Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 µg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.

Phorbol 12-myristate 13-acetate stimulation under hypoxia induces nuclear swelling with DNA outflow but not extracellular trap formation of neutrophils.

Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like extracellular DNA decorated with antimicrobial substances and can trap and eliminate invading microorganisms. Although phorbol 12-myristate 13-acetate (PMA) is a potent NET inducer, previous studies have demonstrated that not all neutrophils exhibit NET formation even if stimulated by PMA at high concentrations. This study first showed that some neutrophils stimulated by PMA displayed a swollen nucleus but not NET formation and that hypoxic environments suppressed the NET release. Next, characterization of PMA-stimulated neutrophils with a swollen nucleus was accomplished by differentiating between suicidal-type NETosis and apoptosis. Furthermore, the significance of the phenomenon was examined using formalin-fixed, paraffin-embedded human lung disease tissues with and without pneumonia. As a result, histone H3 citrullination, DNA outflow, propidium iodide labeling, resistance to DNase I, and suspended actin rearrangement were characteristics of PMA-stimulated neutrophils with a swollen nucleus distinct from neutrophils that underwent either suicidal-type NETosis or apoptosis. Neutrophils stimulated by PMA under hypoxic conditions secreted matrix metalloproteinase-9 cytotoxic to human lung-derived fibroblasts. Further, deposition of neutrophil-derived citrullinated histone H3+ chromatin substances in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and positively correlated with hypoxia-inducible factor-1α expression. The collective findings suggested that neutrophils activated under hypoxic conditions could be putative modulators of hypoxia-related disease manifestations.

Possible implication of intermolecular epitope spreading in the production of anti-glomerular basement membrane antibody in anti-neutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVES: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is sometimes complicated by anti-glomerular basement membrane (GBM) disease. Proteases, including elastase, released from neutrophils activated by ANCA are implicated in the pathogenesis of AAV. Epitopes of anti-GBM antibody exist in the α3-subunit non-collagenous (NC1) domain of collagen type IV [Col (IV)]. This region, called α3(IV)NC1, is structurally cryptic. This study aimed to determine the production mechanism of anti-GBM antibody in AAV. METHODS: We first examined whether α3(IV)NC1 could be revealed by the digestion of formalin-fixed, paraffin-embedded (FFPE) normal kidney sections and Col (IV) by proteases, including neutrophil elastase, using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Next, the reveal of α3(IV)NC1 and the infiltration of CD11c+ macrophages in the affected kidneys were evaluated by IHC and immunofluorescent staining using FFPE sections. Finally, the production of anti-GBM antibody in AAV rats was determined by ELISA. RESULTS: α3(IV)NC1 was revealed by the digestion of FFPE normal kidney sections and Col (IV) by proteases. Although the reveal of α3(IV)NC1 was observed in sclerotic glomeruli regardless of causative diseases, CD11c+ macrophages near α3(IV)NC1 were characteristics of AAV. Anti-GBM antibody was produced subsequent to ANCA in some AAV rats. IHC demonstrated the reveal of α3(IV)NC1 in affected renal tissues and the infiltration of CD11c+ macrophages around the sites. CONCLUSIONS: The collective findings suggest that, in AAV, proteases released from neutrophils activated by ANCA digest Col (IV) and result in the reveal of α3(IV)NC1, CD11c+ macrophages present GBM epitopes, and then the host's immune system produce anti-GBM antibody.

Association of Neutrophil Extracellular Traps with the Development of Idiopathic Osteonecrosis of the Femoral Head.

