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Inflammation & Allergy - Drug Targets, 2008, 7, 41-52 41 Transferrin and the Transferrin Receptor: Of Magic Bullets and Other Concerns Maria F. Macedo*,1,2,3 and Maria de Sousa1,4 1 IRIS, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal 2 ESS Vila Nova de Gaia, Instituto Piaget, Portugal 3 SACS, Secção Autónoma Ciências da Saúde, Universidade de Aveiro, Portugal 4 ICBAS, Instituto de Ciências Biomédicas Abel Salazar Universidade do Porto, Portugal Abstract: Transferrin (Trf) is a highly conserved serum glycoprotein mostly known for its iron transport capacity. As iron is an indispensable nutrient for cell division, Trf and its receptor have long been used as targets of pharmacological intervention mostly for cancer therapy and for diagnosis in inflammation. In recent years several independent pieces of work including data from our group, indicated that Trf can also have an iron independent role in the immune system. In this article new emerging roles of Trf and its receptor on iron independent processes and in drug delivery are reviewed. Keywords: Transferrin, transferrin receptor, immunology, inflammation, drug targets, blood brain barrier, cancer. THE PROTEIN AND ITS PRINCIPAL KNOWN ROLE The main role of Trf is the transportation of iron from the sites of absorption (duodenum) and red blood cell recycling macrophages to virtually all tissues, particularly tissues involved in erythropoiesis and those tissues with actively dividing cells. In addition, Trf accumulates in locals of inflammation due to the leakage of plasma proteins and to the expression of Trf receptors by inflammatory cells. Trf belongs to a group of closely related proteins found in all vertebrates. The Trf family of metal binding proteins, includes lactotransferrin or lactoferrin (found in abundance in milk, in a variety of intracellular fluids and in specific granules of polymorphonuclear leukocytes), ovotransferrin (present in the egg white) and melanotransferrin (present in the majority of human melanomas [reviewed in 1]. All these proteins show a large degree (around 40%) of amino acid sequence homology [reviewed in 2]. Trf vital role is illustrated by the hypotransferrinemic (Trfhpx/hpx) mouse inability to survive without an external source of Trf [3]. Human Trf consists of a polypeptide chain containing 679 amino acids. The Trf molecule is composed of two domains, homologous to each other, the N-terminal sequence (residues 1 to 336) and the C-terminal sequence (residues 337 to 679) [4]. The crystallographic structure of Trf shows two globular lobes, representing the N- and C-terminal sequences [reviewed in 5,6]. Each of the two lobes (C-lobe and N-lobe) can be further divided into two domains of similar size. In each lobe there is an iron-binding site located in the cleft between the two domains. During iron uptake and release the domains suffer conformational changes causing closing and opening of the cleft [reviewed in 5,6]. *Address correspondence to this author at the IRIS, IBMC, Rua do Campo Alegre 823, 4150-180 Porto, Portugal; Tel: +351 22 6074956; E-mail: [email protected] 1871-5281/08 $55.00+.00 SYNTHESIS, CIRCULATING LEVELS AND GENE EXPRESSION The main source of Trf is the liver [reviewed 2,7]. Other organs have been reported to produce Trf, namely the testis (Sertoli cells) [8], and brain (oligodendrocytes, choroid plexus and cerebellum) [9-11]. Trf has also been detected in placenta, stomach, heart, kidney, lung, mammary glands, immune system cells and tumor cells [reviewed in 7, 10, 1214]. Several studies using different techniques reported the presence of Trf in immune system cells. Earlier studies reported low Trf expression in human lymph nodes and peripheral blood lymphocytes, as well as in rodent spleen, newborn thymus, macrophages and peripheral blood leukocytes [reviewed in 7]. In 1980, Nishiya and co-workers using immunofluorescence reported the presence of iron binding proteins in human peripheral blood cell populations. These authors reported that Trf was found in T cells and polymorphs and only very few Trf containing cells were detected in the B cell and monocyte fraction [15]. Lactoferrin was present in polymorphonuclear cells. Later Djeha and coworkers studied Trf synthesis by lymphomyeloid cells. Trf synthesis was observed in mouse macrophages but mouse lymphocytes or thymocytes did not express Trf [16,17]. In humans, contrary to what happened in mice, Trf synthesis was reported in T lymphocytes, specifically CD4+ T lymphocytes [18], but monocyte/macrophages were negative for Trf [17]. Effect of Iron In Vivo Increased serum Trf levels are found in iron deficiency anemia. Decreased plasma Trf is found in association with iron overload, cirrhosis and protein malnutrition [reviewed in 7]. In accordance with the human data, chicken and rat models of iron deficiency have also shown increased Trf mRNA levels in the liver [19-21]. In both cases the increase in liver Trf mRNA results in an increase in serum Trf. In irondeficient rats, however, Tuil and co-workers did not observe © 2008 Bentham Science Publishers Ltd. 42 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 alterations in liver Trf mRNA levels [22]. Discrepant results concerning the influence of iron on Trf levels have also been reported in animals subjected to iron overload. In rats subjected to iron overload, by a diet supplemented with carbonyl iron, no alteration was found in the level of Trf synthesis [19,22]. Opposite results were obtained in mice expressing the human Trf gene. When these mice were subject to iron overload, by intraperitoneal injections of ferric chloride and ferric nitrate, a decrease in both human Trf and in the endogenous mouse Trf levels were observed [23,24]. Such discrepancies should alert us for the necessity of only comparing results of similar experimental protocols. TRANSFERRIN AS A CELL GROWTH NUTRIENT More than thirty years ago several studies were done to identify relevant nutrients for cell growth in vitro [25-30]. Most of the earlier studies were done in mouse or human lymphocyte cell cultures. Trf was also reported as a neurotrophic factor, isolated from peripheral nerves, and acting as a myogenesis inducer [31]. In the central nervous system Trf has marked pro-myelinating effects and induces oligodendroglial cell differentiation [32-35]. Trf was also identified as the main serum component stimulating the metanephric kidney