binding of transferrin, or vice versa. Binding of lactoferrin was not inhibited by 30 mM glucose or fucose nor by incubating the cells with heparinase. Transferrin, but not lactoferrin, was internalized, and 3 mM primaquine caused intracellular accumulation of transferrin but not lactoferrin. The cells rapidly acquired iron from transferrin, but uptake from lactoferrin was 10-fold slower and probably resulted from transfer of 59Fe from lactoferrin to unlabeled transferrin during culture. Lactoferrin, but not transferrin, released iron to the extracellular medium when bound to U937 cells. Lactoferrin inhibited cellular uptake of iron from Fe-nitrilotriacetate but not from transferrin. It is concluded that transferrin, but not lactoferrin, acts as an iron donor to U937 cells. Lactoferrin may regulate uptake of potentially toxic non-transferrin-bound iron.

Binding Sites↗
PubMed1993

Transferrin synthesis by mouse lymph node and peritoneal macrophages: iron content and effect on lymphocyte proliferation.

Transferrin is an essential requirement for lymphocyte proliferation, because it supplies activated lymphocytes with iron needed for cell proliferation. However, during inflammation or an immune response, the iron content of circulating transferrin, which is of hepatic origin, decreases. It is hypothesized that activated lymphocytes may therefore obtain transferrin-iron from an alternative source, and we have investigated the possibility that transferrin is synthesized locally in lymphoid tissues. It was found that lymph node cells from mice stimulated in vivo with Freund's complete adjuvant were able to synthesize transferrin, and this was because of the macrophage rather than the lymphocyte population. Transferrin synthesized by mouse lymph node or peritoneal macrophages contained iron and was able to promote mouse lymphocyte proliferation. Peritoneal macrophages activated in vivo synthesized more transferrin, released more transferrin-bound iron, and were more effective than resident macrophages at enhancing lymphocyte proliferation. These results suggest that transferrin synthesized by macrophages acts in a paracrine manner to support lymphocyte proliferation, thus eliminating possible detrimental effect of hypoferremia on the immune system.

Animals↗
PubMed1993

Effect of heat treatment and other milk proteins on the interaction of lactoferrin with monocytes.

The interaction of lactoferrin from human and bovine milk with the human promonocytic cell line U937 has been studied. Both human and bovine Fe-lactoferrins bound to the cells. Binding of bovine lactoferrin was inhibited by excess bovine lactoferrin but not by human lactoferrin, suggesting that the binding mechanisms for the two proteins are different. Binding of human but not bovine lactoferrin was inhibited by bovine lactoperoxidase, while a 20-fold excess of human IgA inhibited binding of human but not bovine lactoferrin. Human and bovine alpha-lactalbumins, bovine beta-lactoglobulin, and human lysozyme had no effect on binding of lactoferrin from either species. Samples of bovine Fe- and apolactoferrin in capillary tubes were exposed to temperatures of 72 degrees C for 20 s, 85 degrees C for 20 min or 137 degrees C for 8 s. All the heated samples inhibited binding of native Fe- and apolactoferrin, though to a lesser extent than the native proteins. Both heated and native lactoferrins enhanced [3H]thymidine incorporation by U937 cells, except for Fe-lactoferrin heated at 85 degrees C for 20 min, which was inhibitory. These results suggest that heat treatment of lactoferrin under conditions used for industrial processing does not greatly affect its ability to interact with and stimulate monocytic cells, and that other milk proteins in general do not interfere with lactoferrin-monocyte interactions. It may thus be feasible to incorporate biologically active lactoferrin into infant formulas.

Animals↗
PubMed1993

Effect of transferrin concentration on bacterial growth in human ascitic fluid from cirrhotic and neoplastic patients.

Cirrhotic patients with ascites have an unusually high frequency of development of spontaneous bacterial peritonitis. Iron availability is a key factor in bacterial growth and the ability of the host to limit it is associated with resistance to infection. The present study was undertaken to evaluate the influence of iron and transferrin on bacterial growth in ascitic fluid from 25 biopsy-proven cirrhotic and nine neoplastic carcinomatous patients. No significant differences were found when comparing total ascitic fluid iron between the two groups but ascitic fluid transferrin concentration was significantly lower in cirrhotic (29.26 mg dl-1 SD 29.58) than neoplastic (96.57 mg dl-1 SD 76.01) patients. Moreover, a significant negative correlation was found between bacterial growth and transferrin concentration in ascitic fluid (P = 0.039). When the iron concentration in ascitic fluid was experimentally elevated (50 micrograms dl-1 or 150 micrograms dl-1) we observed a progressive increase in bacterial growth. If transferrin concentration is simultaneously elevated (250 mg dl-1) this increase does not occur. These findings indicate that the transferrin level is an important factor in the regulation of bacterial growth in ascitic fluid and that the low concentration found in cirrhotic patients could facilitate spontaneous bacterial peritonitis.

Adult↗
PubMed1992

Isolation of a ferritin from Bacteroides fragilis.

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Amino Acid Sequence↗
PubMed1992

Effect of iron and retinoic acid on the control of transferrin receptor and ferritin in the human promonocytic cell line U937.

The effect of changes in iron availability and induction of differentiation on transferrin receptor expression and ferritin levels has been examined in the promonocytic cell line U937. Addition of iron (as 200 micrograms/ml saturated transferrin) or retinoic acid (1 microM) both caused approx. 70% reduction in the average number of surface transferrin receptors, while the iron chelator desferrioxamine caused an 84% increase. Comparable changes also occurred in the levels of transferrin receptor mRNA. Neither iron nor retinoic acid significantly altered the half-life of transferrin receptor mRNA in the presence of actinomycin D (approx. 75 min) but a 10-fold increase in stability occurred in the presence of desferrioxamine. Iron and retinoic acid both caused an increase in intracellular ferritin levels (approx. 4-and 3-fold, respectively), while desferrioxamine reduced ferritin levels by approx. two-thirds. The effect of iron and retinoic acid added together did not differ greatly from that of each agent alone. None of the treatments greatly affected levels of L-ferritin mRNA. Virtually no H-ferritin mRNA was detected in U937 cells. These results show that changes in ferritin and transferrin receptor caused by treatment with retinoic acid are similar to those induced by excess iron, and suggest that changes in these proteins during cell differentiation are due to redistribution of intracellular iron into the regulatory pool(s), rather than to iron-independent mechanisms.

Cell Differentiation↗