Expression of transferrin receptors during erythroid maturation

A monoclonal antibody, F111/2Dl, produced after immunisation of C3H/He mice with the human erythroleukemia cell line, K562, is described. It detects cell surface determinants of similar distribution to those characterised by the OKT-9 monoclonal antibody, which has been shown to identify the transferrin receptor. The F111/2Dl antibody, as well as OKT-9, has been used to investigate the distribution of transferrin receptors during erythroid maturation in normal bone marrow and peripheral blood, and on the K562 cell line during erythroid differentiation, induced in vitro.     

Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and ¹²⁵l-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor anti-bodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using ¹²⁵l-surface-labeled reticulocytes, it can be shown that the ¹²⁵l-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with ¹²⁵l-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0°C or in phosphate-buffered saline the rate of vesicle release is less than that at 37°C in culture medium. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.     

Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation

The transferrin receptor (TfR) of reticulocytes is released in vesicular form (exosomes) during their maturation to erythrocytes. The heat shock cognate 70-kDa protein (Hsc70) has been demonstrated to interact with the cytosolic domain of the TfR and could thus trigger the receptor toward this secretion pathway. We investigated the characteristics of the interaction between Hsc70 and the TfR in exosomes with an in vitro binding assay using TfR immobilized on Sepharose beads and purified Hsc70. The results show that Hsc70 binds to exosomal TfR with characteristics expected of a chaperone/peptide interaction. We demonstrated that heat-denatured luciferase competed for in vitro binding, dependent on the nucleotide bound to Hsc70, and that this interaction activates the ATPase activity of Hsc70. Moreover, we used immunosuppressive agents that interact with Hsc70, thus decreasing Hsc70 binding to TfR in our in vitro binding assay and enabling us to assess the role of this interaction in vivo during reticulocyte maturation.     

Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach.

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.     

Subcellular localization of the EGF receptor maturation process

The glycosylation and the processing of the epidermal growth factor (EGF) receptor are suggested to play a crucial role(s) in the activation of ligand binding activity. To examine whether the receptor acquires EGF binding activity in the endoplasmic reticulum (ER) or in the Golgi complex, we carried out parallel kinetic analysis of the EGF binding activity and the intracellular transport of the newly synthesized receptor by immunoprecipitation with the anti-EGF receptor antibody B4G7 using the EGF receptor hyperproducing cell line NA. The kinetic analysis revealed that a receptor capable of binding EGF appeared after 30 to 60 min labeling with [35S]methionine. Pulse-chase experiments also indicated that the receptor capable of binding EGF appeared after a 30-min pulse with a 30-min chase. Subcellular fractionation analysis indicated that the newly synthesized receptor was present in the Golgi complex after labeling with [35S]methionine for 30 min. After a 30-min chase, the Mr 170K receptor appeared in the Golgi complex and plasma membrane. Thus, these results together indicated that after a 30-min pulse incubation a fraction of the EGF receptors have been transported from the ER to the Golgi complex; however, the receptor is unable to bind EGF. Although the EGF receptor appeared on the cell surface after a 30-min pulse with a 30-min chase, only half of the receptors are capable of binding EGF. Therefore, the EGF receptor acquires ligand binding activity at a late stage of the maturation process, most likely in the Golgi complex.     

Expression of the chicken transferrin gene in transgenic mice

The chicken transferrin gene was microinjected into the male pronucleus of fertilized mouse eggs, and the eggs were then implanted into foster mothers. Approximately 15%-30% of the offspring from the injected eggs carried chicken DNA sequences; restriction mapping indicated that multiple copies of the chicken gene had integrated into the genome in a tandem arrangement in most of the mice. Six of the seven mice studied expressed the chicken gene, and in five mice there was a 5 to 10 fold preferential expression of chicken transferrin mRNA in liver compared to that in other tissues. Chicken transferrin was secreted into the serum of five of the mice, where it reached steady state concentrations up to 67 micrograms/ml. Offspring from transgenic parents also expressed the chicken gene; in some cases the expression in offspring was very similar to the parent, but in one line expression in offspring had increased 2 to 4 fold.     

Differential immunohistochemical resolution of the human mononuclear phagocyte system

Four new monoclonal antibodies, termed Ki-M1, Ki-M2, Ki-M3, and Ki-M4, were developed for distinguishing macrophage subgroups. Purified lysosomes of cells of stimulated U-937 cell line were used as immunogen. Specificity control was performed by staining unfixed cryostat sections of fresh human tissue with an immunohistochemical method, which allowed reliable recognition of reactive structures. Ki-M1 reacted with macrophages of lymphoid tissue, lung, and serous cavities. Ki-M2 recognized Kupffer cells and splenic macrophages. Both monoclonal antibodies reacted with interdigitating reticulum cells and Langerhans cells, which are thought to be accessory cells of the T-cell immune response. Accessory cells of the B-cell immune response, on the other hand, showed reactivity with Ki-M3 and Ki-M4. Thus, analogous to T lymphocytes, the human mononuclear phagocyte system (MPS) can be subdivided into different subgroups with the aid of appropriate monoclonal antibodies.     

