Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron.

BACKGROUND: In transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. RESULTS: LIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DISCUSSION: The difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

After haemin treatment intracellular non-haem iron increases prior to haem oxygenase-1 induction: A study in human monocytic cell line THP-1.

BACKGROUND: Iron overload is a major health concern for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, in turn, inducing cell death. We previously demonstrated that haemin-induced cell death in human monocytic THP-1 cells is consistent with ferroptosis, an iron-dependent cell death regulation mechanism. However, direct measurement of iron after haemin treatment has not yet been conducted. In this study, we measured intracellular non-haem iron concentration and haem oxygenase levels after haemin treatment. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and the cell lysate was prepared. Non-haem iron concentration of the cell lysate was measured using the Nitroso-PSAP method. Expression of haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) was quantified by western blotting. RESULTS: We measured intracellular non-haem iron and the expression of haem oxygenases post-haemin treatment. Concentration of non-haem iron post-haemin treatment increased dependently with time and dose. HO-1 expression was detected 4 h after haemin treatment, whereas HO-2 expression was constitutive. DISCUSSION: Increase in non-haem iron prior to induction of HO-1 expression suggests the involvement of HO-2 in haem-induced cytotoxicity. (184 words).

Haemin-induced cell death in human monocytic cells is consistent with ferroptosis.

BACKGROUND: Iron overload is a major issue for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, and the haemin in turn induces cell death and the generation of reactive oxygen species (ROS) in both murine macrophages and human monocytic THP-1 cells. This haemin-induced cell death process has been shown to be iron-dependent. Thus, we hypothesized that haemin-induced THP-1 cell death is a result of ferroptosis, an iron-dependent mechanism of cell death regulation. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and haemin-induced cell death and ROS generation were assessed using flow cytometry. RESULTS: Haemin-induced THP-1 cell death showed a necrosis pattern, and treatment with iron chelators suppressed both haemin-induced cell death and ROS generation. Treatment with ferrostatin-1, a ferroptosis inhibitor, suppressed haemin-induced cell death without affecting ROS generation, whereas erastin, a ferroptosis inducer, enhanced both haemin-induced cell death and ROS generation. DISCUSSION: Our findings support haemin-induced cell death as an example of ferroptosis. Therefore, ferroptosis inhibitors may be useful for the treatment or prevention of transfusion iron overload.

Iron-chelating agent, deferasirox, inhibits neutrophil activation and extracellular trap formation.

Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 µmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 µmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

Heme-related molecules induce rapid production of neutrophil extracellular traps.

BACKGROUND: Pulmonary endothelial cell damages caused by neutrophil overactivation could result in acute lung injuries including transfusion-related acute lung injury (TRALI). We previously reported that heme-related molecules derived from hemolysis induced the production of reactive oxygen species from neutrophils. Recently, neutrophil extracellular traps (NETs) have been demonstrated to associate with the onset of TRALI. STUDY DESIGN AND METHODS: In this study, neutrophils' morphologic changes induced by the heme-related molecule hemin were confirmed to be NETs via confocal laser scanning microscopy and electron microscopy (EM). Additionally, concentrations of hemin in red blood cell (RBC) components were measured via enzyme-linked immunosorbent assay and possible contribution of these molecules to the onset of TRALI was discussed. RESULTS: SYTOX green staining observation via confocal laser scanning microscopy revealed that neutrophil morphology changed rapidly upon addition of hemin. The nuclei began to be enlarged and become segmented after 5 minutes, and NET-like structures were released from neutrophils after 15 minutes. In EM observation, NET-like structures appeared after 10 minutes and the nucleoplasm was partially separated from the nuclear membrane, which were consistent with the features of NET formation. These structures stained positively for both myeloperoxidase and histone H3 antibodies. CONCLUSION: Thus, our results suggest that hemin induced NETs in 15 minutes, a quicker reaction than NET induction by phorbol myristate acetate requiring 3 hours. Moreover, since RBC components, especially those with long-term storage, contained sufficient hemin concentration to induce NETs, special attention to hemolysis of stored RBC components is important.

