Effect of Iron on Antigen Presenting Cells: Implications For Transfusion Dependent Hemoglobinopathies.

Ibrahim Mustafa, Duncheng Wang and Mark D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) are characterized by destabilized hemoglobin that can lead to a potentially life-threatening anemia. This anemia arises due to iron-driven destruction of the RBC. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immune consequences such as the observed increased risk of bacterial infections in these patients. Methods: To determine the functional consequences of iron and iron chelators on the MPS, the effects of ferric iron (Fe3+; ferric ammonium citrate, FAC) on dendritic cells (DC) antigen presentation [tetanus toxoid (TT Ag)] and the proliferation of peripheral blood mononuclear cells (PBMC) were examined. The iron chelators tested included Desferal (DFO) and Deferiprone (L1). PBMC were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) to measure cell proliferation. In addition, the effects of iron +/- iron chelators on the expression of CD83, CD80, CD86 and HLA-DR on mature DC were examined. Results: Importantly, iron significantly inhibited antigen presentation and PBMC proliferation. Treatment of DC cells with 200 µM FAC for 24 hours resulted in a ~70% reduction in PBMC proliferation in response to the TT Ag following 14 days culture. However, inclusion of iron chelators (e.g., 200 µM DFO or L1) restored near normal proliferation. Similarly, CD83 an important co-stimulatory molecule expressed on DC cells was also negatively affected by FAC in a dose (0-200 µM) dependent manner. Following 24 hours treatment with 200 µM FAC, a ~30% reduction in the mean fluorescence of CD83 was observed via flow cytometric assay. Treatment with DFO or L1 overcame the effects of iron on CD83 expression. There were no significant effects of iron on CD80 or CD86. Conclusions: As shown, iron has significant immunosuppressive effects on antigen presentation and lymphocyte proliferation. Iron chelators can effectively bind and remove free and complexed iron and reverse iron-mediated immunosuppression. These data suggest that iron chelation may provide a mechanism to diminish the risk of recurrent bacterial infections in patients with unstable hemoglobins or with iron-overload (hemochromatosis or secondary iron overload).

Note: This abstract was published on conference proceedings, of Canadian Society for Transfusion Medicine 2010 held at Vancouver

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Immunological Inhibition Arising From Misplaced Iron: Implications for Thalassemia and Sickle Cell Disease

I. Mustafa, D. Wang and M.D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) arise from mutations to the globin subunits of adult hemoglobin (HbA) resulting in destabilized hemoglobin and, potentially, a life-threatening anemia due in part to iron-driven redox reactions. While transfusions corrects the anemia, secondary iron overload can occur. Thus, both the primary and secondary pathology of thalassemia and SCD arise from “misplaced” iron. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immunoregulatory effects such as the observed increased risk bacterial infections in these patients.

Methods: To determine the functional consequences of iron on the MPS, the effects of ferric iron (Fe3+; ferric ammonium citrate, FAC), heme, purified HbA and oxidized RBC on antigen presentation/proliferation by PBMC and cultured dendritic like (DC) cells was examined. Antigens examined included tetanus toxoid (TT Ag), formalin-fixed Streptococcus mutans (SM Ag) and RhD peptide. PBMC proliferation was determined by 3H-thymidine incorporation or via flow cytometry using carboxyfluorescein diacetate, succinimidyl ester (CFSE) stained cells. To determine if iron-driven immunomodulation could be reversed, an iron shuttle chelation system using Desferal (DFO; shuttle chelator) and S-DFO (a high molecular weight DFO-starch conjugate) was examined.

Results: Importantly, all forms of iron, including oxidized RBC, significantly inhibited antigen presentation and PBMC proliferation. For example, 100 µM hemin resulted in a >98% reduction in proliferation in response to the TT or SM Ag. Similarly, phagocytosis of oxidized RBC virtually abolished the ability of antigen presenting cells within the PBMC to present antigen and abolished the response to the TT and SM antigens. DC cells were similarly affected by FAC (200 µM) exposure (7 days) with a ~78% reduction PBMC response to an immunodominant RhD peptide. Iron chelators could partially overcome the effects of the bioreactive iron. Of interest, prolonged treatment with S-DFO (unlike DFO) did not adversely affect purified hemoglobin.

Conclusions: As shown, iron has significant immunodepressive effects on immune function (antigen presentation and lymphocyte proliferation). Iron chelation can effectively bind and remove free and complexed iron /heme preventing both redox-driven damage and immuosuppression. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage within cells and may also protect immune competency.

Note: This was published Transfusion AABB Annual Meeting, New Orleans, LA USA 24-27 Oct 2009.