Idiopathic osteonecrosis of the femoral head (ONFH) is defined as necrosis of osteocytes due to a non-traumatic ischemia of the femoral head. Iatrogenic glucocorticoid administration and habitual alcohol intake are regarded as risk factors. It has been suggested that glucocorticoid-induced activation of platelets contributes to the local blood flow disturbance of the femoral head. Both activated platelets and alcohol can induce neutrophil extracellular traps (NETs). To determine the association of NETs with the development of idiopathic ONFH, surgically resected femoral heads of patients with idiopathic ONFH and osteoarthritis were assessed for existence of NET-forming neutrophils by immunofluorescence staining. NET-forming neutrophils were present in small vessels surrounding the femoral head of patients with idiopathic ONFH but not osteoarthritis. Moreover, Wistar-Kyoto rats were intravenously injected with NET-forming neutrophils or neutrophils without NET induction, and then the ischemic state of the tissue around the femoral head was evaluated by immunohistochemistry for hypoxia-inducible factor-1α. NET-forming neutrophils circulated into the tissue around the femoral head, and hypoxia-inducible factor-1α expression in the tissue was higher compared with that of rats intravenously administered with neutrophils without NET induction. Furthermore, ischemic change of osteocytes was observed in the femoral head of rats given an i.v. injection of NET-forming neutrophils. The collective findings suggest that NETs are possibly associated with the development of idiopathic ONFH.

Formation and Disordered Degradation of Neutrophil Extracellular Traps in Necrotizing Lesions of Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by the production of ANCAs and systemic necrotizing vasculitis in small vessels. Disordered regulation of neutrophil extracellular traps (NETs) is critically involved in the pathogenesis of AAV. NETs are web-like DNA decorated with antimicrobial proteins; they are extruded from activated neutrophils. The principal degradation factor of NETs in vivo is DNase I; however, NETs resistant to DNase I can persist in tissues and can lead to the production of ANCAs. Deposition of NETs has been demonstrated in glomerular crescents and necrotizing vasculitis in AAV. Here, the amount of NETs in formalin-fixed, paraffin-embedded tissue sections was examined, and the results for AAV were compared with the results for diseases that should be distinguished from AAV. NETs were more abundant in necrotizing vasculitis of AAV than in non-ANCA-associated vasculitis, or in granulomatous angiitis. Pulmonary granulomas in AAV and non-ANCA-associated diseases were further studied. The amount of NETs was significantly greater in necrotizing granulomas of AAV than in granulomas of sarcoidosis without necrosis. Although NETs were formed in necrotizing granulomas of tuberculosis equivalently to those formed in AAV, they were more susceptible to degradation by DNase I than were NETs in AAV. The formation and disordered degradation of NETs in necrotizing lesions are characteristics of AAV and are possibly related to its pathogenesis.

Recombinant thrombomodulin ameliorates autoimmune vasculitis via immune response regulation and tissue injury protection.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by necrotizing vasculitis with the presence of pathogenic ANCA. ANCA can potentially cause neutrophil activation and induce neutrophil extracellular traps (NETs), resulting in endothelial damage as well as activation of autoreactive B cells and alternative complement pathway. Recombinant thrombomodulin (rTM) protects the endothelium from vascular injury during disseminated intravascular coagulation, thus we hypothesized that rTM ameliorates necrotizing vasculitis in AAV. In this study, rTM was administered in an experimental AAV rat model. Treatment of experimental AAV rats with rTM improved pulmonary hemorrhage and glomerulonephritis, with a suppression of ANCA production and NETs formation. In addition, in vitro experiments showed that rTM bound to neutrophils via Mac-1 (macrophage-1 antigen) and inhibited ANCA-induced NETs formation accompanied by a suppression of histone citrullination, leading to a protection of the endothelium from NETs toxicity. Additionally, rTM affected lymphocytes leading to the inhibition of pro-inflammatory cytokine/chemokin in PBMC during the antibody production process, which might indirectly be involved in the reduction of pathogenic ANCA. Our data revealed that the rTM could ameliorate autoimmune vasculitis through a combination of different biological mechanisms.

Fluvastatin prevents the development of arthritis in env-pX rats via up-regulation of Rho GTPase-activating protein 12.

