Beneficial effects of anti-apolipoprotein A-2 on an animal model for coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is usually treated with high-dose intravenous immunoglobulin (IVIg) as severe infectious and other diseases. Due to issues that are associated with immunoglobulin preparation, such as the risk of possible contamination by infectious agents and limited blood banking resources, recombinant immunoglobulins are required. We developed a novel recombinant antibody drug candidate, "VasSF," based on the therapeutic effects it exerted on a mouse spontaneous crescentic glomerulonephritis model (SCG/Kj). Apolipoprotein A-2 (ApoA2) has been identified as one of VasSF's target molecules. METHODS: Here, we tested the potential of anti-apolipoprotein A-2 antibodies (anti-ApoA2) as a new therapeutic drug against KD by examining its effect on a mouse model, in which KD was induced via Candida albicans water-soluble fraction (CAWS). CAWS (2 mg/mouse) was injected intraperitoneally into C57BL/6NCrSlc mice for five consecutive days. The incidence and histological severity of vasculitis in CAWS-induced coronary arteritis in mice administered anti-ApoA2 was examined. The following experimental groups were tested: solvent (only PBS (-) injection); anti-ApoA2 antibodies at dosages of 0.05 mg, 0.1 mg, and 0.5 mg/kg/day; human IgG at 0.1 mg/kg/day. RESULTS: The group treated with anti-ApoA2 0.5 mg/kg/day showed a lower incidence of panvasculitis induced by CAWS, less inflammation of the coronary arteries and aortic roots, and lower levels of serum IL-6, M-CSF, and MIP-1α and 32 cytokines/chemokines compared with those in the solvent group. CONCLUSIONS: The anti-ApoA2 treatment suppressed the development of coronary arteritis in an animal KD model and anti-ApoA2 shows potential as an effective therapeutic candidate for the treatment of KD vasculitis. The use of specific antibodies that display higher vasculitis-suppressing effects, such as anti-ApoA2, may attenuate KD as well as other infectious diseases, with less severe adverse side effects than treatment with IVIg.

Soluble Uric Acid Promotes Atherosclerosis via AMPK (AMP-Activated Protein Kinase)-Mediated Inflammation.

OBJECTIVE: Uric acid is supposed but not yet determined to be associated with atherosclerosis. Uric acid is released from damaged cells to form urate crystal, which is recognized by the immune system to produce IL (interleukin)-1. Danger signals and IL-1 have been shown to play an important role in atherosclerosis. We determined whether the physiological level of soluble uric acid promotes inflammation and develops atherosclerosis. Approach and Results: The secretion of IL-1ß from human peripheral blood mononuclear cells mediated by NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome was promoted by physiological levels in serum uric acid. This augmentation of inflammation was mediated by the regulation of the AMPK (AMP-activated protein kinase)-mTOR (mammalian target of rapamycin) mitochondrial reactive oxygen species and HIF-1α (hypoxia-inducible factor-1α) pathway. In both of uricase transgenic and xanthine oxidase inhibitor-treated mice, decreased levels of uric acid resulted in the activation of AMPK and attenuation of the development of atherosclerotic plaques. Further, acute uric acid reduction by the administration of benzbromarone in healthy humans for 2 weeks significantly decreased plasma IL-18-an inflammasome-dependent cytokine. CONCLUSIONS: The data indicate that the development of atherosclerosis and inflammation is promoted by uric acid in vivo. Moreover, the lowering of uric acid levels attenuated inflammation via the activation of the AMPK pathway. This study provides mechanistic evidence of uric acid-lowering therapies for atherosclerosis.

Role of serum autoantibodies in blood brain barrier damages in neuropsychiatric systemic lupus erythematosus.