Fe3+ as a potent accelerator for hemoglobin oxidation in vitro and prooxidant effects of reduced glutathione (GSH).

Authors: Mustafa, I. and Scott, M.D. Department of Pathology and Laboratory Medicine and the Centre for Blood Research at the University of British Columbia and the Canadian Blood Services.

Abstract

Iron is an essential trace element for all living cells.Indeed, iron cores constitute the functional sites of many enzymes and proteins involved in generating energy, transporting oxygen, and DNA synthesis. Containing approximately 20 mM iron, normal erythrocytes are the most iron and oxygen rich somatic cell.While this important metal is vital, maintaining its biological balance in an organism is far more crucial than virtually any other trace element (with the possible exception of copper). Excess iron, due to its catalysis of one electron redox chemistry, plays a key role in the formation of toxic oxygen radicals.Indeed, this is readily observed in diseases such as thalassemia and sickle cell anemia. This potentially dangerous combination of oxygen and iron within the erythrocyte is kept in check by a number of endogenous mechanisms. These include the hemoglobin (Hb) tetramer itself, as well as a number of antioxidants such as superoxide dismutase (SOD), catalase, glutathione/glutathione peroxidase, and methemoglobin reductase.

Experiments were conducted in vitro using purified hemoglobin (HbA; a2 ß2) exposed to ferric (Fe3+) iron.Upon Fe3+ addition (0-250 µM ferric ammonium sulfate), spectrophotometric shifts in the absorption spectra (500 –700 nm) of purified hemoglobin (~10 µM HbA; pH 7.4) was determined. HAb concentration was determined by the cyanomethemoglobin (Drabkin’s) method. The inhibitory effects of glutathione (GSH), iron chelators (desferrioxamine, DFO; and Deferiprone, L1), SOD and catalase on Fe3+-driven oxidation was assessed.In the absence of Fe3+ no HbA oxidation was noted.Similarly, in contrast to erythrocyte hemolysates, simple addition of Fe3+ to purified HbA also had minimal oxidative effects.However, addition of the “anti-oxidant” GSH, resulted in a potent GSH (0-10 mM) and Fe3+ (0-250 µM) dose-dependent oxidation of the purified HbA to methemoglobin. Methemoglobin is characteristized by a peak (or shoulder) at 630 nm. This data demonstrates that a reducing agent is necessary for iron-driven oxidation.Notably the intact erythrocyte is rich in both GSH and ascorbate. This oxidation was further enhanced by the presence of hydrogen peroxide (H2O2), a normal byproduct of HbA auto-oxidation.Importantly, inclusion of the iron chelators (DFO or L1) inhibited Fe3+-mediated HbA oxidation in a dose-dependent manner.For example, >250 µM DFO, inhibited virtually all iron mediated HbA oxidation induced by the addition of 250 µM Fe3+ and 0. 4 mM GSH.Further experiments were carried out to see if Fe3+ mediated HbA oxidation could be inhibited by SOD or catalase. These studies demonstrated no protective effects, suggesting that Fe3+ – GSH degradation is not due to either O2 or H2O2, or even hydroxy radicals, because SOD or catalase would have blocked hydroxyl radical formation. Consequently this data implicates thiol radicals as the agent of injury.

These findings have significant implications in the mechanisms of injury in disease such as the thalassemias and sickle cell anemia.Furthermore, these results suggest that chelation of bioreactive iron within the abnormal erythrocyte may be an effective therapeutic intervention.My research is currently focusing on a novel iron-shuttle chelation therapy regime utilizing low and high molecular weight iron chelators.

 