The pleiotropic effects of statins, including an antiarthritic potential, have been noted. This study aimed to determine the efficacy of statins on rheumatoid arthritis (RA) and clarify how statins affect its pathogenesis. Fluvastatin (500 µg/kg/day) or vehicle was given per os to env-pX rats, which carry the human T-cell leukemia virus type I env-pX gene and spontaneously develop destructive arthritis mimicking RA, for 30 days. Blood sampling and ultrasonography (US) of the ankle joints were conducted on days 0, 10, 20, and 30. On day 30, all rats were euthanized, and the ankle joints were subjected to histological analysis. To clarify how fluvastatin affects the pathogenesis of RA, comprehensive serum exosomal microRNA (miRNA) analysis was performed. Gene expression in the primary culture of synovial fibroblasts derived from arthritic rat and human and non-arthritic rat periarticular tissues was determined quantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR). As a result, the development of arthritis in env-pX rats was significantly suppressed by fluvastatin, which was evident from the viewpoints of serology, US imaging, and histology. Comprehensive serum exosomal miRNA analysis suggested that the expression of Rho GTPase-activating protein 12 (Arhgap12) was decreased in arthritic env-pX rats but increased with the administration of fluvastatin. Corresponding results were obtained by quantitative RT- PCR using primary culture of synovial fibroblasts. The collective findings suggest that fluvastatin prevents the development of arthritis in env-pX rats via the up-regulation of ARHGAP12. This study suggests that ARHGAP12 can be a possible therapeutic target of RA.

Relationship between lysosomal-associated membrane protein-2 and anti-phosphatidylserine/prothrombin complex antibody in the pathogenesis of cutaneous vasculitis.

OBJECTIVES: We investigated the relationship between lysosomal-associated membrane protein-2 (LAMP-2) and anti-phosphatidylserine/prothrombin (PS/PT) antibody in the pathogenesis of cutaneous vasculitis. METHODS: Cell surface LAMP-2 expression of human neutrophils was measured using flow cytometry. Twenty inbred wild-type Wistar-King-Aptekman-Hokudai (WKAH) rats were divided into four groups: Group 1, rabbit IgG injection only as negative control (n=5); Group 2, both histone and rabbit IgG injection (n=5); Group 3, anti-LAMP-2 antibody injection only (n=5); and Group 4, both histone and anti-LAMP-2 antibody injection (n=5). Ten WKAH rats were divided into two groups: Group A, histone, anti-PS/PT antibody, and anti-LAMP-2 antibody injection (n=5), and Group B, histone, anti-PS/PT antibody, and rabbit IgG injection as control (n=5). RESULTS: LAMP-2 expression on human neutrophils was induced by cell-free histone exposure in a dose- and time-dependent manner. Histopathological examination revealed the recruitment of neutrophils in cutaneous small vessels in all Group 4 rats. These observations were not evident in systemic organs other than the skin. LAMP-2 expression on the surface of vascular endothelial cells was evident in Group 2, exclusively in the skin, but not in Group 1. Thrombi were detected in various organs in all Groups A and B rats. However, no apparent thrombi were observed in the skin. CONCLUSIONS: Anti-PS/PT and anti-LAMP-2 antibodies are responsible for independent effector mechanisms in the rats given intravenous injection of cell-free histones. We considered that undetermined factors other than cell-free histones could be required for the induction of cutaneous vasculitis by anti-PS/PT and anti-LAMP-2 antibodies.

Pharmaceutical immunoglobulins reduce neutrophil extracellular trap formation and ameliorate the development of MPO-ANCA-associated vasculitis.

