Transferrin receptor distribution and iron deposition in the hepatic fobufe of iron-overloaded rats

Under the condition of obvious iron-overload, there is a zonal hernoeiderin (iron) deposition in hepatic lobules. The deposition is heavtest in the periporfal (zone 1) and lightest in the perivenws (zone 3) hepatocytes. However, the mechanism for this pattern of iron deposition is obscure. Hepatic tissues from control, iron-deficlent or ironoverloaded Wistar rats me used to study its pathogenesis. iron-deficiency was Induced by a low Iron regimen. Ironoverload was produced by repeated intraperitoneal injections of ferric nitrilotriace-We (Fe³⁺-NTA) for 1–4 months. Liver tissues of the rats were lmmunohistochemically and histochemically stained for tmnaferrin receptor (TfR), transferrin (Tf), ferritin (Ft), and iron. The staining intensity of TfR, Tf and Ft increased in hepatocytes of iron-deflctent rats and decreased in that of the iromverloaded in comparison with the control rats. TfR atalning was strong in zone 1, with gradual transition into weak staining in zone 3 hepatocytes of the rat liver. TfR located primarily on the hepatocyte membrane. Tf had both membranous and cytoplasmic distribution. Many hepatocytes in group B had strong cytoplasmic Tf staining. Conversely, only a few hepatocytes had weakly stained cytoplasmic Tf in group C. Hepatocytes and Kupffer cells were Ft positive in control rats. Ft was distributed only in the cytoplasm. The staining intensity of Ft was stronger in zone 3 than in zone 1 hepatocytes of iron-deficient rats. In iron-overloaded rats, the iron deposition was severe in zone 1 and mild in zone 3 hepatocytes. These findings suggest that uptake of iron into hepatocytes in vivo is regulated and mediated by TfR and Tf. Gradient TfR distribution from zone 1 to 3 hepatocytes and active TfR-Tf mediated iron uptake resuited in the zonal iron deposition in the hepatic lobule of iron-overloaded rats.     

Selective iron deposition in pancreatic islet B cells of transfusional iron-overloaded autopsy cases

Pancreatic islets of 36 autopsy cases with transfusional iron-overload were examined. Immunohistochemical and histo-chemical stainings were used to clarify the relationship between blood transfusion and iron deposition in the islet. Disease of the lymphohemopoietic system (leukemia, lymphoma, aplastic anemia) or liver (carcinoma and/or cirrhosis) accounted for 86.1% of the patients'main diagnosis. Sixteen of them had slight hemosiderin deposition (Group 1), twenty cases had severe hemosiderin deposition (Group 2). Another ten cases were used as controls (Group 3). The cases had a similar age distribution to Group 1 and 2, with neither blood transfusion nor hemosiderin deposition. The volume of blood transfusion was 6.1 ± 3.6, 17.5 ± 12.2 L for Groups 1 and 2, respectively. The plasma glucose was 137.8 + 54.4 and 170.6 ± 108.4 mg/dL, respectively. Four cases in Group 1 and 14 cases in Group 2 had glycosuria. The number of islet cells with hemosiderin increased with the enlargement of transfusion volume ( r = 0.664, P ± 0.001). Plasma glucose also related with the percentage of hemosiderin positive islet cell (r = 0.386, P < 0.025). In severely iron-overloaded cases, hemosiderin was selectively deposited in B cells of the islet.
It was concluded that large amounts of blood transfusions for non-congenital disease can induce selective hemosiderin deposition and impairment of pancreatic B cell that may result in hyperglycemia and diabetes mellitus of the patients.     