[Effects of four bisphosphonates on macrophage phagocytosis: quantitative measurement by flow cytometry using high-fluorescence particles and human monocytic cell line THP-1].

Impairment of macrophage phagocytosis is a major cause of chronic inflammation. Bisphosphonates (BPs) are widely used as anti-osteoclastic agents. The effects of BPs on monocyte-macrophage lineage cells are being increasingly reported; however, the detailed effects of BPs on macrophage phagocytic activity are still unclear. We examined the effects of four BPs: clodronate as a non-nitrogen containing BP (non-N-BP), and pamidronate, alendronate, and zoledronate as nitrogen-containing BP(N-BP), on macrophage phagocytic activity. The uptake of high fluorescence-labeled polystyrene beads by the human monocytic cell line THP-1 was investigated by flow cytometry. All three N-BPs suppressed the phagocytosis of macrophages more potently than the non-N-BP, clodronate. Pamidronate and zoledronate were more potent than alendronate. BP induced the apoptosis of THP-1. Pamidronate and zoledronate induced apoptosis more effectively than clodronate. The method described to observe phagocytosis was simple and quantitative, and might be useful in screening for the effects of drugs, such as N-BP and non-N-BP, on phagocytic activity.

Deferasirox, an iron-chelating agent, alleviates acute lung inflammation by inhibiting neutrophil activation and extracellular trap formation.

OBJECTIVE: Reactive oxygen species (ROS) production by neutrophils induces pulmonary endothelial cell damage and results in acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits the ROS production and neutrophil extracellular trap (NET) formation induced by phorbol myristate acetate and formylmethionylleucylphenylalanine in vitro. In the present study, we investigated the effects of DFS in vivo using a mouse model of lipopolysaccharide (LPS)-induced ALI. METHODS: After DFS administration for 7 days, ALI was induced in mice by LPS via intratracheal administration. RESULTS: LPS treatment induced neutrophil invasion in the lung tissues, along with NET formation and a significant increase in the quantity of double-stranded DNA in the bronchoalveolar lavage fluid, while pre-administered DFS inhibited these phenomena. However, alteration of neutrophil morphology in the cytoplasm in terms of shape and vacuolization was not inhibited by the pre-administration of DFS, possibly through ROS production. CONCLUSIONS: DFS suppressed neutrophil invasion into lung tissues and reduced the double-stranded DNA content released by the neutrophils. These results suggest that DFS can potentially be used to prevent diseases related to neutrophil activation including ALI, thrombosis, and vascular endothelial dysfunction.

[Neutrophil function tests and oxidative stress detection as clinical examinations].

We herein introduce several clinically available methods to detect neutrophil function and oxidative stress. The flowcytometric detection of adhesive protein expression, such as CD11b(Mac-1), assessment of phagocytosis activity, and measurement of reactive oxygen species (ROS) production are relatively easy to apply as tools for laboratory medicine. A new device to simultaneously detect superoxide and calcium ion influx is also introduced. Oxidative stress induced by ROS produced not only from phagocytic cells but also from the mitochondria or endoplamic reticulum of all kinds of living cells is etiologically related to many disorders and also aging. A simple method using the FRAS4 instrument is demonstrated. These methods are expected to be clinically beneficial, especially in hematology, transfusion medicine, and the public health field.

[Laboratory medicine for blood transfusion and transplantation medicine].