OBJECTIVES: The present study was carried out to elucidate the roles of serum autoantibodies in the development of blood-brain barrier (BBB) damages in neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: Paired serum and CSF samples were obtained from 101 SLE patients when they presented active neuropsychiatric manifestations (69 patients with diffuse psychiatric/neuropsychological syndromes [diffuse NPSLE] and 32 patients with neurologic syndromes or peripheral neuropathy [focal NPSLE]). IgG anti-NR2 subunit of NMDA receptor (anti-NR2), anti-Sm, anti-ribosomal P and IgG anti-cardiolipin in sera and albumin in CSF and sera were measured by ELISA. Blood-brain barrier (BBB) function was evaluated by Q albumin (CSF/serum albumin quotient x 1,000). RESULTS: Q albumin was significantly higher in acute confusional state (ACS) than in non-ACS diffuse NPSLE (anxiety disorder, cognitive dysfunction, mood disorder and psychosis) or in focal NPSLE. Anti-Sm, but not anti-NR2, anti-P or anticardiolipin, was significantly elevated in ACS compared with the other 2 groups of NPSLE, although serum anti-NR2 was significantly higher in ACS than that in focal NPSLE. Multiple regression analysis confirmed the significant contribution of anti-Sm (p=0.0040), but not anti-NR2 (p=0.5023), anti-P (p=0.2651), or anti-cardiolipin (p=0.6769) in the elevation of Q albumin. CONCLUSIONS: The data demonstrate that serum anti-Sm antibodies play a most important role in the disruption of BBB in NPSLE.

Association of cerebrospinal fluid anti-Sm antibodies with acute confusional state in systemic lupus erythematosus.

INTRODUCTION: Neuropsychiatric manifestation in systemic lupus erythematosus (NPSLE) is one of the most serious complications of the disease. Previous studies revealed the strong association between serum anti-Sm and organic brain syndrome, consisting mainly of acute confusional state (ACS) of diffuse psychiatric/neuropsychological syndromes (diffuse NPSLE). However, the precise mechanism by which anti-Sm causes diffuse NPSLE remains unclear. Of note, recent studies demonstrated that anti-U1 RNP antibodies (anti-RNP) in cerebrospinal fluid (CSF) are associated with NPSLE. The present study was designed to explore the association of anti-Sm antibodies in CSF with NPSLE. METHODS: Paired serum and CSF specimens were obtained from 72 patients with NPSLE (49 with diffuse NPSLE, 23 with neurological syndromes or peripheral neuropathy (focal NPSLE) and from 22 control patients with non-SLE neurological diseases. Sera were also obtained from 41 patients with active SLE without neuropsychiatric manifestations (non-NPSLE). Anti-Sm and anti-RNP were measured by enzyme-linked immunosorbent assay (ELISA). Blood-brain barrier (BBB) function and intrathecal anti-Sm production were evaluated by Q albumin and CSF anti-Sm index, respectively. Binding of anti-Sm to neuroblastoma cell lines SK-N-MC and Neuro2a was examined by flow cytometry and by cell ELISA. RESULTS: Anti-Sm and anti-RNP in CSF and sera were elevated in NPSLE compared with non-SLE control. CSF anti-Sm, but not CSF anti-RNP, was significantly elevated in ACS compared with non-ACS diffuse NPSLE or with focal NPSLE. By contrast, there were no significant differences in serum anti-Sm or anti-RNP among subsets of NPSLE and non-NPSLE. Whereas there were no significant differences in CSF anti-Sm index, Q albumin was elevated in ACS compared with non-ACS or with focal NPSLE. Notably, CSF anti-Sm was correlated with Q albumin (r = 0.2373, P = 0.0447) or with serum anti-Sm (r = 0.7185, P <0.0001) in 72 patients with NPSLE. Finally, monoclonal anti-Sm and purified human anti-Sm bound to the surface of SK-N-MC and Neuro2a. CONCLUSIONS: These results demonstrate that the elevation of CSF anti-Sm through transudation from systemic circulation due to damaged BBB plays a critical role in the pathogenesis of ACS. More importantly, the data indicate that anti-Sm is yet another autoantibody with presumed neural toxicity, but might not be the last.

Blood-brain barrier damages and intrathecal synthesis of anti-N-methyl-D-aspartate receptor NR2 antibodies in diffuse psychiatric/neuropsychological syndromes in systemic lupus erythematosus.