differentiation in an in vitro organ culture model. Experiments with Trf-depleted serum suggested that other factors in serum could not replace Trf in the stimulation of kidney differentiation [36]. Several studies indicate that media in the absence of Trf do not support lymphocyte proliferation or if they do, cell growth occurs at a lower rate [28,37]. Phillips and Azari, using human lymphocytes cultured in serum-free medium showed lymphocyte proliferation in response to phytohemagglutinin (PHA) when iron-bound Trf was added to the medium, but not if apo-Trf was used, nor if the iron was added as ferric citrate [28]. However, the K562 cell line was grown for many years in Trf-free media containing ferrosulphate as a source of iron [38]. Other studies reported good lymphocyte proliferation with other iron donors rather than Trf [39] underlying once more the need to examine and evaluate results and conclusions in the context of the experimental protocols used. THE TRANSFERRIN RECEPTOR Iron-bound Trf is internalized by cells expressing receptors specific for Trf by receptor-mediated endocytosis [reviewed in 40]. The Trf receptor is a disulfide-linked homodimer. In humans, each monomer consists of 760 amino acids. Each monomer possesses a short cytoplasmic Nterminus region, a single transmembranar domain and a large glycosylated extracellular region. The extracellular domain is involved in ligand binding. Each monomer can bind to one molecule of Trf. The intracellular segment contains a site for receptor phosphorylation triggered by protein kinase C activators [41]. For several proteins phosphorylation is an important mechanism of signaling. However, Trf receptor phosphorylation does seem to play an important role in receptor endocytosis as mutations of the Trf receptor phosphorylation site do not prevent Trf internalization [42,43]. Transferrin Receptor Expression The Trf receptor is highly expressed on the surface of proliferating normal and neoplastic cells [44], erythrocyte Macedo and de Sousa precursors and placenta cells [44, 45, reviewed in 46]. Trf receptor is also present in other tissues like the endocrine pancreas, liver, muscle cells, kidney, testis and pituitary glands [44, 47, reviewed in 48]. In general, proliferating cells express Trf receptor contrasting with resting cells in which the expression is absent or extremely rare [49]. During inflammation monocytes leave the blood and differentiate into macrophages at the extra vascular tissues, macrophages express surface Trf receptors [50-53]. T cell differentiation is characterized by steps of intensive proliferation both in fetal [54], and in adult thymus [55]. Approximately, 10 to 20% of all thymocytes are dividing [56, 57]. In accordance with the percentage of dividing thymocytes, the proportion of thymocytes expressing Trf receptor is between 15 to 20%, both in mouse and humans [58, 59]. Some mature cells also require increased amounts of iron like fully differentiated muscle cells and terminally differentiated syncytiotrophoblast in the placenta. Completely differentiated muscle cells in vitro express high levels of Trf receptor [47]. This may be related to its increased iron need because of myoglobulin production. The presence of the Trf receptor on the terminally differentiated syncytiotrophoblast in the placenta is key to the iron transfer function of this maternal-fetal barrier layer [45]. Developing erythroid cells need large amounts of iron for hemoglobin synthesis. Trf receptor increases before the peak of hemoglobin synthesis and stays elevated for a period of time during erythrocyte development as iron uptake remains high [60,61]. As hemoglobin production shuts down, during erythroid maturation, the number of surface Trf receptors also decreases [60,61]. Fully mature erythrocytes no longer express Trf receptors [reviewed in 46]. The regulation of Trf receptor expression differs in erythroid and non-erythroid cells. In non-erythroid cells, Trf receptor expression is controlled at the post-transcriptional level by the iron status of the cell [reviewed in 48, 62]. Five iron response elements (IREs) are located in the 3’untranslated region (UTR) of the Trf receptor mRNA. IREs are nucleotide sequences that form a stem-looped secondary structure. Iron regulatory proteins (IRPs) are RNA binding proteins that regulate the expression of mRNAs containing IREs. In iron-starved cells, the IRPs bind to the IREs. The IRE/IRP interaction results in stabilization of the otherwise unstable Trf receptor mRNA. This results in a subsequent increase in Trf receptor protein levels and expression at the cell surface. Conversely, when intracellular iron is high, IRPs do not bind to the IREs. Trf receptor mRNA degradation is thus enhanced, decreasing Trf expression and limiting iron uptake by the cell. IREs are also present in the mRNAs coding for other iron related proteins like ferritin, DMT1 and ferroportin [63-65]. Transferrin Receptor and Lymphocyte Proliferation As mentioned earlier the Trf receptor is present on the surface of proliferating normal cells and on neoplastic cells [44]. Several studies reported the importance of Trf receptor for lymphocyte cell division. Drugs that inhibit lymphocyte proliferation, like the ion channel-blocking agent diltiazem, interferon and cyclosporin, also down regulate Trf receptor expression in activated lymphocytes [66-68]. The results of the use of monoclonal antibodies against Trf receptor give Transferrin and the Transferrin Receptor Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 43 further support to the importance of Trf on cell growth, an effect reviewed below in the “Transferrin and Transferrin receptors as targets of pharmacological intervention” section of this paper. central level, namely in a specific step of early T cell differentiation in the thymus, and in the periphery (Table 1). THE TRANSFERRIN RECEPTOR 2 Results of studies done by us in Trfhpx/hpx mice [3] show that Trf via its interaction with the Trf receptor is important in a specific step of early T cell differentiation in the thymus [84]. Trfhpx/hpx mice carry a spontaneous point mutation in a splice domain of the Trf gene leading to a severe deficiency of serum Trf [85]. These mice present less than 1% of normal serum Trf values and need to be treated with a source of Trf during at least the first four weeks of life [85]. Our analysis of the Trfhpx/hpx mice immunologic system revealed no alteration in the number