铁过剩时铁在肝小叶内区域性沉积的机理

用Ｗｉｓｔａｒ大鼠通过词以缺铁词料或腹腔内反复注射铁。氨三乙酸络合物４个月制成铁缺乏或铁过剩大鼠模型。用ＡＢＣ法及铁染色法观察、分析铁缺乏（Ａ组）、铁过剩（Ｂ组）与对照（Ｃ组）大鼠肝脏转铁蛋白（Ｔｆ）及其受体（ＴｆＲ）的表达与铁沉积的关系。与Ｃ组相比，ＴｆＲ的表达在Ａ组增强、Ｂ组减弱。三组动物ＴｆＲ在肝小叶第１区表达最强，第２区次之，第３区最弱。三组动物Ｔｆ表达差异与ＴｆＲ类似。Ｂ组肝小叶内可见铁沉积。以１区分布最多，３区最少；与ＴｆＲ表达在肝小叶分布一致。作者认为体内肝细胞摄取铁主要经ＴｆＲ与Ｔｆ介导，ＴｆＲ和Ｔｆ的区域性分布可导致铁过剩时肝小叶内铁从第１区至第３区的顺序递减型区域性沉积。     

Transferrin receptor expression in normal, iron-deficient and iron-overloaded rats

The distribution of transferrin receptor (TfR)-positive cells and their staining intensity were examined in the liver, duodenum, pancreas, spleen, kidney and brain of iron-deficient, iron-overloaded and normal Wistar rats to elucidate the regulatory effects of iron on TfR expression in vivo. Iron deficiency was produced by an iron-deficient food and water regimen, and iron overload by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3(+)- NTA) for 12 weeks. In iron-deficient rats, levels of hemoglobin (Hb = 5.9 +/- 0.7) and serum iron (SI = 29.9 +/- 4.4) were lower, and total iron-binding capacity (TIBC = 624.4 +/- 72.7) was higher than in normal rats (Hb = 15.6 +/- 0.9, SI = 206.5 +/- 20.5, TIBC = 416.0 +/- 56.0), and vice versa for SI (217.7 +/- 15.5) and TIBC (307.1 +/- 45.4) in iron-overloaded rats. In normal rats, TfR-positive granules were observed in hepatocytes and Kupffer cells of the liver, absorptive epithelium of the duodenum, acinar and Langerhans islet cells of the pancreas, macrophages and red pulp erythroblast of the spleen, and distal convoluted tubular epithelium of the kidney. Although the tissue distribution pattern of TfR-positive cells was similar in normal, iron-deficient and iron-overloaded rats, the staining intensity and number of TfR-positive cells were obviously higher in iron-deficient, and lower in iron-overloaded rats. We conclude that TfR expression is negatively regulated by the tissue concentration of iron.     

Deferasirox and deferiprone remove cardiac iron in the iron-overloaded gerbil

Introduction: Deferasirox effectively controls liver iron concentration; however, little is known regarding its ability to remove stored cardiac iron. Deferiprone seems to have increased cardiac efficacy compared with traditional deferoxamine therapy. Therefore, the relative efficacy of deferasirox and deferiprone were compared in removing cardiac iron from iron-loaded gerbils. METHODS: Twenty-nine 8- to 10-week-old female gerbils underwent 10 weekly iron dextran injections of 200 mg/kg/week. Prechelation iron levels were assessed in 5 animals, and the remainder received deferasirox 100 mg/kg/D po QD (n = 8), deferiprone 375 mg/kg/D po divided TID (n = 8), or sham chelation (n = 8), 5 days/week for 12 weeks. RESULTS: Deferasirox reduced cardiac iron content 20.5%. No changes occurred in cardiac weight, myocyte hypertrophy, fibrosis, or weight-to-dry weight ratio. Deferasirox treatment reduced liver iron content 51%. Deferiprone produced comparable reductions in cardiac iron content (18.6% reduction). Deferiprone-treated hearts had greater mass (16.5% increase) and increased myocyte hypertrophy. Deferiprone decreased liver iron content 24.9% but was associated with an increase in liver weight and water content. CONCLUSION: Deferasirox and deferiprone were equally effective in removing stored cardiac iron in a gerbil animal model, but deferasirox removed more hepatic iron for a given cardiac iron burden.     

Transferrin binding and iron uptake by mouse lymph node cells during transformation in response to concanavalin A.

