Poly(I:C) promotes neurotoxic amyloid ß accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid ß (Aß) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aß-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aß degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aß toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aß was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aß1-42. Altogether, these results suggest that viral infections induce Aß neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aß degradation.

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.

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.

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).

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.

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.

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.

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.

[The Utility of XE-2 100 Analyzer's NEUT-X and NEUT-Y Parameters for Detecting Neutrophil Dysplasia in Myelodysplastic Syndromes].

Morphological abnormalities of blood cells are the typical characteristics of myelodysplastic syndromes (MDS). Recently, the usefulness of multiparameter automatic hematology analyzer XE-2100 for detecting neutrophil dysplasia has been reported by using parameters of NEUT-X and NEUT-Y, reflecting neutrophil cytoplasmic granularity and the cellular content of nucleic acid and protein, respectively. We evaluated the utility of these parameters by analyzing the blood samples of fifty MDS patients consulting Kakogawa West Municipal Hospital between Jan, 2010 and Jun, 2014, as well as 100 persons undergoing medical examinations during the same period as controls. Neutrophil granulation level was classified as normal, hypo-granular, or agranular under microscopic observation, and degranulation index was calculated using the following formula. The relationship between NEUT-X, NEUT-Y values and degranulation index was studied as below. Degranulation index = agranular (%) x 2+ hypo-granular (%) x 1 + normal (%) x 0 Neut-X values of MDS patients were 1,350 (mean), 1,345 (median), and NEUT-Y values, 430 (mean) and 432 (median). The NEUT-X and NEUT-Y values of control patients were 1,350, 1,349, 446 and 445, respectively. Correlation efficiency between degranulation index and NEUT-X or NEUT-Y were r = 0.62 or 0.52, respectively. Relationship between NEUT-X and NEUT-Y for all patients was r = 0.90. All the 10 patients showing NEUT-X lower than 1,315 and NEUT-Y lower than 400 simultaneously were MDS. Hence, we conclude that NEUT-X and NEUT-Y information is useful for quantitative evaluation of neutrophil morphological abnormalities.

Successful Treatment of Leukemic Mature B-Cell Lymphoid Neoplasm with Similar Features to Splenic Marginal Zone Lymphoma Possessing Aberrant Myeloid Markers.

In splenic marginal zone lymphoma (SMZL), there are cases that cannot accurately be classified as such because of overlapping morphologic and/or immunophenotypic features. We report here a 76-year-old Japanese female, who showed leukemic B-cell lymphoproliferative disease possessing characteristic features identified for SMZL. The patient was leukemic with white blood cell counts 49,400/µL (abnormal cells, 78.5%) and neoplastic cells were characterized by aberrant expression of myeloid markers with CD19(+)CD13(+) (64.2%) and CD20(+)CD11c(+) (25.1%). Considering her history of previous chemotherapy and systemic leukemic phase of the disease, we treated the patient without performing splenectomy, with successful use of a combination of rituximab/bendamustine hydrochloride and of rituximab/cladribine. The patient has been in a complete remission longer than 44 months, with no detectable M-protein.

Bortezomib Causes ER Stress-related Death of Acute Promyelocytic Leukemia Cells Through Excessive Accumulation of PML-RARA.