of T cells in the lymph nodes. The thymus, however, had a reduced cellularity with no major differences in the proportion of its four major thymic populations: Triple Negative, TN (CD4-CD8-CD3-), Double positive, DP (CD4 +CD8 +), Single Positive for CD4 CD4SP (CD4+CD8-) and CD8, CD8SP (CD4-CD8+) [84]. Analysis of the proliferation/apoptosis levels did not explain the reduced thymocyte numbers. The proliferation of the four major thymocyte populations was not diminished and there was no alteration in apoptosis. The TN thymocytes, the most immature of the four major thymocyte populations can be further subdivided into four subpopulation according to the expression of the CD44 and CD25 cell surface markers: TN1 (CD44+CD25-), TN2 (CD44+CD25+), TN3 (CD44-CD25+) and TN4 (CD44-CD25-). A more detailed analysis of the TN thymocytes in Trfhpx/hpx mice revealed a specific leaky block at early thymocyte differentiation from the passage of TN3 to TN4 [84]. A human gene homologue of the Trf receptor, designated Trf receptor 2, was identified and mapped on chromosome 7 [69]. This gene generates two different transcripts, the and the transcript. The transcript is mostly expressed in the liver whereas the transcript is expressed ubiquitously at low levels. The deduced amino acid sequence corresponding to the transcript exhibits 45% identity, in its extracellular domain, with the classical Trf receptor [69]. The transcript encodes for a protein lacking the N-terminal portion of the transcript. In contrast with what happens for the classical Trf receptor, Trf receptor 2 expression is not altered by iron levels [70-72]. In accordance with these results, it was shown that the Trf receptor 2 mRNA does not contain IREs [70]. Trf receptor 2 protein levels are regulated by the levels of diferric Trf available [73, 74]. The affinity of Trf receptor 2 for Trf is 25-fold lower than the affinity of the classical Trf receptor [75]. A recent study indicates that Trf receptor 2 mediates uptake of non-Trf bound iron besides the Trf bound iron [76]. In the erythroid cell line K562, the co-precipitation of the two Trf receptors was observed and Western-blot analysis showed that they can form heterodimers [77]. Several mutations have been described in Trf receptor 2 associated with an iron overload phenotype [78-83]. TRANSFERRIN AND THE TRANSFERRIN RECEPTOR: EMERGING ROLES IN THE IMMUNE SYSTEM There is growing evidence that Trf and Trf receptor have immunologic roles unrelated to its iron transport capacity. Trf, and its receptor, have been shown to play important roles at a Table 1. Central Effects Trfhpx/hpx mice present different defects in addition and related to the low levels of serum Trf, namely anemia and massive iron deposition in almost every organ. Transferrin and Transferrin Receptor: Emerging Roles in the Immune System Role Central role Model Ref. Trf deficiency leads to a leaky block on early T cell differentiation (from the TN3 to the TN4 stage) Hypotransferrinemic mice [84] Trf receptor heterozygosity leads to a leaky block on early T cell differentiation (from the TN3 to the TN4 stage) Trf receptor heterozygous mice [84] Trf receptor deficiency is associated with deficient lymphopoiesis more dramatic in the T than in the B compartment. Trf receptor knockout chimeric mice [87] Trf receptor is a receptor for IgA [88] Trf receptor over expression on mesangial cells is associated with IgA nephropathy IgA nephropathy [88] Trf receptor expression on enterocytes, via IgA, is responsible for the transport of the gliadin peptides (peptides known to induce the celiac disease) Celiac disease [95] Comitogenic activity of the Trf receptor Peripheral role T lymphocyte stimulation occurs, after incubation with Trf or with anti-Trf receptor antibody, with phosphorilation of the TCR chain and association of Trf receptor with the TCR chain. [89, 90] In vitro models of T cell activation Relocation of Trf receptor to lipid rafts after T cell activation [96] Anti-Trf receptor antibody A24 induces apoptosis in T cells from ATL patients Cells that fail to cluster the Trf receptor after galectin-3 binding do not die Anti-Trf receptor antibodies induces apoptosis in chicken lymphoid cells [91] [97] In vitro models of T cell death [93] [98] 44 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 T cell differentiation was also studied in Trf receptor heterozygous animals [84], as the Trf receptor knockout mice are not viable dying at embryonic day 12.5 [86]. Trf receptor heterozygous mice were examined and shown to present a similar defect in thymocyte differentiation, namely a leaky block in the differentiation from TN3 to TN4 indicating a specific function of Trf in early T cell differentiation, a defect mediated by the Trf receptor [84]. Furthermore, studies of Trf receptor knockout chimeric animals, generated by introducing Trf receptor knockout embryonic stem cells (ESC) into wild-type blastocysts, also demonstrated the importance of the Trf receptor in the differentiation of T lymphocytes with a less dramatic effect on the production of B cells [87]. In the RAG2 knockout background two out of six chimeric mice showed Trf receptor knockout ESC contribution at the bone marrow level, these two mice had B cells in the spleen and lymph nodes [87]. None of the mice had T cells in the spleen or lymph nodes stressing the seemingly greater importance for T cells of Trf and its receptor. In the thymus the chimeric mice had cells negative for CD4 and CD8, corresponding to the DN thymocytes, representing the most immature stage of thymocyte differentiation, or/and corresponding to other cells rather than thymocytes. Peripheral Effects The Trf receptor is a receptor for the IgA1 class of antibodies [88]. There is consisting data showing the role of Trf receptor in the activation of T cells by a process independent of its iron uptake capacity [89-91]. Trf receptor has also been found to be a marker that enables to discriminate between activated (higher Trf receptor expressing cells) and anergic T cells (lower Trf receptor expressing cells) [92]. In addition Trf receptors have been connected with galectin-3 induced T cell apoptosis [93]. IgA1 Nephropathy Trf receptor over expression was associated with the pathogenesis of IgA1 nephropathy [88]. Indeed, the discovery of Trf receptor as an IgA1 receptor was first demonstrated in IgA1 nephropathy. IgA1 nephropathy is a condition in which there is deposition of IgA1 and IgA1 immune