Mouse lymph node cells cultured with Concanavalin A (Con A) in serum-free medium containing 59Fe-transferrin took up 59Fe more rapidly than cells cultured without Con-A. Uptake of iron commenced rapidly and preceded the onset of DNA synthesis in stimulated cells. Total uptake of transferrin during culture was much lower than that of iron, indicating that cells could remove iron from transferrin. The released transferrin appeared to be undergraded. Lymphoblasts bound six times more transferrin per cell than small lymphocytes. Lymphocytes also took up iron from citrate and nitrilotriacetate complexes, and iron so acquired was not readily removed by desferrioxamine, indicating that it was not bound extracellularly.     

Increased expression of transferrin receptors and iron in amoeboid microglial cells in postnatal rats following an exposure to hypoxia

This study was aimed to ascertain the effects of hypoxia on regulation of iron in the brain of newborn rats. At 3 h and 1 day after hypoxic exposure transferrin receptor expression as detected immunohistochemically with the antibody OX-26, and the iron content as shown by Perls' staining of amoeboid microglial cells was markedly increased. The induced changes, however, were not evident at 10 min and in longer surviving rats killed at 3 and 7 days. It is suggested that the upregulation of transferrin receptor expression coupled with iron uptake by amoeboid microglial cells in the periventricular regions is a protective mechanism to facilitate the sequestration of excess iron that may have been released either from the iron-rich oligodendrocytes, or accumulated due to a disruption of its normal transportation following the hypoxic insult. This would help protect the brain from harmful effects of iron.     

Abnormal iron deposition in renal cells in the rat with chronic angiotensin II administration.

Acute experimental iron loading causes iron to accumulate in the renal tissue. The accumulation of iron may play a role in enhancing oxidant-induced tubular injury by producing increased amounts of reactive oxygen species. From findings in cells from heme oxygenase-1 (HO-1)-deficient mice, HO-1 is postulated to prevent abnormal intracellular iron accumulation. Recently, it has been reported that HO-1 is induced in the renal tubular epithelial cells, in which iron is deposited after iron loading, and that this HO-1 induction may be involved in ameliorating iron-induced renal toxicity. We previously showed that chronic administration of angiotensin II to rats induces HO-1 expression in the tubular epithelial cells. These observations led us to investigate whether there is a link between iron deposition and HO-1 induction in renal tubular cells in rats undergoing angiotensin II infusion. In the present study, rats were given angiotensin II for continuously 7 days. Prussian blue staining revealed the distinct deposits of iron in the proximal tubular epithelial cells after angiotensin II administration. Electron microscopy demonstrated that iron particles were present in the lysosomes of these cells. Histologic and immunohistochemical analyses showed that stainable iron and immunoreactive ferritin and HO-1 were colocalized in the tubular epithelial cells. Treatment of angiotensin II-infused rats with an iron chelator, deferoxamine, blocked the abnormal iron deposition in kidneys and also the induced expression of HO-1 and ferritin expression. Furthermore, deferoxamine treatment suppressed the angiotensin II-induced increase in the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase, a marker of tubular injury; however, deferoxamine did not affect the angiotensin II-induced decrease in glomerular filtration rate. These results suggest that angiotensin II causes renal injury, in part, by inducing the deposition of iron in the kidney.     

Signaling in B cells via Toll-like receptors

Toll-like receptors (TLRs) and their ligands have emerged as important regulators of immunity, relevant to a wide range of effector responses from vaccination to autoimmunity. The most well-studied ligands of TLRs expressed on B cells include the lipopolysaccharides (for TLR4) and CpG-containing DNAs (for TLR9), which induce and/or co-stimulate B cells to undergo proliferation, class switching and differentiation into antibody-secreting cells. Recent developments in this area include advancements into our understanding of the role of these receptor pathways in B cells, and in particular the relevance of TLR9, which has received substantial attention as both a Th1-like inflammatory immunomodulator and a pathogenic co-stimulator of autoreactive B cell responses.     

Chronic iron overload in rats induces oval cells in the liver.