INTRODUCTION: Although neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the recalcitrant complications of the disease, its pathogenesis still remains unclear. Previous studies revealed that antibodies reactive with NMDA (N-methyl-D-aspartate) receptor NR2 (anti-NR2) are elevated in cerebrospinal fluid (CSF) of patients with diffuse psychiatric/neuropsychological syndromes (diffuse NPSLE), which is usually more recalcitrant than neurologic syndromes of NPSLE (focal NPSLE). Two mechanisms have been implicated for the elevation of CSF IgG, including intrathecal synthesis and transudation through the damaged blood-brain barrier (BBB). The present study was designed in order to elucidate the roles of BBB function and intrathecal synthesis of anti-NR2 in the elevation of CSF anti-NR2 with regard to the severity in NPSLE. METHODS: Paired serum and CSF samples were obtained from 81 systemic lupus erythematosus (SLE) patients when they presented active neuropsychiatric manifestations, and from 22 non-SLE control patients with non-inflammatory neurological diseases. The 81 SLE patients consisted of 55 patients with diffuse NPSLE, including 23 patients with acute confusional state (ACS), the severest form of diffuse NPSLE, and 26 patients with neurologic syndromes or peripheral nervous system involvement (focal NPSLE). IgG anti-NR2 and albumin were measured by ELISA. BBB function and intrathecal synthesis of anti-NR2 were evaluated by Q albumin and by CSF anti-NR2 index, respectively. RESULTS: CSF anti-NR2 levels, Q albumin and CSF anti-NR2 index were significantly higher in NPSLE than in non-SLE control. CSF anti-NR2 levels and Q albumin were significantly higher in ACS than in non-ACS diffuse NPSLE (anxiety disorder, cognitive dysfunction, mood disorder and psychosis) or in focal NPSLE, whereas there was no significant difference in CSF anti-NR2 index among the 3 groups. CSF anti-NR2 levels were significantly correlated with Q albumin in diffuse NPSLE (r = 0.3754, P = 0.0053). CONCLUSIONS: These results demonstrate that the severity of BBB damages plays a crucial role in the development of ACS, the severest form of diffuse NPSLE, through the accelerated entry of larger amounts of anti-NR2 into the central nervous system.

Molecular determinants of sterile inflammation.

Necrotic cell death alerts the acquired immune system to activate naïve T cells even in the absence of non-self derived molecules (e.g. pathogens). In addition, sterile necrosis leads to innate immune-mediated acute inflammation. The dying cells still represent a threat to the body that should be eliminated by the host immune response. Although the inflammatory response plays important roles in protecting the host and repairing tissues, it can also cause the collateral damage to normal tissues that underlies disease pathogenesis. Tissue resident macrophages recognize the danger signals released from necrotic cells via the pattern recognition receptors and secrete IL-1 that results in acute neutrophilic inflammation. This article will review our current knowledge especially focusing on the role of IL-1 in the sterile necrotic cell death induced inflammation.

Increased generation of pre-plasmacytoid dendritic cells in bone marrow of rheumatoid arthritis.

OBJECTIVE: Plasmacytoid dendritic cells (pDCs) have been found to be accumulated in synovial tissues in rheumatoid arthritis (RA). Since pDCs originate from bone marrow (BM), we explored the differentiation of pDC in BM in RA and osteoarthritis (OA). METHODS: BM mononuclear cells (BMMNCs) of the posterior ileac crest from 25 RA patients and 22 OA patients were examined for the expression of BDCA2 and CD34 by flow cytometry. The degree of synovial proliferation was assessed on light microscopy in 10 of 25 RA patients. RESULTS: There were no significant differences in percentages of CD34 + cells or BDCA2 + cells within BMMNC between RA and OA. However, RA BMMNC contained higher percentages of BDCA2 + CD34 + cells (pre-pDCs) than OA BMMNCs. Accordingly, percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly higher in RA than in OA. Finally, the percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly correlated with the degree of synovial proliferation in RA. CONCLUSION: These results indicate that the generation of pre-pDC from BM CD34 + cells is increased in RA compared with OA. Moreover, the data suggest that the increased output of pDC from BM might be involved in the synovial proliferation in RA.