complexes in the kidney mesangial region. In kidney biopsies of patients with IgA1 nephropathy Trf receptor is overexpressed and co-localizes with IgA1 mesangial deposits. Anti-Trf receptor antibody and Trf block IgA binding to the mesangial cells. Purified polymeric IgA1 is a major inducer of Trf receptor expression in quiescent human mesangial cells. IgA-induced human mesangial cell proliferation is dependent on Trf receptor engagement [88, reviewed in 94]. Celiac Disease Celiac disease is an inflammatory disease induced by gluten-derived peptides. In this disease Trf receptor was shown recently to be highly expressed and colocalize with the IgA on the apical pole of enterocytes [95]. Furthermore the intestinal transport of the gliadin peptides p31-49 and 33mer (peptides known to induce the celiac disease) was blocked by soluble Trf receptor in ex-vivo experiments using human intestinal biopsies [95]. These data indicate a role for the Trf receptor, which is abnormally expressed on the apical pole of enterocytes, in the retrotranscytosis of IgA/gliadin peptide complexes in the celiac disease. Macedo and de Sousa T Cell Activation Studies with antibodies against Trf receptor present consistent data indicating a role of the Trf receptor in T cell activation by a process independent of its iron uptake capacity, as most of the studies were done with anti-Trf receptor antibodies and not with Trf [89-91]. Early studies, using anti-Trf receptor antibodies, show a co-mitogenic capacity of this molecule. J64 is an anti-Trf receptor antibody that binds to an epitope different from the Trf binding site. This antibody in combination with PHA or calcium ionophores induces IL2 secretion by the human T cell line HUT 78 [89] a marker of T cell activation. The anti-Trf receptor antibody FG 1/6, in the presence of submitogenic doses of phorbol esters, induced cell proliferation and IL-2 secretion by normal and transformed human T lymphocytes [90]. Later in 1995 Salmerón and co-workers went further in the characterization of the Trf receptor function in T cell activation. In that study it was shown that T lymphocyte stimulation occurred using Trf or the anti-Trf receptor antibody FG 1/6, without the use of other source of stimulation. Lymphocyte stimulation resulted in tyrosine phosphorylation of the TCR chain. More importantly, the Trf receptor was shown to associate physically with the TCR chain as well as ZAP70 tyrosine kinase [91]. In addition to the lymphocyte activation observed by stimulation with the Trf receptor, lymphocyte stimulation with a mitogenic mixture of anti-CD3 antibodies leads to the relocation of Trf receptor into lipid rafts (membrane microdomains that participate in the building of the immunological synapse) [96]. Cell Death The balance between lymphocyte cell death and survival is critical for normal immune development and homeostasis. Galectins are a family of mammalian proteins that positively and negatively regulate cell death. The Trf receptor, on the surface of T cells, is able to bind galectin-3 [93]. Galectin-3 induces clustering of the Trf receptor on dying cells. Cells that fail to cluster the Trf receptor, after galectin-3 binding, do not die. These data suggest a role of Trf receptor in the galectin-3 induced T cell death pathway [93] and illustrate another possible role for the receptor in lymphocyte homeostasis, seemingly iron independent. In addition, two independent pieces of work reported apoptosis induction, in vitro, by anti-Trf receptor antibodies [97,98]. Anti-Trf receptor antibody A24 blocked proliferation and induce apoptosis in human T cells from acute and chronic adult T cell leukemia/lymphoma [97]. A recent paper, aiming to identify cytotoxic antibodies against chicken lymphoid cells, identified the Trf receptor as a cell-death receptor as incubation with the anti-Trf receptor antibody induced apoptosis in chicken lymphoid cells [98]. TRANSFERRIN AND TRANSFERRIN RECEPTORS AS TARGETS OF PHARMACOLOGICAL INTERVENTION Once it became clear that the Trf receptor is expressed in inflammation and in proliferating malignant cells as briefly reviewed above it was natural to try to make use of Trf receptor binding in diagnosis and in therapy of both conditions. As described in the next sections a great deal more work has been done in cancer than in inflammation, nevertheless, studies in both situations are reviewed. Transferrin and the Transferrin Receptor Diagnosis 111 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 45 In Vitro Studies 111 Indium chloride ( InCl3) is a radionucleotide used clinically in imaging. It competes for the iron-binding site of Trf and therefore can be used to identify cells expressing Trf receptors. Indium has been used in the diagnosis of cancer and inflammatory arthritis. Radioimaging experiments with 111InCl3 were conducted early in animal models of arthritis and tested in patients with rheumatoid arthritis. In a rabbit model of antigen-induced knee monoarthritis [99]. Rabbits with monoarticular disease were injected with 111InCl3 and imaged at 30 minutes and 24, 48, 72 and 96 hours. In control knee joints, the ratio of activity in the knee to a soft-tissue region in the thigh remained constant (1.5-1.85:1); in arthritic knees, the 111InCl3 uptake ratio increased from 2:1 to 3.2, 3.5, 3.9, and 4.6:1 at the later time points. The difference between the knee-to-soft-tissue ratios observed in the control and involved joints were significant (p less than 0.01, 48-96 hr). Tissue distribution measurements demonstrated that the synovium and intraarticular structures covered by synovium in arthritic joints had the highest concentration of 111InCl3. In control joints, these tissues were found to take up tenfold less activity [99]. In a rat collagen model of proliferation synovitis 111InCl3 imaging revealed significant differences between the detection levels of affected joints in rats immunized with collagen than in non-immunized rats [100]. The efficacy of 111InCl3 was also tested in rheumatoid arthritis patients to establish the pattern of joint involvement, and response to treatment. Positive images correlated significantly with conventional clinical measures of arthritis [100-102]. In the 80s and 90s several studies were also published describing the use of 111InCl3 in tumor detection. In a rat tumor model of lymph node metastases 111InCl3 labeling correlated significantly with the incorporation of tritiated thymidine by H-4-II-E