Objectives: Intravenous immunoglobulin (IVIG) therapy is effective against some autoimmune diseases. We examined the effects of pharmaceutical immunoglobulins on the development of MPO-ANCA-associated vasculitis (MPO-AAV).Methods: Peripheral blood neutrophils were pretreated with 5 mg/ml sulfo-immunoglobulins (IVIG-S) and then exposed to 100 nM phorbol myristate acetate (PMA). Thereafter, neutrophil extracellular traps (NETs) were detected by flow cytometry. Next, Wistar-Kyoto rats were given oral administration of 10 mg/kg/day propylthiouracil for 28 days and intraperitoneal (i.p.) injection of 1 µg PMA on days 0 and 7. These rats were divided into two groups: Group 1 with i.p. injection of 400 mg/kg IVIG-S on days 8-12 and Group 2 with vehicle similarly. ANCA titers were chronologically determined by indirect immunofluorescence. On day 28, all rats were killed to examine NET formation in the peritoneum and the development of AAV.Results: IVIG-S significantly inhibited NET formation induced by PMA in vitro. NET amounts in the peritoneum in Group 1 were significantly smaller than in Group 2, and ANCA titers in Group 1 were significantly lower than in Group 2. The degree of pulmonary hemorrhage in Group 1 was also smaller than in Group 2.Conclusion: IVIG-S reduce NET formation and ameliorate the development of MPO-AAV.

The presence of anti-neutrophil extracellular trap antibody in patients with microscopic polyangiitis.

OBJECTIVE: Although ANCA is the major autoantibody in patients with ANCA-associated vasculitis, previous studies have suggested the presence of anti-neutrophil extracellular trap (NET) antibody in patients with microscopic polyangiitis (MPA), one type of ANCA-associated vasculitis. In this study, we aimed to determine the prevalence and pathogenic role of anti-NET antibody (ANETA) in MPA. METHODS: We examined the presence or absence of ANETA in sera obtained from 19 MPA patients by indirect immunofluorescence. We compared the clinical parameters, including age, sex, MPO-ANCA, creatinine, CRP, MPO-DNA complexes and vasculitis activity, in ANETA-positive and ANETA-negative MPA patients. We investigated the serum NET induction and degradation abilities of ANETA-positive and ANETA-negative MPA patients with reference to healthy controls (n = 8). Furthermore, we assessed the relationship between ANETA and the effect of IgG depletion on the serum NET degradation ability. RESULTS: ANETA was present in 10 of the 19 MPA patients. There was no significant difference in the clinical parameters in ANCA-positive and ANCA-negative MPA patients. Although the NET induction ability was higher and the NET degradation ability was lower in MPA sera than those in healthy controls, these abilities were not different between ANETA-positive and ANETA-negative MPA sera. Interestingly, the NET degradation ability in some sera with ANETA was markedly increased by IgG depletion. CONCLUSION: Some MPA patients produce ANETA and some ANETA possess an inhibitory function against the serum NET degradation ability. Although further studies are needed, ANETA is worthy of attention in order to understand the pathophysiology of MPA.

Epitope recognized by anti-glomerular basement membrane (GBM) antibody in a patient with repeated relapse of anti-GBM disease.

The major epitopes recognized by autoantibodies in anti-glomerular basement membrane (GBM) disease are found in the α3-subunit non-collagenous domain of type IV collagen [α3(IV)NC1], which is present in the glomerular and alveolar basement membranes. These epitopes are structurally cryptic, owing to the hexamer formation of the non-collagenous domain of α3, α4, and α5 subunits and are expressed by the dissociation of the hexamer. Anti-GBM disease usually manifests as a single attack (SA), and we rarely see patients who repeatedly relapse. We recently treated a patient with anti-GBM disease who exhibited repeated relapse (RR). Here, we conducted immunohistochemistry of formalin-fixed paraffin-embedded normal kidney sections and immunoblotting using recombinant human α3(IV)NC1 to compare the epitopes recognized by anti-GBM antibodies in the RR patient and SA patients. Although a clear staining of GBM especially in the connecting basement membrane of Bowman's capsule was observed when IgGs of SA patients were used as primary antibodies, such staining was not obtained when IgG of the RR patient was employed. In immunoblotting of α3(IV)NC1 using the IgG of the RR patient as a primary antibody, an 18-kDa band was detected besides the 56.8-kDa band corresponding to the whole-size α3(IV)NC1. Whereas the 56.8-kDa band disappeared after digestion of the recombinant α3(IV)NC1 by protease, the 18-kDa band remained. Furthermore, the 18-kDa band was not detected by a commercially available anti-α3(IV)NC1 monoclonal antibody. These findings suggest that the IgG of the RR patient recognizes the epitope distinct from that recognized by the anti-α3(IV)NC1 monoclonal antibody.

