Yoshitaka Fukunaga

Implications for Induction of Autoimmunity via Activation of B-1 Cells by Helicobacter pylori Urease

ABSTRACT Besides various gastroduodenal diseases, Helicobacter pylori infection may be involved in autoimmune disorders like rheumatoid arthritis (RA) or idiopathic thrombocytopenic purpura. Such autoimmune disorders are often associated with autoreactive antibodies produced by B-1 cells, a subpopulation of B lymphocytes. These B-1 cells are mainly located in the pleural cavity or mucosal compartment. The existence of H. pylori urease-specific immunoglobulin A (IgA)-producing B cells in the mucosal compartment and of their specific IgM in the sera of acutely infected volunteers suggests the possibility that urease stimulates mucosal innate immune responses. Here, we show for the first time that purified H. pylori urease predominantly stimulates the B-1-cell population rather than B-2 cells, which produce antigen-specific conventional antibodies among splenic B220+ B cells. The fact that such stimulation of B-1 cells was not affected by the addition of polymyxin B indicates that the effect of purified H. pylori urease was not due to the contamination with bacterial lipopolysaccharide. Furthermore, the production of various B-1-cell-related autoreactive antibodies such as IgM-type rheumatoid factor, anti-single-stranded DNA antibody, and anti-phosphatidyl choline antibody was observed when the splenic B cells were stimulated with purified H. pylori urease in vitro. These findings suggest that H. pylori components, urease in particular, may be among the environmental triggars that initiate various autoimmune diseases via producing autoreactive antibodies through the activation of B-1 cells. The findings shown here offer important new insights into the pathogenesis of autoimmune disorders related to H. pylori infection.

ANG II receptor blockade enhances anti-inflammatory macrophages in anti-glomerular basement membrane glomerulonephritis

Macrophages are heterogeneous immune cell populations that include classically activated and alternatively activated (M2) macrophages. We examined the anti-inflammatory effect of ANG II type 1 receptor (AT(1)R) blocker (ARB) on glomerular inflammation in a rat model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). The study focused on infiltrating CD8(+) and CD4(+) cells and macrophages, as well as the heterogeneity of intraglomerular macrophages. Wistar-Kyoto rats were treated with high-dose olmesartan (3 mg.kg(-1).day(-1)), low-dose olmesartan (0.3 mg.kg(-1).day(-1)), or vehicle (control) 7 days before induction of anti-GBM GN. Control rats showed mainly CD8(+) cells and ED1(+) macrophages, with a few CD4(+) cells infiltrating the glomeruli. Necrotizing and crescentic glomerular lesions developed by day 7 with the increase of proteinuria. AT(1)R was expressed on CD8(+) and CD4(+) cells and on ED1(+) macrophages. Low-dose ARB had no anti-inflammatory effects in anti-GBM GN. However, high-dose ARB reduced glomerular infiltration of CD8(+) cells and ED1(+) macrophages and suppressed necrotizing and crescentic lesions by days 5 to 7 (P < 0.05). In addition, high-dose ARB reduced the numbers of ED3(+)-activated macrophages, suppressed glomerular TNF-alpha and IFN-gamma production, and downregulated M1-related chemokine and cytokines (monocyte chemoattractant protein type 1, IL-6, and IL-12). High-dose ARB also enhanced ED2(+) M2 macrophages by day 7 with upregulation of glomerular IL-4 and IL-13 and augmented CCL17, IL-1 receptor antagonist, and IL-10. We concluded that high-dose ARB inhibits glomerular inflammation by increasing the numbers of M2 macrophages and upregulation of anti-inflammatory cytokines and by suppressing M1 macrophage development with downregulation of M1-related proinflammatory cytokines.

Generation of a Chimeric Mouse Reconstituted with Green Fluorescent Protein-Positive Bone Marrow Cells: A Useful Model for Studying the Behavior of Bone Marrow Cells in Regeneration In Vivo

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow—derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for a smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells, as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.

Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

A comparative study of nitric oxide, glutathione, and glutathione peroxidase activities in cerebrospinal fluid from children with convulsive diseases/children with aseptic meningitis.