rat hepatoma cells in vivo and with expression of Trf receptor by the cells when placed into culture [103]. Later Watanabe and co-workers reported the tumor specific localization of 111InCl3 in several human malignant tumor xenografts in nude mice, including human malignant neuroblastoma SK-N-MC, pulmonary papillary adenocarcinoma NCI-H441, pulmonary squamous cell carcinoma PC 9, and colon adenocarcinoma LS 180 [104]. In humans 111 InCl3 was also useful to visualize the gastric involvement of adult T-cell leukemia [105]. More recently 111InCl3 has been used coupled with monoclonal antibodies against tumor antigens to increase the specificity of the signal [reviewed in 106 - 108]. Therapy The association of Trf receptor expression with cell proliferation has for many years motivated research into the possibility of using Trf and Trf receptors as targets of pharmacological intervention both in diagnosis and in therapy. Particularly in the area of cancer therapy as proliferating malignant cells express Trf receptor at high levels. Three types of studies have been conducted: in vitro, using cell lines and also primary cells; in vivo in experimental models of disease and in human clinical trials (Table 2). Several studies with a variety of cell lines and also primary cells were done showing in vitro inhibition of cell proliferation after incubation with anti-Trf receptor antibodies. These studies included: murine myeloma cells [109], mouse T lymphoma cell lines [110], human T, B and myeloid cell lines as well as activated peripheral blood lymphocytes [66,111-116], granulocyte/ macrophage progenitors (CFUGM) isolated from normal volunteers and patients with chronic myelogenous leukemia [115], T lymphocytes isolated from patients with acute and chronic adult T cell leukemia [97], Mantle cell lymphoma cells [117], erythroid and melanoma cell lines [118]. Some studies, however, reported lack of cell growth inhibition by anti-Trf receptor antibodies, indicating that the cell proliferation inhibitory capacity is dependent on the antibody and the cell type used and pointing once again to the need to examine carefully results of distinct experimental protocols. For example, the anti-mouse Trf receptor antibody R17 208 prevented cell growth of a mouse T lymphoma cell line but other antibodies (R17 217, RL34-14 and RR24) did not significantly inhibit cell growth in a careful study done by the same group, using the same cells [110]. In separate experiments by another group using a different anti-human Trf receptor antibody (code 128.1) did not inhibit the growth of the human T-Leukemia CCRFCEM or the erythroid K562 cell line [118]. According to Taetle and Honeysett hematopoietic cells and cell lines appeared to be more sensitive to the growth inhibitory effects of anti-Trf receptor antibodies than non- hematopoietic cell types [119]. In addition to the anti-proliferative proprieties of these antibodies there is evidence that Trf receptor binding can induce apoptosis (see galectin induced cell death). The anti-Trf receptor antibody A24 induced apoptosis of T lymphocytes isolated from patients with acute and chronic adult T cell leukemia [97]. A recent study with chicken cells identified anti-Trf receptor antibodies able to induce apoptosis in chicken lymphoid tumor cells [98]. Studies with the gambogic acid, a xanthone derived from Garcinia hanburyi, demonstrated the role of Trf receptor in inducing apoptosis after binding to gambogic acid [120] an effect similar to that referred to above with galectin [93]. In the case of binding of gambogic acid to the Trf receptor the mechanism seems to involve stimulation of TNF-induced apoptosis by inhibition of the NF-kB signaling pathway [121]. In Vivo Experimental Models of Disease The efficacy of these antibodies was soon tested in vivo. In an early study of the action of the anti-Trf receptor antibody, it was shown that it was effective in prolonging survival in a murine leukemia model [122]. In accordance with this result a combination of two anti-Trf receptor antibodies impaired the growth of a mouse B cell lymphoma [123]. In order to test the in vivo efficacy of the anti-Trf receptor antibodies against human neoplastic cells xenograf transplanted tumor models were tested in nude mice. Nude mice fail to develop the thymus and therefore do not have T cells at the periphery [124] and can tolerate permanently xenograf transplants [125]. Two examples of these studies will be described here. Treatment of nude mice bearing established human T leukemia tumors (CCRF-CEM cell line) with a 46 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 Table 2. Macedo and de Sousa Target Transferrin Receptor with Antibodies Inhibits Cell Proliferation and Induces Apoptosis Study Category In vitro studies In vivo experimental models Clinical trials Cell/Tumor Model Effect Ref. Mouse myeloid cell lines Cell proliferation inhibition [109] Rat myeloid cell line (Y3-Ag1.2.3) Cell proliferation inhibition Apoptosis [127] Rat T lymphoid cell line (C58) Cell proliferation inhibition Apoptosis [127] Mouse T lymphoid cell line (AKR1) Cell proliferation inhibition [110] Human T lymphoid cell lines (CCRF-CEM, 255.88, JURKAT, HPB, MOLT-4, 8402, HSB2) Cell proliferation inhibition [111, 116, 118] Human B lymphoid cell lines (8866, SB, GM4672, UC7296) Cell proliferation inhibition [116] Human myeloid cell line (HL60, 8226, U266, U937, KG-I) Cell proliferation inhibition [113-116] Activated peripheral blood lymphocytes Cell proliferation inhibition [66, 112, 116] Granulocyte/ macrophage progenitors (CFU-GM) Cell proliferation inhibition [115] T lymphocytes from patients with acute and chronic adult T cell leukemia Cell proliferation inhibition Apoptosis [97] Mantle cell lymphoma cells Cell proliferation inhibition Apoptosis [117] Erythroid cell line (K562) Cell proliferation inhibition [118] Melanoma cell line (M21) Cell proliferation inhibition [118] Murine leukemia model Prolonging survival [122] Mouse B cell lymphoma Inhibit tumor growth [123] Nude mice bearing established human TLeukemia tumors (CCRF-CEM cell line) Inhibit tumor growth and in some cases led to complete tumor regression [118] Nude mice bearing established human mantle cell lymphoma Prevent tumor establishment and delay tumor progression of established tumors, prolonging mice survival [117] 27 patients with advance cancer of different origins (epithelial, mesenchymal and hematopoietic) 3 patients with tumors from