Type II Natural Killer T Cells that Recognize Sterol Carrier Protein 2 Are Implicated in Vascular Inflammation in the Rat Model of Systemic Connective Tissue Diseases.

We previously generated a rat model that developed systemic connective tissue diseases, including synovitis, myositis, and small-vessel vasculitis (SVV), and established a vascular endothelial cell-reactive T-cell clone, VASC-1, from the model. VASC-1 was determined to be a type II natural killer T-cell clone. In this study, we attempted to identify the antigen recognized by VASC-1. The monkey-derived cell line COS-7 was used because VASC-1 does not bind naturally to COS-7, although the amino acid sequences are well conserved between monkey CD1d and rat CD1d. We generated 98 COS-7 clones transfected with miscellaneous rat cDNA and screened them for VASC-1 binding. Consequently, we found one clone, 4D2, which could bind to VASC-1. Sequencing identified the rat cDNA introduced into 4D2 as sterol carrier protein 2 (SCP2). When VASC-1 was co-cultured with SCP2 knockdown rat vascular endothelial cells, VASC-1 binding was reduced significantly. Moreover, we designed a series of rat SCP2 peptides and introduced them into COS-7 cells. On the basis of VASC-1 binding and proliferation, we revealed that the peptide rSCP2518-532 included the epitope recognized by VASC-1. Furthermore, immunization with rSCP2518-532 accelerated the development of SVV in the rat model. The collective findings suggest that type II natural killer T cells reactive with autologous SCP2 are implicated in vascular inflammation in the rat model.

Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patients with Vasculitis.

NKT cells are defined as T cells that recognize hydrophobic antigens presented by class I MHC-like molecules, including CD1d. Among CD1d-restricted NKT cells, type I and type II subsets have been noted. CD1d-restricted type I NKT cells are regarded as pro-inflammatory cells in general. On the contrary, accumulated evidence has demonstrated an anti-inflammatory property of CD1d-restricted type II NKT cells. In our earlier study using a rat model with vasculitis, we demonstrated the pro-inflammatory function of CD1d-restricted type II NKT cells and identified that one such cell recognized P518-532 of rat sterol carrier protein 2 (rSCP2518-532 ), which appeared on vascular endothelial cells presented by CD1d. Based on this evidence, we attempted to detect human CD1d-restricted type II NKT cells in peripheral blood using hSCP2518-532 , the human counterpart of rSCP2518-532, together with a CD1d tetramer in flow cytometry. First, we determined the binding of hSCP2518-532 to CD1d. Next, we detected CD3-positive hSCP2518-532 -loaded CD1d (hSCP2518-532 /CD1d) tetramer-binding cells in peripheral blood of healthy donors. The abundance of TGF-ß-producing cells rather than TNF-α-producing cells in CD3-positive hSCP2518-532 /CD1d tetramer-binding cells suggests the anti-inflammatory property of SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between healthy individuals and patients with vasculitis in a pilot study. Interestingly, the percentage of TGF-ß-producing cells in SCP2/CD1d tetramer-binding type II NKT cells in vasculitic patients was significantly lower than that in healthy controls despite the greater number of these cells. Although further studies to clarify the mechanism and significance of this phenomenon are needed, SCP2/CD1d tetramer-binding type II NKT cells in peripheral blood should be examined in more detail to understand the pathophysiology of vasculitides in humans. © 2018 International Society for Advancement of Cytometry.

Anti-neutrophil extracellular trap antibody in a patient with relapse of anti-neutrophil cytoplasmic antibody-associated vasculitis: a case report.