It has been reported that active oxygen and/or free radicals are produced in the central nervous system (CNS) compartment in patients with bacterial meningitis, so it is supposed that the levels of endogenous antioxidative scavengers in the cerebrospinal fluid (CSF) are elevated as an adaptive reaction to bacterial meningitis, which exerts severe stress on the human body. We assumed that they are also elevated in patients with convulsive diseases. Nitric oxide (NO) and endogenous antioxidative scavengers (glutathione (GSH), glutathione peroxidase (GPX), (total) superoxide dismutase (T-SOD), manganese superoxide dismutase (Mn-SOD), and catalase) were measured in CSF from a group of child patients with various neurological diseases and a control group. NO, GSH, and GPX activities in CSF from the patients with convulsive diseases were significantly higher than in those with aseptic meningitis or in the controls. Furthermore, all parameters in CSF from patients with bacterial meningitis were significantly higher than in any other group. The present study suggests that oxidative stress may be associated with the pathophysiology of convulsion and that its clinical attenuation will lead to improvement in the prognosis for convulsive diseases.

Rescue of severe infantile hypophosphatasia mice by AAV-mediated sustained expression of soluble alkaline phosphatase.

Hypophosphatasia (HPP) is an inherited disease caused by a deficiency of tissue-nonspecific alkaline phosphatase (TNALP). The major symptom of human HPP is hypomineralization, rickets, or osteomalacia, although the clinical severity is highly variable. The phenotypes of TNALP knockout (Akp2(-/-)) mice mimic those of the severe infantile form of HPP. Akp2(-/-) mice appear normal at birth, but they develop growth failure, epileptic seizures, and hypomineralization and die by 20 days of age. Previously, we have shown that the phenotype of Akp2(-/-) mice can be prevented by enzyme replacement of bone-targeted TNALP in which deca-aspartates are linked to the C-terminus of soluble TNALP (TNALP-D10). In the present study, we evaluated the therapeutic effects of adeno-associated virus serotype 8 (AAV8) vectors that express various forms of TNALP, including TNALP-D10, soluble TNALP tagged with the Flag epitopes (TNALP-F), and native glycosylphosphatidylinositol-anchored TNALP (TNALP-N). A single intravenous injection of 5×10(10) vector genomes of AAV8-TNALP-D10 into Akp2(-/-) mice at day 1 resulted in prolonged survival and phenotypic correction. When AAV8-TNALP-F was injected into neonatal Akp2(-/-) mice, they also survived without epileptic seizures. Interestingly, survival effects were observed in some animals treated with AAV8-TNALP-N. All surviving Akp2(-/-) mice showed a healthy appearance and a normal activity with mature bone mineralization on X-rays. These results suggest that sustained alkaline phosphatase activity in plasma is essential and sufficient for the rescue of Akp2(-/-) mice. AAV8-mediated systemic gene therapy appears to be an effective treatment for the infantile form of human HPP.

Role of Bone Marrow Cells in the Healing Process of Mouse Experimental Glomerulonephritis

Recent studies have shown bone marrow (BM) cells to differentiate into a variety of cell types and to thereby participate in the reconstitution of damaged organs. In the present study, we examined the extent to which BM-derived cells are incorporated into glomeruli during recovery from experimentally induced nephritis. To investigate the localization of BM cells in glomeruli, chimeric mice were prepared by transplanting BM cells from green fluorescent protein (GFP) transgenic mice into wild-type mice. Five weeks later, glomerulonephritis was induced by intravenous injection of Habu snake venom. Groups of mice were then killed every few days for 42 d, and harvested kidney samples were subjected to immunohistochemical and immunoelectron microscopic analyses with the aim of detecting the presence of GFP(+) cells within glomeruli. Chimeric animals injected with Habu venom developed proliferative glomerulonephritis within 1–3 d. The lesion gradually subsided and the glomerular structure returned to normal within 42 d. Consistent with the disease course, large numbers of GFP(+) cells were present within glomeruli on d 1–3, but most had disappeared by d 7. Nevertheless, some GFP(+) cells did remain within glomeruli showing mesangial proliferative changes, and were found to express thrombomodulin (TM), a specific endothelial cell marker. These GFP-TM–double-positive cells accounted for a mean of 1.31–2.24% of the total glomerular nuclei from d 7 through d 42, levels that remained stable for at least 12 mo. It thus appears that BM cells can give rise to endothelial cells that participate in the remodeling of glomeruli.