hematopoietic origin show a mixed anti-tumor response [126] combination of two anti-Trf receptor antibodies (D65.30 and A27.15) inhibited tumor growth and in some animals led to complete tumor regression [118]. The anti-human Trf receptor A24 totally prevented xenografted mantle cell lymphoma establishment in nude mice and delayed tumor progression of established tumors, prolonging survival [117] and thus corroborating the in vitro results obtained with the same antibody [97]. Clinical Trials In a phase I clinical trial with anti-Trf antibody (42/6) the antibody was administered to 27 patients with advanced cancer of different origins (epithelial, mesenchymal and hematopoietic). The treatment was well tolerated and three patients, with tumors of hematopoietic origin, demonstrated a mixed anti-tumor response [126]. In the in vivo experiments in humans, introducing a mouse anti-human Trf receptor antibody provokes the production of anti-mouse antibodies by the patient thus reducing the efficacy/activity of the anti-Trf receptor antibody. Moreover the immune effector system, like the complement and the Fc (Fragment crystallizable) receptors at the surface of phagocytic cells, does not effectively interact with the murine antibody constant region. To circumvent this, chimeric anti-Trf receptor antibody fusion proteins were prepared in which the constant region of the antibody is substituted by a human constant region. Chimeric anti-human Trf receptor antibodies were produced with the human Fc region. These antibodies inhibit proliferation and directly induced apoptosis in hematopoietic derived cell lines [127]. Moreover using the human tumor cells expressing Trf receptor as target cells, and normal human Peripheral Blood Mononuclear Cells (PBMC) as effector cells, the human Fc fragment of the chimeric anti-human Trf receptor can perform both the antibody-dependent cell-mediated cytotoxicity and the complement-dependent cytotoxicity [128]. In vivo studies in nude mice-bearing human liver cancer xenografts demonstrated tumor specific localization of the chimeric antibody [128]. Transferrin and the Transferrin Receptor ANTI-TRANSFERRIN RECEPTOR ANTIBODIES AND TRANSFERRIN AS DRUG CARRIERS Several drugs have been conjugated to Trf and tested for its efficacy against cancer cells in vitro and in vivo. The conjugates were significantly less toxic, in vivo, than the free drugs due to lack of cytotoxicity affecting normal cells avoiding in this way some of the non–specific side effects observed by the drug alone. In Vitro Studies Adriamycin (ADR) conjugated with Trf has been shown to be toxic for several human cell lines namely, leukemia, erythroleukemia, colorectal carcinoma, breast adenocarcinoma, mesothelioma, liver carcinoma, cervical adenocarcinoma [129-133]. The same drug (ADR) was also effective when conjugated to anti-Trf receptor antibody against human Daudi B and Raji Burkett’t lymphoma cell lines [134]. Cisplatin is another drug that was used conjugated to Trf to inhibit the growth of the human epidermoid carcinoma cell line (A431) [135]. The drugs chlorambucil and daunorubicin were tested as conjugated with Trf showing increased cytotoxicity in human breast cancer cell (MCF-7) and small cell lung carcioma (NCI-H69) compared with drug alone [136,137]. A number of toxins, with capacity to inhibit protein synthesis, were used conjugated with Trf or with Trf receptor into therapeutic strategies against malignant cells [for detailed review see 138]. Among the plant derived toxins it is worth noting the following: ricin from the seeds of Ricinus communis, saporin from Saponaria officinalis, gelonin from Gelonium multiflorum, pokeweed anti-viral protein from Phytolacca americana, Luffa toxin from Luffa aegyptica. In addition fungal and bacteria toxins were tested as conjugates with Trf receptor antibodies: restrictocin from the fungus Aspergillus restrictus, -sarcin from the Aspergilus giganteus, pseudomonas exotoxin from Pseudomonas aeruginosa and diphtheria toxin, which is secreted by Corynebacterium diphtheria. Ricin was the toxin more extensively tested in this context. Both ricin Trf conjugated [139,140] and ricin anti-Trf receptor conjugated were tested in vitro. Ricin Trf receptor conjugates were toxic for leukemic, cervical adenocarcinoma, mesothelioma, glioma, medulloblastoma, neuroblastoma, ovarian and breast cancer human cells lines and primary cells isolated from patients [139-146]. The saporin Trf and saporin anti-Trf receptor antibody conjugates inhibit the proliferation of human hepatoma and erythroleukemia cell lines and primary cells isolated from glioma patients [147-149]. The gelonin Trf receptor antibody conjugate had cytotoxic effects on several leukemias, Burkitt’s lynphoma and cervical carcinoma cell lines [150]. The pokeweed anti-viral and the Luffa toxin were also conjugated with anti-Trf receptor antibodies and shown to be toxic for leukemia cells [151,152]. The fungal toxins restrictocin and -sarcin were conjugated with anti-Trf receptor antibodies and these compounds inhibit proliferation of leukemia, lymphoma, lung carcinoma, breast cancer, epidermoide carcinoma and colon carcinoma human cell lines [153-156]. Pseudomonas exotoxin was conjugated with anti-Trf receptor antibody and was then toxic to the ovarian cancer, breast cancer, leukemia, colon and prostate cells lines [157,158]. Diphtheria exotoxin is able to bind to the cell surface being internalized, a mutant Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 47 form of this toxin (CRM107) without the capacity to bind to the cell surface was constructed. Trf and anti-Trf receptor antibody CRM107 conjugates were cytotoxic to medulloblastoma, glioblastoma, neuroblastoma, leukemia, ovarian cancer and breast human cancer cell lines and primary medulloblastoma cells isolated from patients [143,144]. Ribonuclease, from bovine and human origin, is another toxin with anti protein synthesis proprieties that have been tested as an anticancer therapy in conjugation with Trf and anti-Trf receptors antibodies against the K562 cell line [159]. Due to the disadvantages of using non-human antibodies in human treatments chimeric anti-human Trf receptor antibodies, with the constant region of the antibodies of human origin, were produced and fused with human ribonuclease. This ribonuclease anti-Trf receptor chimeric antibody was tested against human erythroleukemia, epidermoid carcinoma, breast adenocarcinoma, colon