Liver damage induced by a variety of agents including hepatocarcinogens, alcohol, and virus induces proliferation of oval cells. In this study, iron overloading of the liver is used as a means of inducing liver damage over an extended period to ascertain whether it promotes the appearance of oval cells. Rats were fed a 2% carbonyl-iron-supplemented diet for 3 or 6 months. Extensive iron deposits appeared periportally in hepatocytes and some Kupffer cells. Iron deposition was less pronounced pericentrally. Small oval-like cells, morphologically and immunocytochemically similar to CDE-derived oval cells, were identified and quantified. They first emerged periportally and subsequently in small tracts or foci nearer central regions and stained positively for alpha-fetoprotein, pi-class glutathione S-transferase, and the embryonic form of pyruvate kinase. They contained very few iron deposits and were classified as iron free. The major difference between CDE- and iron-overload-derived oval cells was that the latter were negative for transferrin. This study shows that cellular changes occurring in iron-overloaded rat liver are similar to those observed in rats placed on a hepatocarcinogenic diet and in rats chronically exposed to alcohol.     

Immunohistochemical localization of glucocorticoid receptors in anterior pituitary cells of rats.

Adenohypophysial cells having a glucocorticoid receptor were immunohistochemically determined in rats. For detecting the presence of the glucocorticoid receptor, we used the monoclonal antibody for a glucocorticoid receptor (BuGR-2), and immunohistochemically examined the phenotypes of the cells that exhibited BuGR-2-immunoreactivity. The immunoreaction for the glucocorticoid receptor was confined to the nuclei of the majority of corticotrophs (70%) and of some somatotrophs in intact animals. Following adrenalectomy, all corticotrophs became significantly hypertrophic, losing their immunoreactivity for the glucocorticoid receptor. In contrast, somatotrophs that had also lost the immunoreactivity for the glucocorticoid receptor in the nuclei greatly diminished in size. Intraperitoneal administration of corticosterone was performed in adrenalectomized animals to supplement glucocorticoids. This treatment restored BuGR-2-immunoreactivity in the nuclei of some corticotrophs. In intact rats, immunolabeled corticotrophs were classified into two types, stellate and polyhedral. However, the immunoreaction for the glucocorticoid receptor was equally evident in the cell nuclei of these different types of cells. It is concluded that, in rats, both corticotrophs and somatotrophs are target cells of glucocorticoids, although these cell types display opposite growth responses to the removal of glucocorticoids.     

Transferrin receptors on tumor and bone marrow cells: Lack of involvement as target structure for natural killer cells

Summary Two different experimental approaches based on the specificity of monoclonal antibodies (mAbs) have been taken to verify the hypothesis that the transferrin receptor (TfR) on proliferating cells serves as a common target structure for natural killer (NK) cells. Thus, by the lysostrip technique the TfR was removed from the surface of K562 and Molt4 tumor cells by incubation with two different anti-TfR mAbs. The effect of removal of the TfR was controlled by uptake of radiolabeled transferrin, and by binding of non-cross-reacting monoclonal anti-TfR receptor antibodies. Though the modulation of TfR on the membrane of viable cells was nearly complete, the cells remained fully susceptible to NK lysis. The second approach consisted in removal of TfR-bearing cells from bone marrow cell suspensions by an indirect rosetting technique. Using mAbs bound to ox erythrocytes the rosetted TfR-bearing cells could be removed from bone marrow cell suspension by density centrifugation with an efficiency of >99%. It could be shown that both fractions, TfR⁺ and TfR^– cells, could be lysed to the same degree by NK cells. Thus, the evidence obtained speaks against a role of TfR as a recognition structure for NK cells.Abbreviations BM Bone marrow - mAb Monoclonal antibody - NK cell Natural killer cell - PBL Peripheral blood mononuclear cell - TfR Transferrin receptor This work is dedicated to Professor Hans Dierk Waller on the occasion of his 60th birthdayThis work was supported in part by the Deutsche Forschungsgemeinschaft, Bonn, SFB 217     

Effects of D-mannoheptulose upon D-glucose metabolism in pancreatic B and non-B islet cells.