BACKGROUND: Neutrophil extracellular traps (NETs) are web-like DNA decorated with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. Although NETs are essential in innate immunity, an excessive formation of NETs has adverse effects, e.g., induction of anti-neutrophil cytoplasmic antibody (ANCA), to the hosts. Since ANCA can induce NET formation in the primed neutrophils, a positive feedback loop can be formed between NETs and ANCA, which is called "ANCA-NETs vicious cycle." CASE PRESENTATION: A 79-year-old Japanese woman developed hydralazine-induced pauci-immune necrotizing crescentic glomerulonephritis with MPO-ANCA. Although the illness improved after cessation of hydralazine, MPO-ANCA-associated vasculitis relapsed 16 months later. Remission was achieved 5 months after beginning of administration of prednisone. In order to determine the involvement of ANCA-NETs vicious cycle in this patient, we examined NET degradation and induction activities in sera obtained at the disease onset (Serum A; MPO-ANCA, 107 IU/ml), at relapse (Serum B; MPO-ANCA, 195 IU/ml), at 3 months after treatment (Serum C; MPO-ANCA, 4.5 IU/ml), and at remission (Serum D; MPO-ANCA, 2.4 IU/ml). NET degradation activity was low in the all sera. NET induction activity was high in Sera A, B, and C but not in D. Additionally, we demonstrated the presence of anti-NET antibody (ANETA) in Sera B and C but not in A or D. CONCLUSIONS: The collective findings suggest NET induction potential of ANETA in the present patient and that the ANETA could contribute to the enhancement of NETs resulting in amplification of the ANCA-NETs vicious cycle.

Measurement of NET formation in vitro and in vivo by flow cytometry.

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane-impermeable DNA-binding dye, SYTOX Green. SYTOX Green-positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Green-positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co-localization of MPO and plasma membrane-appendant DNA of SYTOX Green-positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green-positive cells induced by PMA. The collective evidence suggests that SYTOX Green-positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well-used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Establishment of a rat model of thrombosis induced by intravenous injection of anti-phosphatidylserine-prothrombin complex antibody.

Objective: Recent studies have suggested that aPS-PT antibody is one of the most relevant autoantibodies to APS. This study aimed to demonstrate the pathogenicity of aPS-PT antibody in vivo . Methods: At first, cultured rat vascular endothelial cells (RECs) were exposed to calf thymus-derived histones. Two hours later, lactate dehydrogenase release from the RECs and expression of PS on the cell surface were assessed. Next, we administered an i.v. injection of calf thymus-derived histones into Wistar rats (12.5 µg/g weight of 8-week-old female rats), and 2 h later they were given an i.v. injection of aPS-PT mAb (1.25 mg/g weight, n = 6) or an equal dose of rat IgM as controls (n = 5). Three days later, histological examination was conducted. Results: Calf thymus-derived histones (>12.5 µg/ml) could injure RECs in vitro . Simultaneously, annexin V could bind to the RECs; thereby, this result indicated that cell-free histone exposure of vascular endothelial cells induced cell surface expression of PS, which is naturally present inside the plasma membrane. Thrombosis developed with higher frequency in the rats given an i.v. injection of aPS-PT mAb than in controls. Conclusion: We established a rat model of thrombosis induced by i.v. injection of aPS-PT mAb.

Establishment of a vascular endothelial cell-reactive type II NKT cell clone from a rat model of autoimmune vasculitis.