We discussed the usefulness of routine technologies of laboratory medicine in blood transfusion and transplantation medicine. New parameters that can be measured by automated hematology analyzers have been clinically evaluated and proven to be useful so far. Based on our experience, detection systems for fragmented red cells (FRC), immature platelets (immature platelet function, IPF), and hematopoietic progenitor cells (HPC) are useful for the diagnosis of thrombotic microangiopathy, differential diagnosis of thrombocytopenia, and decision regarding the optimal timing to collect peripheral stem cells, respectively. Moreover, IPF were suggested to be an indicator of the platelet transfusion requirement. The establishment of non invasive assaying technology has been eagerly anticipated. We evaluated a hemoglobin measurement tool, and revealed that it might be applicable in predeposited, autologous blood donation. Some adverse transfusion reactions are related to neutrophil activation. Thus, we investigated the effects of serum from patients and blood donors, in the context of adverse reactions, on adhesion molecule expressions of neutrophils from volunteers using flow-cytometry. This kind of simple technology is expected to be useful in future studies to clarify the mechanisms and prevent adverse reactions.

[Activation of human neutrophils by hemin].

Activation of neutrophils by free heme is considered as one of the mechanisms for cellular dysfunction under the conditions of hemorrhage or tissue damage. We studied about the effects of hemin, ferriprotoporphyrin IX, on human neutrophil activation by measurements of adhesion molecule expression and reactive oxygen species (ROS) production. Human neutrophils purified from heparinized blood of healthy volunteers were stimulated with hemin. Surface expression of CD11b and L-selectin were evaluated by flow cytometry, and superoxide production was detected by chemiluminescence. Hemin increased the expression of CD11b and produced superoxide accompanying by increase in intracellular free calcium concentration. Thus, free heme-molecule is suggested to possess the activity to initiate or aggravate tissue injuries. Since neutrophils do not express CD163, scavenger receptor for hemoglobin-haptoglobin complex, the mechanisms by which hemin exerts these effects are still to be studied.

Detection of activated neutrophils by reactive oxygen species production using a hematology analyzer.

Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles. This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer. Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general-purpose conventional flow cytometer. In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained some vacuole-like structures of vacuoles or invagination of cell membrane. Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity.

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon.

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe3+ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress. NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs. Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.

Cyclosporin neurotoxicity with Epstein-Barr virus-associated hemophagocytic syndrome.

In September 2000, a 22-year-old female was admitted to our hospital due to high grade fever, liver enzymes elevation and pancytopenia. Bone marrow aspiration was performed, and hemophagocytosis was present. Epstein-Barr virus (EBV) DNA was positive in her peripheral blood, and we diagnosed the case as EBV-associated hemophagocytic syndrome (EB-VAHS) after excluding other malignancies. The initial therapy including etoposide and dexamethasone was started. As severe leukocytopenia developed, etoposide was stopped and cyclosporin A (CsA) was administered continuously. Four days after administration of CsA, she developed convulsive seizures with loss of consciousness. An MRI demonstrated decreased signal with T1-weighting and high signal with T2-weighting in the subcortical white matter including the posterior lobe. We stopped CsA infusion, and glycerol was administered. Soon the symptom disappeared. When patients developed an episode of convulsive seizure, other diagnostic possibilities were central nervous system (CNS) involvement of hemophagocytosis, EBV encephalitis and acute disseminated encephalomyelitis (ADEM). CsA neurotoxicity must be considered even in the case of EB-VAHS with administration of CsA. As previously reported, Fluid-attenuated Inversion Recovery (FLAIR) imaging improved diagnostic confidence and conspicuity of the T2 hyper intense lesions of CsA neurotoxicity, as well as tacrolimus encephalopathy, typically in the subcortical white matter. Key words; Cyclosporin neurotoxicity; Epstein-Barr virus associated-Hemophagocytic syndrome; Magnetic Resonance Image (MRI).

Usefulness of particle image analyzer for evaluation of fragmented red cells.

Quantitative determination of fragmented red cells (FRCs) in peripheral blood is useful in various clinical fields. We have previously reported an automatic detection method using a hematology analyzer essentially based on detection of small red cells. We then tried to use another instrument, the FPIA 2100, to reflect the characteristic morphology of red cells. As the circularity obtained by FPIA 2100 correlated with the FRC%, this type of instrument might be helpful to establish better automatic detection methods.