BACKGROUND/AIM: The success of proteasome inhibitors in therapy of multiple myeloma has led to their use for other malignancies. For the proteasome inhibitor bortezomib, combination therapies with histone deacetylase inhibitors, which up-regulate ubiquitin-proteasome system (UPS)-related enzymes, produce a beneficial effect. However, the mechanisms underlying the effect of bortezomib are not completely understood. We hypothesized that bortezomib causes excessive accumulation of aberrant proteins, which augments endoplasmic reticulum (ER) stress, leading to death of malignant cells. MATERIALS AND METHODS: The NB4 cell line established from a patient with acute promyelocytic leukemia (APL) expressing the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) fusion protein was used to assess changes in cell viability and apoptosis caused by bortezomib, as well as alterations in PML-RARA and UPS-related enzymes via western blotting and immunoprecipitation assays. RESULTS: Bortezomib time- and dose-dependently reduced cell viability and induced apoptosis. Bortezomib significantly increased the abundance of ubiquitinated-PML-RARA (Ub-PML-RARA), ubiquitin-conjugating human enzyme 8 (UbcH8), and Ub-UbcH8, indicating that UbcH8 is the E2 ubiquitin-conjugating enzyme for PML-RARA. Moreover, UbcH8 abundance was dose-dependently increased in the culture supernatant of bortezomib-treated cells. CONCLUSION: UbcH8 may have a utility as a biomarker of treatment response to bortezomib in patients with APL. Furthermore, bortezomib impairs the UPS that controls normal protein homeostasis by causing excessive accumulation of PML-RARA augmenting ER stress and leading to APL cell death. The study provides a rationale for incorporating proteasome inhibitors in the treatment of diseases expressing aberrant proteins. Furthermore, monitoring of UPS-related enzymes might have use in predicting the treatment response to proteasome inhibitors and in assessing their therapeutic effects.

Two cases of primary cold agglutinin disease associated with megaloblastic anemia.

We report two cases of primary cold agglutinin disease (CAD) associated with megaloblastic anemia in Japanese elderly patients. Case 1 was a 67-year-old male and Case 2 was a 55-year-old male. Both patients were diagnosed with primary CAD, with continuously high cold agglutinin titers (1 : >8,192 and 1 : 16,834, resp.), monoclonal IgM-kappa light chains, and no underlying disease. In addition, both patients had megaloblastic anemia due to vitamin B12 deficiency. One patient received rituximab and both received vitamin 12 supplementation. To date, no cooccurrence of primary CAD and megaloblastic anemia has been emphasized. Thus, the association of these hematological diseases may be incidental; however, given that CAD is an autoimmune disease which may show antibodies against intrinsic factor and gastric parietal cells, this association was thought to be probably not a coincidence. Clinicians should be aware of the possible simultaneous presence of autoimmune hemolytic/megaloblastic anemia in patients with primary CAD.

Genotoxicity-suppressing effect of aqueous extract of Connarus ruber cortex on cigarette smoke-induced micronuclei in mouse peripheral erythrocytes.

INTRODUCTION: According to published information, it has not been determined whether the inhalation of cigarette smoke can induce chromosome aberrations and/or point mutations in mice, though cigarette smoke is clearly carcinogenic to mice. We tested clastogenicity of inhaled cigarette smoke in mouse by a micronucleus test using peripheral erythrocytes. Since it is important to determine the in vivo anti-genotoxic effect against inhaled cigarette smoke to reduce the risk of tobacco carcinogenesis, we also tested in vivo anti-gnotoxic effect against inhaled cigarette smoke of a Connarus extract whose in vitro anti-genotoxic effect was shown. RESULTS: Male ICR mice were exposed for 1 min to a 6-fold dilution of the smoke once a day for up to 14 consecutive days. Although the frequencies of reticulocytes with micronucleus (MNRETs) and erythrocytes with micronuclei (MN erythrocytes) did not increase within 72 h after a single inhalation of cigarette smoke, the frequency of MN erythrocytes increased significantly upon inhalation for 7 and 14 days. When the Connarus extract was fed to mice at >23.7 ppm during the inhalation period of 14 days, frequency of MN erythrocytes was significantly lower than that at 0 ppm. In vitro antioxidant activity of Connarus extract was almost same to that of vitamin C. The antioxidant activity of the Connarus extract might play an important role in its anti-genotoxic effect against cigarette smoke in vivo, like vitamins C. CONCLUSIONS: Consecutive inhalation of cigarette smoke is clastogenic to mouse bone marrow as shown by the increased frequency of MN erythrocytes. Also, it was shown the possibility that the Connarus extract reduces the risk of tobacco carcinogenesis.