We previously generated a rat model that spontaneously developed small vessel vasculitis (SVV). In this study, a T cell clone reactive with rat vascular endothelial cells (REC) was established and named VASC-1. Intravenous injection of VASC-1 induced SVV in normal recipients. VASC-1 was a TCRαß/CD3-positive CD4/CD8 double-negative T cell clone with expression of NKG2D. The cytokine mRNA profile under unstimulated condition was positive for IL-4 and IFN-γ but negative for IL-2 and IL-10. After interaction with REC, the mRNA expression of IL-2, IL-5 and IL-6 was induced in VASC-1, which was inhibited by blocking of CD1d on the REC surface. Although the protein levels of these cytokines seemed to be lower than the detection limit in the culture medium, IFN-γ was detectable. The production of IFN-γ from the VASC-1 stimulated with LPS-pre-treated REC was inhibited by the CD1d blockade on the REC. These findings indicated VASC-1 as an NKT cell clone. The NKT cell pool includes two major subsets, namely types I and II. Type I NKT cells are characterized by expression of semi-invariant TCRs and the potential to bind to marine sponge-derived α-galactosylceramide (α-GalCer) loaded on CD1d; whereas, type II NKT cells do not manifest these characteristics. VASC-1 exhibited a usage of TCR other than the type I invariant TCR α chain and did not bind to α-GalCer-loaded CD1d; therefore, it was determined as a type II NKT cell clone. The collective evidence suggested that REC-reactive type II NKT cells could be involved in the pathogenesis of SVV in rats.

Abnormal conformation and impaired degradation of propylthiouracil-induced neutrophil extracellular traps: implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: Neutrophil extracellular traps (NETs) are composed of DNA and antimicrobial proteins, including myeloperoxidase (MPO). Recent studies have demonstrated that impaired regulation of NETs could trigger an autoimmune response. Propylthiouracil (PTU), an antithyroid drug, is associated with a risk of MPO antineutrophil cytoplasmic antibody (ANCA) production and MPO ANCA-associated vasculitis (MPO AAV). This study was undertaken to clarify the mechanism of MPO ANCA production, using the PTU-induced model of MPO AAV. METHODS: NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats. RESULTS: When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci-immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum. CONCLUSION: Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU-induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

Possible implication of Fc γ receptor-mediated trogocytosis in susceptibility to systemic autoimmune disease.

Leukocytes can "gnaw away" the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fc γ receptor-(Fc γ R-) mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of Fc γ R-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via Fc γ R-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via Fc γ R-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of Fc γ R-mediated trogocytosis as a means to remove antibodies (Abs) that bind with self-molecules rather than to extract molecules from other cells. This concept means that Fc γ R-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of Fc γ R-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that Fc γ R-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases.

Demonstration of equivocal anti-glomerular basement membrane antibody positivity as a non-specific reaction through multiple immunologic assays in a case of pediatric asymptomatic hematuria.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody is essential for the diagnosis of anti-GBM disease. The major epitope consists of the α3 subunits of type IV collagen non-collagenous domain (α 3(IV)NC1). There have been only a few reports of patients false-positive for anti-GBM antibody. CASE REPORT: We experienced an 8-year-old boy who presented with asymptomatic hematuria followed by positivity for anti-GBM antibody as evaluated by a commercially available chemiluminescent enzyme immunoassay (CLEIA). While his condition remained stable other than continuing hematuria, his anti-GBM antibody titer increased. Further examination of another anti-GBM antibody assay (fluoroenzyme immunoassay) showed negative results. Thus, evaluation of the accuracy of his positivity for anti-GBM antibody was required. We conducted the following examinations: A) enzyme-linked immunosorbent assay, B) immunoblotting for recombinant α 1-5(IV)NC1, and C) immunohistochemical analysis of normal kidney tissue sections. Specimens used for the analysis were sera in A and IgG from the patient in B and C, respectively. As a result, no anti-GBM antibody was detected in A. In B, no band specific to α 1-5(IV)NC1 was observed. In C, the kidney tissue was not stained. Taken together, these results led us to judge the positive anti-GBM result in CLEIA of our patient to be a non-specific reaction. CONCLUSION: The commercial assays for anti-GBM antibody can lead to false-positive results. We recommend confirmation of anti-GBM antibody positivity through the use of multiple assays in patients demonstrating an atypical clinical course for anti-GBM disease.