Iron Shuttle Chelation Therapy: A Novel Approach To Treating Hemoglobinopathies

I. Mustafa, N. Rossi, J. N. Kizhakkedathu and M.D. Scott
Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: Thalassemias arise from deficiency of the globin subunits of adult hemoglobin (HbA) and results in ineffective erythropoiesis and the rapid destruction of RBC in the periphery. These iron-driven events give rise to anemia. While transfusion therapy corrects the anemia, it give rise to secondary iron overload. Thus, both the primary and secondary pathology of thalassemia arise from “misplaced” iron. Our previous studies suggest that chelation of bioreactive iron within the thalassemic/sickle RBC may be an effective therapeutic intervention. Our research is focused on a novel iron-shuttle chelation therapy utilizing both low (shuttle) and high (docking) molecular weight iron chelators.
Methods: The effect of shuttle and docking chelators on ferric iron (Fe3+) – driven Hb and lipid oxidation was assessed singularly and in combination. Experiments were conducted in vitro using HbA exposed to Fe3+ (0-175µM). HbA oxidation was quantitated spectrophotometrically. Shuttle chelators included DFO, L1, HBED and ICL-670.The docking chelators consisted of S-DFO (a starch conjugate of DFO) and P-DFO (a novel poly(ethylene glycol)-acrylate based copolymer of DFO).Lipid peroxidation was measured by thiobabutaric acid reactive substances (TBARS) formation
Results: In the absence of Fe3+ no HbA oxidation was noted. Addition of 175µM Fe3+ resulted in rapid methemoglobin formation. Importantly, inclusion of any of our chelators inhibited HbA oxidation in a dose-dependent manner. For example,175 or 200 µM DFO , P-DFO or S-DFO respectively, inhibited >90% HbA oxidation induced by 175 µM Fe3+. Similarly, lipid peroxidation was inhibited in a chelator dose dependent manner: 400 µM DFO or P-DFO equivalents resulted in a 58 or 70% reduction TBARS formation (respectively).
Conclusions: As shown, both shuttle and docking chelators can effectively bind and remove free and complexed iron /heme from aqueous and lipid environments and prevent redox-driven damage. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage and improve both effective erythropoiesis and the viability of abnormal RBC within the periphery. Thus this iron shuttle system may have therapeutic importance in the treatment of hemoglobinopathies.

Note: This abstract was published on AABB Transfusion journal 2008 special edition for the anual conference in Montreal.

Importance of transfusion medicine in Maldives

Over the past many years I have been impressed by the prevalence of blood dyscrasias In Maldives. Reduced levels of hemoglobin are common among all the Maldivians and the consequences thereof seem to be a household story. Then there are various blood cancers, many bleeding disorders and such prevalent hereditary disorders as thalassaemias, sickle cell anemias, and enzyme deficiencies. In the comparative analysis of diseases, it is obvious that blood diseases are the commonest ailments in man. This is compounded by the fact that abnormalities in blood are encountered as an important secondary manifestation in large number of non-haematological disorders.

At educational level in the Maldives there is no degree college, medical school or university where structured courses in Haematology are offered at any level. Yet haematological investigations account for almost 40 % of the laboratory workload. The irony is that there are no personnel specifically trained in the field of Haematology and specifically Immunohaematology to handle this workload. This also includes Blood transfusion services where an error may cost a patient his life.

The Haematology Department should provide comprehensive laboratory services for the diagnosis and management of both haematological and oncological disorders. Most of the routine haematology and coagulation tests have to be performed in the laboratory. Specialist laboratory services should be established in the Haematology laboratories include immunophenotyping of leukaemia and lymphoma, thrombophilic screening, coagulation factor assays, platelet function tests, bone marrow culture and special assays such as those for vitamin B12, erythropoietin and red cell enzymes.

Transfusion/Immunohaematology services are also controlled by the Haematology Department. As well as providing the routine services of blood grouping, cross matching and the issuing of various blood products, the Transfusion/Immunohaematology section plays an important role in the diagnosis and treatment of immunohaematological disorders, such as haemolytic disease of the newborn, and in the management of haemophilia and thalassaemia major.

Though the above-mentioned things have to be there with in the infrastructure of health system for the management of blood disorders in the Maldives. We don’t have enough locals who has been trained in the following field to meet the demand to establish a National blood bank. This is of special interest to the Maldives as the incidence of Thalassaemia is one of the highest in the world. Prevalence of Thalassaemia in the Maldives is about 18.5% of the whole population, which is perhaps the highest in the world.

There are about 600 registered thalassaemia major patients in National Thalassaemia Center in the capital Male’ where they receive regular blood transfusions. These patients they require only packed erythrocytes to correct the deficiency of hemoglobin. Yet we are unable to provide them these blood components in appropriate manner due to unavailability of equipments and lack of personnel. Blood component therapy is the modern concept of administration of blood and blood products. It is aimed at providing specific blood components for specific deficiency states effectively, economically and with greater safety. The procedures are easy to adopt, provided the technical know how for fractionation of blood.

Component therapy enhances the number of users without the need of increasing the demand on blood supply. With modest fractionation one unit of fresh whole blood can now be used by at least four different recipients; more can be benefited with further fractionation. Blood component therapy also protects the recipients from the risks, which are inherent in whole blood transfusions.

I have found Immunohaematology or Transfusion Medicine is an important field to a country like Maldives where the prevalence of blood diseases are extremely high. Therefore it is mandatory to train qualified people in this field for the national interest and for the betterment of family health.