In vivo evaluation of neutrophil recruitment in response to sterile particulates.

Sterile particulates such as monosodium urate crystals induce inflammasome activation resulting in activation of caspase-1, secretion of IL-1α, and processing of IL-1ß. Local production and activation of IL-1 leads to neutrophil recruitment in vivo. Here we describe two quick and simple methods for the evaluation of neutrophil recruitment in the peritoneal cavity and skin in response to sterile particulates, which are dependent on IL-1 receptor signaling.

Anti-ribosomal P protein antibody induces Th1 responses by enhancing the production of IL-12 in activated monocytes.

Autoantibodies to ribosomal P proteins (anti-P) are detected in 12-16% of patients with systemic lupus erythematosus (SLE), and have been found to be associated with some manifestations, including lupus psychosis, nephritis and hepatitis. We have recently disclosed that anti-P react with activated human peripheral blood monocytes, and enhance their production of tumor necrosis factor-α and interleukin (IL)-6. It is also possible that anti-P might regulate other monocyte functions, including the regulation of T helper (Th) responses. The current study was therefore undertaken to explore the effects of anti-P on the induction of Th1 responses. Peripheral blood mononuclear cells (PBMC) from healthy donors were cultured with affinity-purified anti-P or control IgG. Highly purified monocytes were cultured with interferon (IFN)-γ in the presence of anti-P or normal IgG. Anti-P significantly enhanced the production of IFN-γ by PBMC. Of note, anti-IL-12 monoclonal antibodies almost completely abrogated the anti-P-mediated upregulation of the IFN-γ production of PBMC. Accordingly, anti-P significantly enhanced the production of IL-12 by activated monocytes. These results indicate that anti-P induce Th1 responses by upregulating the production of IL-12 by activated monocytes. The data therefore suggest that anti-P play an important role in the pathogenesis of SLE through the promotion of Th1 responses.

Differential influences of bucillamine and methotrexate on the generation of fibroblast-like cells from bone marrow CD34+ cells of rheumatoid arthritis patients.

We have recently demonstrated that bone marrow CD34+ cells from rheumatoid arthritis (RA) patients displayed abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1 (type B synoviocyte -like cells). The current study examined the effects of representative potent disease-modifying antirheumatic drugs, including bucillamine (BUC) and methotrexate (MTX) on the in vitro generation of fibroblast-like cells from RA bone marrow CD34+ cells. CD34+ cells purified from bone marrow specimens of 8 patients with active RA were cultured in the presence or absence of pharmacologically attainable concentrations of intramolecular disulfide form of bucillamine (BUC-ID, 3 microM), a major metabolite of BUC or MTX (20 nM). After incubation for 28 days, the generation of fibroblast-like cells was assessed under phase-contrast light microscopy and the concentrations of MMP-1 and VEGF in the culture supernatants were measured by ELISA. BUC-ID, but not MTX, significantly suppressed the generation of fibroblast-like cells from RA bone marrow CD34+ cells stimulated with SCF, GM-CSF and TNF-alpha (p=0.024 as determined by Wilcoxon signed rank test). Accordingly, BUC-ID, but not MTX, significantly suppressed the production of MMP-1 (p=0.017) and VEGF (p=0.017) by RA bone marrow CD34+ cells, without inhibition of beta2-microglobulin production. These results demonstrate that BUC-ID, but not MTX, is a potent inhibitor of differentiation of fibroblast-like cells from RA bone marrow CD34+ cells. Since MTX, but not BUC, has been previously shown to influence on type A synoviocytes, the data provide rationale of combination of BUC and MTX in the treatment of RA.