adenocarcinoma, renal carcinoma and glioma cell lines demonstrating cytotoxic effects [160-163]. Artemisinin, produced by the plant Artemisia annua, is a well-known antimalaria drug. Artemisinin is activated inside the parasite by the intraparasitic heme-iron to form free radicals. Besides the antimalaria proprieties artemisinins have inhibitory effects on cancer cell growth, as the neoplastic cells express high levels of Trf receptor [reviewed in 164]. Artemisinin conjugated to Trf has been shown to be a potent killer of prostate cancer and lyphobastoid cell lines [reviewed in 164,165]. In Vivo Experimental Models of Disease Trf-ADR conjugated prolonged the lifespan of human mesothelioma tumor bearing nude mice when compared to ADR alone or ADR plus free Trf [132]. ADR conjugated with anti-Trf receptor antibody was also effective against xenograft tumor models of human Daudi B and Raji Burkett’t lymphoma cells in nude mice [134]. Cisplatin conjugated to Trf was also tested in vivo showing capacity to block the growth of mammary carcinoma in rats and melanoma growth in mice [166]. Moreover Ricin Trf receptor conjugates inhibit human cervical adenocarcinoma, melanoma, mesothelioma, gliomas and ovarian xenograft growth in nude mice [139,140,167,168]. Gelonin Trf receptor antibody conjugate prolonged the life of nude mice with Burkitt’s lynphoma xenografts [150]. Trf-CRM107 compound was tested in nude mice with human glioma xenografts causing tumor regression in all of the treated mice (n=5) [168]. Clinical Trials Clinical trials with Cisplatin conjugated to Trf demonstrated safety and a partial response rate of 87% (7/8) in patients with advanced breast cancer [166]. In addition a clinical trial was done with ricin anti-Trf receptor conjugate in leptomeningeal neoplasia, the results show more than 50% reduction of tumor cell count in cerebrospinal fluid in 4 out of 8 patients treated, but tumor removal was never achieved [169]. The Trf-CRM107 compound was tested in clinical trials in patients with several types of malignant brain tumors. The compound was delivered by intratumoral infusion. In phase I, 9 out of 15 patients with different forms of brain tumors presented a 50% decrease in tumor volume and 2 demonstrated complete tumor regression [169]. In the phase II patients with glioblastoma multiform or anaplastic astro- 48 Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 cytoma were treated, at the one-year evaluation 34 patients received two treatment doses and 13 received one dose [170]. Five of the patients that received the two doses treatment showed complete responses while seven showed partial responses, the patients that receive only one infusion demonstrated progressive disease at the one year evaluation [170]. The Use of New Vectors New vectors that have been already tested after incorporation with Trf or/and anti-Trf receptor antibodies are polymers/polyplexes, liposomes, viral vectors and nanoparticles. Trf conjugated polyethylenimine containing radioactive nuclide Rhenium-188 induced tumor necrosis in human Burkitt’s lymphoma xenografts in nude mice [171]. Trf polyethylenimine, polyethylenglycol, delivering mouse TNF gene inhibited tumor growth in 3 murine neuroblastoma tumor models after systemic administration [172]. Both Trf and anti-Trf receptor antibodies conjugated liposomes were tested for the delivery of chemotherapeutic drugs (like ADR or cisplatin), therapeutic oligonucleotides (like endostatin gene, Bcl-2 antisence oligonucleotides, or the p53 gene) into various human malignant cell lines [reviewed in 138]. One of the most promising results was obtained with Trf conjugated liposomes containing the wild type p53 gene, this therapy in conjunction with radiotherapy led to complete regression of human prostate cancer DU145 xenografts in nude mice [173]. In addition some studies have been made with Trf conjugated to viral vectors (adenovirus [174]) and in the new field of nanoparticles using Trf conjugated with nanoparticles [175]. These new fields represent new opportunities of therapies but more studies need to be conducted in order to achieve efficient therapies. THE QUESTION OF DRUG DELIVERY TO THE BRAIN The brain is sheltered from the blood by the blood brain barrier (BBB). This barrier however acts also as a barrier to the delivery of drugs to the brain impeding the therapy of brain tumors and neurodegenerative disease. Interestingly in studies of the monoclonal antibody OX 26 in the rat Jefferies et al. found the expression Trf receptors restricted to brain capillaries [176]. Consequently several strategies have been designed for drug delivery to the brain using Trf and the anti-Trf receptor antibodies. Cerebral Malaria Cerebral malaria is the major cause of death by malaria infection. Preliminary results of a therapeutic strategy for cerebral malaria, using Trf conjugated solid lipid nanoparticles to deliver quinine to brain, have been published recently [177]. The Trf conjugated solid lipid nanoparticles enhanced in vivo brain uptake of quinine compared with unconjugated solid lipid nanoparticles or drug solution alone in rats [177]. Lysosomal Storage Diseases A paper describing the use of anti-Trf receptor antibody conjugated with Trojan horse liposomes for the delivery of enzyme replacement therapy to mucopolysaccharidosis type VII (a lysosomal storage disorder) has recently been published [178]. In this disorder there is a deficiency in the lysosomal enzyme, -glucuronidase (GUSB). The DNA encoding this enzyme was inserted into anti-Trf receptor anti- Macedo and de Sousa body conjugated liposomes, this compound was administrated to GUSB null mice and a 10-fold increase in activity of this enzyme in the brain was observed [178]. Viral Infections The antiviral drug azidothymidine (AZT) has been brain targeted using nanoparticles conjugated with Trf in the rat. The systemic administration of this compound resulted in an increase in the amount of AZT delivered to the brain when compared to the AZT nanoparticles alone [179]. One other strategy to conjugate the drug agent to the anti-Trf receptor antibody vector utilizes the high affinity streptavidin/biotin complex. Delivery of Neuroprotective Compounds The delivery of neuroprotective agents such as brainderived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) have been tested for brain delivery using the streptavidin/biotin complex [180,181]. BDNF was coupled to biotin and the anti-Trf antibody was