D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, the heptose (10 mM) decreased the steady state content of dispersed rat pancreatic islet cells in D-[U-(14)C]glucose, and inhibited to a greater relative extent the utilization of D-[5-(3)H]glucose, the oxidation of D-[U-(14)C]-glucose and its conversion to radioactive amino acid when the dispersed islet cells were incubated at 16.7 mM rather than 2.8 mM D-glucose. A comparable situation was found in purified islet B-cells, whereas D-mannoheptulose only exerted minor to negligible effects upon the metabolism of D-glucose in non-B islet cells. This coincided with a much higher uptake of D-[(3)H]mannoheptulose by B, as distinct from non-B, islet cells. These findings indicate that the unexpectedly greater relative inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated islets (or dispersed islet cells) observed at high rather than low hexose concentration cannot be accounted for solely by differences in the relative contribution of non-B cells to total D-glucose metabolism by islets incubated at increasing concentrations of D-glucose. A comparable metabolic response to D-mannoheptulose is indeed observed in purified B cells. It could be attributable, in part at least, to D-glucose and D-mannoheptulose countertransport, resulting inter alia in a greater net uptake of the heptose by B cells exposed to a high concentration of the hexose.     

Ferritin and intestinal iron absorption: pancreatic enzymes and free iron.

Rat intestinal mucosa gave low yields of ferritin purified by standard procedures. The resulting ferritin had less protein relative to iron and migrated faster electrophoretically than ferritin from other rat tissues. Pancreatic duct ligation reduced these differences, suggesting digestive enzyme attack during ferritin isolation. Even in ligated rats, ferritin accounted for only 5-10% of mucosal iron. However, shortly after giving 59FeCl3 orally, 50% of mucosal radioactivity occurred in cell sap, about equally distributed between ferritin and low-molecular-weight (chelated?) iron. No other cell sap components were 59Fe labeled. Iron may thus be transported as a chelate with which ferritin is in rapid equilibrium. Mucosal ferritin content increased with age and iron treatment and decreased with iron deficiency. The iron-deficient rats showed accelerated 59Fe uptake into blood with little mucosal retention. One day after administering parenteral iron to deficient rats, 59Fe transfer to blood became retarded but 59Fe now accumulated excessively in the mucosa, suggesting that iron status affects transport more rapidly at the serosal than at the mucosal cell surface. A scheme for control of iron absorption is presented.     

Transferrin receptors and natural killer cell lysis. A study using Colo 205 cells exposed to 60 Hz electromagnetic fields

The role of target cell transferrin receptors (TfR) in natural killer (NK) cell-induced cytolysis has been studied using Colo 205 cells in which prior exposure to 60 Hz electromagnetic fields produced constitutive expression of maximum numbers of TfR. It was found that NK cell cytolysis was decreased in those cells expressing the highest levels of TfR, while cells expressing the lowest level of TfR were lysed to the greatest extent. Furthermore, pretreatment of target cells with, and the continued presence in the assay medium of, iron transferrin produced, in general, only a slight decrease in cytolysis. Incubation of cells with apotransferrin also produced only a slight decrease in cytolysis, while incubation with zinc transferrin produced a greater decrease in cytolysis. This latter effect was particularly dramatic for cells that had been exposed previously to either a 60 Hz magnetic field alone (1.0 gauss) or to a combined electric + magnetic field (current density 300 mA/m2, 1.0 gauss). In addition to indicating a discordance between TfR expression on target cells and their sensitivity to NK cell cytolysis, the data presented provide a further indication of cellular membrane changes produced by exposure to 60 Hz electromagnetic fields.     

Nonrandom positioning of Golgi apparatus in pancreatic B cells

The Golgi apparatus has a polarized distribution in a variety of cell types, and in certain epithelia its intracellular position correlates with the direction of vectorial transport and release of secretory products. We have studied quantitatively the orientation of the Golgi apparatus in pancreatic islet B cells by examining its location with respect to the juxtacapillary face of the cell. This was done a) at the light microscope level on semithin sections of osmium-impregnated islets, and b) at the ultrastructural level on thin sections of intact pancreas. The data obtained with both methods show that in pancreatic B cells the Golgi apparatus is preferentially located in the position opposite to the juxtacapillary face of the cell. These results suggest a structural polarization of B cells, which may be important for the coordinate function of islets of Langerhans.     