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.

A case of acute promyelocytic leukemia with morphologic multilineage dysplastic changes.

Although reports of typical acute promyelocytic leukemia (APL) cases rarely mention dysplastic changes, this report concerns a rare case of APL with tri-lineage dysplastic changes resembling the characteristic features of myelodysplastic syndrome (MDS). The patient, a 77-year-old Japanese male, was diagnosed as having pancytopenia with hematologic morphological abnormalities comprising micro - megakaryocytes, neutrophils with hypo-granulation and negative peroxidase activity, and erythroblasts containing nuclei with abnormalities such as karyorrhexis. Although there is one report of a case of transformation of de novo MDS into APL and several reports of cases of therapy-related MDS transformed into APL, our patient had no history of cytopenia or of either chemo or radiation therapy. Our case can thus be considered to constitute a rare case of APL with dysplastic morphology.

Deferasirox reduces oxidative stress in patients with transfusion dependency.

BACKGROUND: Iron chelation therapy is useful against the over-accumulation of iron and is expected to reduce oxidative stress resulting from the Fenton reaction and Haber-Weiss reaction. We monitored oxidative status and serum ferritin levels after in vivo administration of deferasirox (DFS) and studied the in vitro effects of iron chelators on neutrophil function. METHODS: Nine patients suffering from transfusion dependency were recruited for this study, and derivatives of reactive oxygen metabolite (dROM) tests to detect serum hydroperoxide levels were evaluated in addition to serum ferritin levels. Human neutrophil reactive oxygen species (ROS) production was determined with flow cytometry. RESULTS: Ferritin levels decreased after DFS treatment (P = 0.068), and a significant reduction in dROM levels was measured (P = 0.031). Fifty microM DFS significantly inhibited ROS production induced by fMLP in vitro (P < 0.0001), and tended to inhibit that induced by PMA. On the other hand, deferioxamine failed to inhibit ROS production even at high concentrations. CONCLUSIONS: In vivo administration of DFS resulted in the reduction of oxidative stress, and this effect was considered to depend not only on a reduction in iron storage but also on the ability of DFS to inhibit neutrophil ROS production in vitro at clinically relevant plasma levels. Further studies are needed to examine the effects of iron chelators.

Morphological and flow-cytometric analysis of haemin-induced human neutrophil activation: implications for transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is associated with vascular endothelial cell injury following neutrophil activation. Recently, it has been suggested that haem-related molecules induce activation of neutrophils and that erythrocyte-derived substances contained in blood preparations are involved in TRALI. We observed the morphological effects and reactive oxygen species (ROS) production of haem-related molecules and investigated the effects of signal transduction inhibitors on haem-induced neutrophil activation. MATERIALS AND METHODS: The polymorphonuclear cell fraction was isolated and stimulated using a control stimulant, PMA or fMLP, or by haem-related molecules, haemin, ferric citrate, or protoporphyrin IX. After stimulation, neutrophil was analysed using electron microscopy, a flowcytometer (FCM) and confocal laser scanning microscope to determine the fluorescent intensity of aminophenyl fluorescein (to detect ROS). RESULTS: In FCM analysis, haemin and protoporphyrin IX, both of which have a porphyrin ring, induced ROS production in neutrophils. Ferric citrate, which has no porphyrin ring, did not induce neutrophil activation. Haemin alone induced ROS production at relatively high concentrations, whereas low-level fMLP acted as an agonist in the presence of low concentrations of haemin. Haem-related molecules induced ROS production in neutrophil granules through signal transduction in a way similar to PMA. However, in electron microscopy studies, haemin stimulated neutrophils showed minute process at their surface and did not show the vacuolation observable following stimulation with PMA or fMLP. DISCUSSION: We suggest that low concentrations of haem-related molecules with porphyrin rings in the presence of other stimulating agent are important for ROS production and possibly the onset of TRALI. The ROS production induced by these molecules is dependent on a signal transduction pathway in a way similar to PMA.