Endocytosis by liver cells during stimulation of the mononuclear phagocyte system by yeast polysaccharides]

Endocytosis (phagocytosis, fluid-phase- and receptor-mediated endocytosis) by liver cells, lysosomal enzyme activities have been studied during macrophages stimulation by yeast polysaccharides. It was shown that like macrophages stimulator zymosan, yeast polysaccharides cryelan and rhodexman increased the carbon particles phagocytosis. The most effective was intravenous administration of yeast polysaccharides. Compared to rhodexman, the effect of cryelan was more prominent. Macrophages stimulation was followed by suppression of fluid-phase endocytosis by liver cells. Increased activity of cathepsin B was discovered on day 5 after macrophages stimulation (proteinase, most typical for macrophages enzymes).     

Modulation of mononuclear phagocyte function by intravenous gamma-globulin

To assess the effects on mononuclear phagocyte function of i.v. gamma-globulin treatment in idiopathic thrombocytopenic purpura, we examined in vivo and in vitro mononuclear phagocyte function in 11 patients before and after therapy. All patients, both splenectomized and non-splenectomized, demonstrated a prolongation of in vivo clearance of autologous IgG-sensitized erythrocytes (p less than 0.01). Concurrent in vitro assessment of blood monocyte function showed decreased IgG-sensitized erythrocyte (EA) rosette formation (mean +/- SD: 31.6% +/- 8.2 vs 24.5% +/- 9.5; p less than 0.03) and decreased affinity of Fc receptor-specific IgG oligomer binding (9.9 +/- 16.3 vs 1.8 +/- 2.1 X 10(8) M-1; p less than 0.008), but no consistent change in the estimate of the maximum number of binding sites. Phagocytosis of two different EA probes was decreased (EhuA:0.49 +/- 0.26 vs 0.25 +/- 0.14 erythrocyte/monocyte/hr; p less than 0.02, EoxA: 1.76 +/- 0.66 vs 1.27 +/- 0.67 erythrocyte/monocyte/hr, p less than 0.05). The change in in vivo mononuclear phagocyte system clearance was significantly correlated with the change in the association constant for oligomer binding (r = 0.98, p less than 0.05). These data demonstrate that i.v. gamma-globulin infusions induce alterations of mononuclear phagocyte function that are not dependent on the presence of autologous serum containing infusate. The change in apparent Fc receptor affinity rather than receptor number may reflect an altered Fc receptor population with different binding properties.     

The valence for ligand of the human mononuclear phagocyte 72 kD high-affinity IgG Fc receptor is one

The valence for ligand of the 72 kD high-affinity IgG FcR present on human mononuclear phagocytes was evaluated. Lysates of U937 cells whose high-affinity FcR had been saturated with equivalent quantities of 125I-IgG1 kappa and unlabeled IgG1 lambda or with 125I-IgG1 lambda and unlabeled IgG1 kappa were incubated with Sepharose-anti-kappa. Eighty-nine percent of the applied 125I-IgG1 kappa was bound, whereas 0.35% of the applied 125I-IgG1 lambda bound (mean of two experiments), indicating that if the receptors are occupied with ligand, the receptors bind only one ligand molecule at a time. Two experiments were performed to show that the receptors were ligand-occupied. First, a monoclonal antibody directed against the 72 kD FcR (FcRmab32) was added to lysates of U937 cells saturated with equal quantities of 125I-IgG1 lambda and IgG1 kappa. This anti-FcR antibody caused a dose-dependent sevenfold increase in the amount of 125I-IgG1 lambda bound to the anti-kappa immunoadsorbent (presumably by cross-linking receptors bearing 125I-IgG1 lambda with receptors bearing IgG1 kappa), whereas monoclonal antibodies (MMA and IV3) directed against two other determinants on U937 caused no such increase. In the second experiment, Sepharose-FcRmab32 adsorbed 60% of the 125I-IgG1 kappa and 46% of the 125I-IgG1 lambda applied in a U937 lysate (bearing high-affinity FcR), whereas only 3% of 125I-IgG1 kappa and 6% of 125I-IgG1 lambda applied in a K562 lysate (bearing no high-affinity FcR) were adsorbed. We interpret these data to indicate that in detergent solution the valency of the high-affinity FcR on U937 cells is one.     

The response of the mononuclear phagocyte system to chronic hyperglycemia

In this study we revealed a common reaction of cells of the mononuclear phagocyte system in the central and peripheral immunopoietic organs and pancreas in rats with chronic hyperglycemia. The activation of monocytopoiesis and recruitment of mononuclear phagocytes in peripheral tissues were observed. The modulation of the functional activity of mononuclear phagocytes by 3-aminophthal-hydrazide contributed to the normalization of monocytopoietic intensity and a decrease in the level of macrophagal infiltration in the thymus, pancreas, and peripancreatic lymph nodes. These changes indicate that mononuclear phagocytes are involved in the adaptive response to chronic hyperglycemia.     

The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1

The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.