coupled to streptavidin. The conjugate was administrated intravenously into rats subjected to permanent middle cerebral artery occlusion. The rats that received the conjugate had a 243% increase in motor performance relative to BDNF alone [181]. In the case of bFGF the growth factor was coupled to biotin and the anti-Trf antibody was coupled to streptavidin. The conjugate was also administered intravenously to rats with permanent cerebral artery occlusion, resulting in an 80% reduction in infarct volume [180]. In addition, the glialderived neurotrophic factor (GDNF) using Trojan horse liposomes linked to anti rat Trf receptor antibodies were used in the treatment of a Parkinson disease model in the rat, causing a lasting reduction in apormorphine-induced rotation [182]. Neuroinflammation The BBB controls also the extravasation of leukocytes from the blood. Strategies have been adopted to deal with chronic and acute neuro inflammation. Bhattacharya and coworkers reported recently the delivery of NF-kB decoys to brain derived endothelial cells (bEnd5) in vitro, using biotin polyethylenglycol-polyethylenimine complexed to streptavidin anti-Trf receptor antibody. The complex was internalized and the NF-kB decoys induced potentially antiinflammatory effects by significantly inhibiting the expression of mRNA of cell adhesion molecules VCAM-1 and ICAM-1 [183]. CONCLUDING REMARKS: TRANSFERRIN AND TRANSFERRIN RECEPTOR OF MAGIC BULLETS AND OTHER CONCERNS Progress in unraveling the roles of Trf and its receptor reflects general progress in knowledge in the biomedical sciences spanning from protein biochemistry [40] to gene targeting [184], the development of monoclonal antibodies [176,185] and of gene knock-out mice [186]. The protein biochemistry years established Trf as the iron binding and transport protein, identified polymorphisms compared Trf in different species [187] an exercise much improved by advances in molecular biology [188,189]. More recently the interest in the Trf field moved to testing the ef- Transferrin and the Transferrin Receptor Inflammation & Allergy - Drug Targets, 2008, Vol. 7, No. 1 49 fectiveness of diminishing tumor cell proliferation by targeting the receptor with antibodies (reviewed in Table 2). Gulbenkian Foundation, the Universities of Porto and the Piaget Institute. The availability of the Trfhpx/hpx [3] and the attempt to generate Trf receptor knock out mice [86] corroborated the principal role of Trf first established from the previous protein biochemistry years, and demonstrated Trf and its receptor as vital for survival, in addition it extended clarification of their roles in T cell differentiation and lymphopoiesis [84,87]. The discovery of a role in T cell differentiation is an illustration of the principle that one only finds new answers if one has new questions. The question of the interface of the immune system and the metabolism of iron has dominated much of the questioning by our group in the last 20 years [reviewed in 190]. Molecular evolution studies using cDNA cloning have clearly established Trf as a protein responding to bacterial infection in the insects: Drosophila melanogaster, Aedes aegypti and Aedes albopictus [188,189] illustrating an early evolutionary functional interface between the two systems. ABBREVIATIONS With the discovery of Mason’s group that the OX 26 monoclonal antibody “labeled capillaries in the brain but not in other tissues” [176] a tool became available to dissect iron transport across the BBB [191]. Taking into consideration that one major problem in the treatment of brain cancer and neurodegenerative diseases is drug delivery, the use of anti-Trf receptor antibodies as carriers of therapeutic agents could be of major importance for the treatment of these conditions. Expression of Trf receptor in brain endothelial cells is being used for delivery of newly developed drugs such as siRNA and DNA based therapies [178,182,192, reviewed in 193]. With the discovery by Monteiro’s group that both in IgA nephropathy and in celiac disease overexpression of the Trf receptor is crucial for the development of the pathologies [88,95] one can easily foresee other new pharmacological forms of intervention in diseases with an immunological basis. The scarcity of studies in some areas, the deficient numbers reported in some clinical trials speak perhaps to the little importance attributed to basic research in this field by pharmaceutical companies and funding agencies, naturally anxious (as we all) to see magic bullets to work as Paul Ehrlich imagined them. Ehrlich had the Nobel Prize for Physiology and Medicine in 1908 [194]. Two of the authors cited in the present concluding remarks, Capecchi and Smithies had the Nobel Prize in 2007. One hundred years have passed since Ehrlich started his Nobel lecture discussing the need to review the progress that led to the cell as the central unit in pathology and the microscope as the tool that made it possible. Presently we do not lack tools. Progress in understanding Trf and its receptor as pharmacological tools does not depend only on tools or on innovation; it depends also on new questions. This review took us to portray the differences between the numbers of potentially magic bullet papers and papers opening new windows of opportunity to look at new functions, with potential applications in the treatment of inflammation and neuroinflammation. We hope that this review will contribute to correcting such imbalance. ACKNOWLEDGEMENTS We acknowledge support by the following funding agencies and universities: the FCT, the APBRF, the Calouste ADR = Adriamycin AZT = Azidothymidine BBB = Blood brain barrier BDNF = Neurotrophic factor bFGF = Basic fibroblast growth factor ESC = Embryonic stem cells GDNF = Glial-derived neurotrophic factor GUSB = -Glucuronidase 111 = IREs = Iron response elements IRPs = Iron regulatory proteins PBMC = Peripheral blood mononuclear cells PHA = Phytohemagglutinin Trf = Transferrin InCl3 Trf hpx/hpx UTR 111 Indium chloride = Hypotransferrinemic = Untranslated region REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Kwok, J.C.; Richardson, D.R. Crit. Rev. Oncol. Hematol., 2002, 42, 65. Arosio, P.; Cairo, G.; Levi, S. (1989) The molecular biology of iron-binding proteins. In: De Sousa, M., and Brock, J.H. (eds). Iron and Immunity, Cancer and Inflammation., John Wiley & Sons Ltd, New York Bernstein, S.E. J. Lab. Clin. 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