Stress-induced cell surface expression and antigenic alteration of the Ro/SSA autoantigen.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous. There are two isoform families; the 60 kD forms (Ro60) and the 52 kD forms (Ro52). Recently we have found that autoantibodies to the Ro/SSA proteins are conformation dependent. Anti-Ro60 antibodies are mainly directed to the native protein and conversely anti-Ro52 antibodies are directed only to the denatured protein. It has been known that UV irradiation to cultured keratinocytes induces cell surface expression of Ro/SSA and this phenomenon has been thought to be related with photosensitivity in patients with anti-Ro/SSA antibodies. We studied the quantitative and qualitative changes of the Ro/SSA protein induced by stress, such as with heat shock and UV irradiation, and found that only Ro52 could be expressed on the cell surface of human peripheral lymphocytes by either heat shock or UV irradiation. Moreover, flow cytometric analysis revealed that HS-treated and UV-treated lymphocytes could be stained with patient sera, and by using a technique which combined immunoprecipitation and Western immunoblotting, it has been confirmed that Ro52 expressed on the cell surface can be recognized by anti-Ro/SSA antibodies in native form while cytoplasmic Ro52 cannot be recognized. These data suggest that Ro52 can be antigenic in vivo when expressed on the cell surface and may explain the mechanism of direct tissue damage by anti-Ro/SSA antibodies.

Successful Gene Therapy In Utero For Lethal Murine Hypophosphatasia

Hypophosphatasia (HPP), caused by mutations in the gene ALPL encoding tissue-nonspecific alkaline phosphatase (TNALP), is an inherited systemic skeletal disease characterized by mineralization defects of bones and teeth. The clinical severity of HPP varies widely, from a lethal perinatal form to mild odontohypophosphatasia showing only dental manifestations. HPP model mice (Akp2(-/-)) phenotypically mimic the severe infantile form of human HPP; they appear normal at birth but die by 2 weeks of age because of growth failure, hypomineralization, and epileptic seizures. In the present study, we investigated the feasibility of fetal gene therapy using the lethal HPP model mice. On day 15 of gestation, the fetuses of HPP model mice underwent transuterine intraperitoneal injection of adeno-associated virus serotype 9 (AAV9) expressing bone-targeted TNALP. Treated and delivered mice showed normal weight gain and seizure-free survival for at least 8 weeks. Vector sequence was detected in systemic organs including bone at 14 days of age. ALP activities in plasma and bone were consistently high. Enhanced mineralization was demonstrated on X-ray images of the chest and forepaw. Our data clearly demonstrate that systemic injection of AAV9 in utero is an effective strategy for the treatment of lethal HPP mice. Fetal gene therapy may be an important choice after prenatal diagnosis of life-threatening HPP.

IL-8 in Cerebrospinal Fluid from Children with Acute Encephalopathy is Higher than in that from Children with Febrile Seizure

We identify possible differences in the cytokine/chemokine profiles in cerebrospinal fluid (CSF) from children with encephalopathy and febrile seizure. Interleukin (IL)‐1β, 2, 4, 5, 6, 7, 8, 10, 12, 13, 17, interferon‐γ, tumour necrosis factor‐α, granulocyte colony‐stimulating factor, granulocyte monocyte colony‐stimulating factor, monocyte chemoattractant protein‐1 and macrophage inflammatory protein‐1β were measured simultaneously in CSF supernatants from children with encephalopathy (n = 8), febrile seizure (n = 16) and fever without neurological complications (n = 8). IL‐8 in CSF from children with encephalopathy was significantly elevated compared to that in CSF from children with febrile seizure and fever without neurological complications. IL‐8 in CSF was also higher than serum IL‐8, suggesting that increased IL‐8 was generated from glia cells or astrocytes, not by leakage from serum. Increased IL‐8 in CSF in encephalopathy may protect against severe brain damage.