Albumin attenuates neutrophil activation induced by stimulators including antibodies against neutrophil-specific antigens.

As neutrophil activation is related to several adverse transfusion reactions, we studied about the activation induced by anti-neutrophil antibodies and the stabilizing effects of albumin pretreatment by means of flow cytometry. Anti-neutrophil monoclonal antibody (anti-HNA-1a, 1b, 2a) alone induced CD11b expression and shedding of L-selectin, and anti-HNA-1a reinforced reactive oxygen species (ROS) production. Albumin pretreatment significantly reduced CD11b expression and L-selectin shedding induced by fMLP and ROS production induced by PMA, G-CSF combined with PMA or LPS-fMLP, or anti-HNA-1a combined with PMA. These findings suggest that anti-HNA-1a is related to adverse reactions and albumin has a regulating effect on neutrophil activation.

Noninvasive monitoring of hemoglobin. The effects of WBC counts on measurement.

The efficacy of a noninvasive hemoglobin monitoring device (Astrin, Sysmex, Kobe, Japan) was evaluated for healthy volunteers and for patients with hematologic disorders. At the same time, the effects of WBC counts on noninvasive monitoring were studied by clinical evaluation and in ex vivo experiments. The hemoglobin levels determined by the device (Ast-Hb) and a conventional analyzer (T-Hb) were compared. The coefficient of correlation between findings with the Ast-Hb and the T-Hb for healthy volunteers was r = 0.626, whereas that for patients with hematologic disorders was r = 0.762. A comparison of the ratios of measurement errors in hemoglobin levels by Ast-Hb and T-Hb indicated that the number of WBCs had no effect on hemoglobin monitoring. Moreover, ex vivo studies using isolated WBCs and an optical model that imitates blood vessels and tissue in human fingers confirmed these results. Therefore, this new hemoglobin monitoring device can be expected to be useful for continuous hemoglobin monitoring.

[Two cases of acute lymphoblastic leukemia with cytoplasmic granules].

The presence of cytoplasmic granules in blastoid cells of patients with acute leukemia is generally accepted as a useful morphological marker for differentiation of myeloid leukemia from lymphoblastic leukemia. We diagnosed two cases of acute lymphoblastic leukemia(ALL) with cytoplasmic granulation. Surface marker analysis of leukemic cells revealed they were positive for CD10, 19, 20, 33, 34 and HLA-DR. Immunoglobulin gene rearrangement was detected by means of Southern hybridization with an Ig-JH probe for both patients. On the basis of these findings, the patients were diagnosed as having B-precursor ALL. Electron microscopic observation showed no myeloperoxidase activity, so that the granules were considered to be related to autophagolysosomes. This experience demonstrates that the recognition of the presence of granular ALL is necessary for making an accurate differential diagnosis of acute leukemias.

[Invasive pulmonary aspergillosis complicated by complete atrioventricular block and aspergillus pericarditis after induction chemotherapy in a patient with acute lymphoblastic leukemia].

A 50-year-old man developed invasive pulmonary aspergillosis after induction chemotherapy for acute lymphoblastic leukemia. He was treated with 5-fluorocytosine and intravenous amphotericin B (AMPH-B). During antifungal therapy, he developed aspergillus pericarditis and complete atrioventricular (A-V) block. The pericardial effusion was decreased and the A-V block was improved after treatment with intravenous and intrapericardial instillation of AMPH-B. Because the patient's renal function deteriorated, AMPH-B was replaced with itraconazol after the latex agglutination (LA) test for an aspergillus-specific antigen showed a negative result. The patient, however, died from disseminated aspergillosis. Aspergillus DNA was detected in retrospective analysis of the serum which had been negative with the LA test. This case indicates that LA is not sufficient for diagnosis and post therapy evaluation of invasive aspergillosis. PCR or other methods should be used concomitantly with LA. Intrapericardial instillation of AMPH-B might be effective for patients with aspergillus pericarditis in whom surgical treatment is not indicated.