大鼠胰腺导管上皮细胞及转分化胰岛样细胞免疫学特性研究

目的:通过对大鼠胰腺导管上皮细胞、转分化的胰岛样细胞与天然胰岛细胞进行免疫原性比较,了解胰腺导管上皮细胞及转分化胰岛样细胞的免疫学特性.方法:大鼠胰腺导管上皮细胞,转分化胰岛样细胞和天然胰岛在体外与淋巴细胞共培养,检测淋巴细胞MHCⅠ、MHCⅡ抗原决定簇,IFN-γ和IL-2、IL-4水平.将三组细胞分别植入到大鼠糖尿病模型皮下,流式细胞技术检测外周血MHCⅠ、MHCⅡ表达、ELISA检测IL-2、IL-4水平和机体对其反应的病理学变化.结果:三组细胞与淋巴细胞共培养,MHCⅠ的表达未见明显差异(P＜0.05),MHCⅡ的表达胰岛样细胞(29.5%±3.1%)和天然胰岛细胞(32.6%±3.6%)较胰腺导管上皮细胞(10.8%±0.9%)明显升高(P＜0.05).淋巴细胞分泌IL-2水平和Elispot检测分泌IFN-γ的细胞数,胰腺导管上皮细胞较胰岛样细胞和天然胰岛细胞组显著性降低(P＜0.01).植入糖尿病大鼠模型的外周血IL-2水平和淋巴细胞MHCⅡ表达均随着植入时间逐渐升高,胰岛样细胞和天然胰岛细胞组较胰腺导管上皮细胞组升高明显,IL-4水平在三组细胞无明显差异.病理结果显示胰腺导管上皮细胞组淋巴细胞浸润较胰岛样细胞和天然胰岛细胞组为轻.结论:胰腺导管上皮细胞具有低免疫原性,转分化的胰岛样细胞免疫原性近似天然胰岛.     

Strategies for selective priming of memory B cells

Long-term humoral immunity elicited by pathogens and vaccines alike relies upon the generation of both memory B cells (B(mem)) and long-lived plasma cells (PCs). Virtually all vaccine formulations induce the concomitant emergence of both B(mem) and PCs, suggesting that the emergence of these two differentiated B cells subsets is commonly controlled. Evidence presented shows specific Toll-like receptor (TLR) agonists coupled with soluble protein antigen (sAg) can selectively induce the expansion of antigen specific B(mem) in the absence of PC generation. The co-administration of either TLR 3 or 9 agonists with sAg induced germinal centre (GC) formation, antigen-specific B(mem), but failed to substantively induce the generation of long-lived bone marrow (BM) PCs. Upon re-challenge, high levels of PCs were induced with concomitant high titres of antigen-specific serum IgG. Hence, vaccines can be developed that can prime and protect the host to subsequent infectious agents without initial, high levels of antibody production. Furthermore, these studies suggest that the signals that govern the expansion and differentiation of B(mem) can be uncoupled from those that induce long-lived BM PCs.     

The selective localization of B lymphocytes in the spleen and the role of complement receptors

The role of complement receptors on the localization of T and B cells in the spleen of mice was studied using short-term homing experiments in cobra venom factor (CoF)-treated animals. The localization ratio of B and T cells in the spleen of CoF-treated mice decreased significantly compared to control recipients. No changes could be found in the relative distribution of resident T and B cells in the spleen or other lymphoid organs of CoF-treated animals and when their spleen or lymph node cells were transferred, the localization pattern was normal. When cells were incubated in serum prior to transfer a disturbed localization ratio in the spleen of untreated recipients was observed. This was due to a blockade of complement receptors as determined by the inability of the incubated cells to form EAC rosettes. No blockade of EAC rosettes and no changes in localization ratios upon transfer could be observed when the cells were incubated in functionally C3-depleted serum. The results suggest a role for the complement-receptor on B cells in the initial localization in the spleen, whereas no influence upon the selective localization in high endothelial venules-bearing organs was found.