Kouhei Yamashita

Involvement of activation of toll-like receptors and nucleotide-binding oligomerization domain-like receptors in enhanced IgG4 responses in autoimmune pancreatitis.

OBJECTIVE IgG4-related disease is a recently recognized entity affecting multiple organs, including the pancreas, biliary tracts, and salivary glands. Although IgG4-related disease is characterized by systemic IgG4 antibody responses and by infiltration of IgG4-expressing plasma cells, the innate immune responses leading to adaptive IgG4 antibody responses are poorly understood. The aim of this study was to clarify the innate immune responses leading to IgG4 antibody production. METHODS IgG4 and cytokine responses to various nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) and Toll-like receptor (TLR) ligands were examined using peripheral blood mononuclear cells (PBMCs) from healthy control subjects and patients with IgG4-related autoimmune pancreatitis. RESULTS Activation of NOD-2 in monocytes from healthy control subjects induced IgG4 production by B cells in a BAFF-dependent and T cell-independent manner. In addition, PBMCs from patients with IgG4-related disease produced a large amount of IgG4 upon stimulation with NLR and TLR ligands; this enhanced IgG4 production was associated with the induction of BAFF by NLR and TLR ligands. Monocytes from patients with IgG4-related disease induced IgG4 production by B cells from healthy control subjects upon stimulation with NLR and TLR ligands. CONCLUSION The results of these studies suggest that abnormal innate immune responses against microbial antigens may underlie the immunopathogenesis of IgG4-related disease.

Singlet oxygen is essential for neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

Toll-like receptor activation in basophils contributes to the development of IgG4-related disease

BackgroundIgG4-related disease (IRD) is characterized by systemic IgG4 antibody responses and by infiltration of IgG4-expressing plasma cells into the affected organs. Although T helper type 2 (Th2) cytokines are implicated in enhanced IgG4 responses, molecular mechanisms accounting for the development of IgG4 antibody responses are poorly defined. Since basophils function as antigen-presenting cells for Th2 responses, we tried to clarify the role of basophils in the development of IgG4 responses in this study.MethodsIgG4 and cytokine responses to various nucleotide-binding oligomerization domain-like receptor and Toll-like receptor (TLR) ligands were examined by using basophils isolated from healthy controls and from patients with IgG4-related disease.ResultsActivation of TLRs in basophils from healthy controls induced IgG4 production by B cells, which effect was associated with enhanced production of B cell activating factor (BAFF) and IL-13. In addition, activation of TLRs in basophils from patients with IRD induced a large amount of IgG4 by B cells from healthy controls. This enhancement of IgG4 production was again associated with BAFF and IL-13.ConclusionsThese data suggest that innate immune responses mediated through TLRs may play a role in the development of IgG4-related disease, in part by production of BAFF from basophils.

Serum Neutrophil Extracellular Trap Levels Predict Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

Ozone production by amino acids contributes to killing of bacteria

Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.

The expression of co-stimulatory molecules and their relationship to the prognosis of human acute myeloid leukaemia : poor prognosis of B7-2-positive leukaemia

We examined the expression of co‐stimulatory molecules on leukaemic cells of 52 adult patients with acute myeloid leukaemia (AML) (34 men and 18 women) and analysed the relationship between these expressions and the patients prognosis. B7‐1 was not expressed in any of the 23 patients investigated, whereas B7‐2 was expressed in 26/52 patients (50.0%). B7‐2 was expressed in all AML patients with monocytic morphology (M4 or M5) and in 16/42 cases without monocytic morphology. CD54 was expressed in 28/37 patients examined (75.7%), and CD58 was expressed in all of the AML patients except one (M7). The overall survival of the 26 B7‐2‐positive leukaemia patients (1–24 months, median survival 11.5 months) was significantly shorter than that of the 26 B7‐2‐negative leukaemia patients (1–71+ months, median 35.1 months) (P = 0.0080). In addition, the B7‐2‐positive patients exhibited significantly shorter disease‐free survival periods compared to the B7‐2‐negative patients (P = 0.021). There was no significant difference in age, sex, haematological data and complete remission rate between the B7‐2‐positive and B7‐2‐negative patients. Our results indicated that B7‐2 is one of the most crucial factors in the prognosis of adult acute leukaemia and can be expected to have an important role in tumour immunity.

Pretransplant serum ferritin and C-reactive protein as predictive factors for early bacterial infection after allogeneic hematopoietic cell transplantation.

Although fluoroquinolones or other antibiotics are commonly used to prevent bacterial infections after hematopoietic cell transplantation (HCT), because of the growing presence of multidrug-resistant microorganisms, it is important to identify patients who are more likely to benefit from antibacterial prophylaxis. To evaluate risk factors for early bacterial infection after allogeneic HCT, we retrospectively analyzed clinical data for 112 consecutive adult patients with hematological malignancies who received transplants without any antibacterial prophylaxis. The cumulative incidence of bacterial infection at 30 days after transplantation was 16%. Among various pre-transplant factors, only high serum ferritin (>700 ng/mL, 47 patients) and high C-reactive protein (CRP) (>0.3 mg/dL, 28 patients) levels were significantly associated with the development of bacterial infection in a multivariate analysis (hazard ratio (95% confidence interval): ferritin, 4.00 (1.32–12.17); CRP, 3.64 (1.44–9.20)). In addition, septic shock and sepsis with organ failure were exclusively observed in patients who had high ferritin and/or high CRP levels. These results suggest that pretransplant serum ferritin and CRP levels can be useful markers for predicting the risk of early bacterial infection after allogeneic HCT. It may be prudent to limit antibacterial prophylaxis to patients with predefined risk factors to ensure the safety of HCT with the use of fewer antibiotics.

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation.

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.

Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C

Granulocyte/macrophage colony‐stimulating factor (GM‐CSF) inhibits Fas‐induced apoptosis of neutrophils. However, the exact step in the apoptotic pathway blocked by GM‐CSF remained unclear. Here, we found that pretreatment of neutrophils with GM‐CSF inhibits the recruitment of Fas‐associated protein with death domain (FADD) to Fas, abolishing the formation of the death‐inducing signaling complex required for Fas‐induced apoptosis. Two‐dimensional electrophoresis revealed that GM‐CSF modifies the ratio of FADD subspecies. These GM‐CSF‐triggered changes were abrogated, and Fas‐induced apoptosis was restored by an inhibitor of classical protein kinase C (PKC), Gö6976, and by the combination of a phosphatidylinositol 3‐kinase (PI‐3K) inhibitor, LY294002, and an inhibitor of mitogen‐activated protein kinase kinase (MEK)1, PD98059. Gö6976 blocked GM‐CSF‐elicited phosphorylation of Akt/PKB and extracellular signal‐regulated kinase (ERK)1/2. These results indicated that GM‐CSF suppresses Fas‐induced neutrophil apoptosis by inhibiting FADD binding to Fas, through redundant actions of PI‐3K and MEK1‐ERK1/2 pathways downstream of classical PKC.