Association of cerebrospinal fluid anti-NR2 glutamate receptor antibodies with diffuse neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: To explore the association of antibodies directed against N-methyl-D-aspartate receptor subunit NR2 (anti-NR2) in cerebrospinal fluid (CSF) with neuropsychiatric manifestations in systemic lupus erythematosus (NPSLE). METHODS: Paired serum and CSF specimens were obtained from 56 patients with NPSLE (38 with diffuse psychiatric/neuropsychological syndromes [diffuse NPSLE] and 18 with neurologic syndromes or peripheral neuropathy [focal NPSLE]) and from 20 control patients with noninflammatory neurologic diseases. IgG anti-NR2 antibodies were measured by enzyme-linked immunosorbent assay using synthetic peptide containing the extracellular ligand-binding domain of NR2. The binding of affinity-purified anti-NR2 to human neuroblastoma cell line SK-N-MC was examined by flow cytometry. RESULTS: Purified anti-NR2 bound to the surface of SK-N-MC cells. Levels of anti-NR2 antibodies in CSF were significantly elevated in patients with diffuse NPSLE compared with levels in control patients or those with focal NPSLE, whereas there were no significant differences in serum anti-NR2 levels among the 3 groups. In 31 of the 38 patients with diffuse NPSLE (81.6%) and 8 of the 18 patients with focal NPSLE (44.4%), CSF anti-NR2 levels were more than 3 SD above the mean level in the control patients (P=0.0120 by chi-square test). CONCLUSION: These results indicate that anti-NR2 is a constituent of antineuronal antibodies and, more importantly, that anti-NR2 antibodies in CSF, but not in serum, are associated with diffuse NPSLE.

Anti-ribosomal P protein antibody in human systemic lupus erythematosus up-regulates the expression of proinflammatory cytokines by human peripheral blood monocytes.

OBJECTIVE: Autoantibodies to ribosomal P proteins (anti-P antibodies) are detected in 12-16% of patients with systemic lupus erythematosus (SLE) and have been found to be associated with some manifestations of the disease, including lupus psychosis and hepatitis. Recent studies have disclosed that anti-P antibodies react with activated T cells but not with B cells, suggesting possible direct effects of anti-P antibodies on immune regulation. The present study was designed to explore the presence of the epitope recognized by anti-P antibodies on human peripheral blood monocytes. METHODS: Highly purified peripheral blood monocytes obtained from healthy donors were cultured with or without interferon-gamma (IFNgamma) in the presence of either anti-P antibodies purified by affinity chromatography from the sera of patients with SLE or control IgG. RESULTS: Flow cytometry analysis disclosed that fresh (day 0) monocytes did not express the ribosomal P epitope, whereas expression of the ribosomal P epitope was induced on annexin V-negative monocytes after activation through plastic adherence for 48 hours. More important, anti-P antibodies (compared with normal IgG or IgG from SLE patients devoid of anti-P antibodies) enhanced the production of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) by activated monocytes. Accordingly, anti-P antibodies also up-regulated the expression of TNFalpha and IL-6 messenger RNA in activated monocytes. Of note, F(ab')(2) fragments of anti-P antibodies, which do not result in Fcgamma receptor (FcgammaR) crosslinking, also effectively up-regulated the expression of TNFalpha and IL-6. CONCLUSION: These results indicate that human peripheral blood monocytes express the ribosomal P epitope upon activation, irrespective of induction of apoptosis. Moreover, the data suggest that anti-P antibodies might modify a variety of inflammatory responses through up-regulation of the expression of proinflammatory cytokines in monocytes, in a manner that does not involve FcgammaR crosslinking.

Enhanced generation of endothelial cells from CD34+ cells of the bone marrow in rheumatoid arthritis: possible role in synovial neovascularization.

OBJECTIVE: To examine the capacity of bone marrow CD34+ cells to generate endothelial cells, in order to assess the role of bone marrow in neovascularization in the synovium of rheumatoid arthritis (RA). METHODS: CD34+ cells purified from the bone marrow of 13 patients with active RA and 9 control subjects (7 osteoarthritis [OA] patients and 2 healthy individuals) were cultured in the presence of stem cell factor (10 ng/ml) and granulocyte-macrophage colony-stimulating factor (1 ng/ml). After 18 days of incubation, the generation of endothelial cells was assessed by flow cytometry. The generation of endothelial cells was compared with the degree of vascularization in the synovial tissues and with the microvessel densities in the synovium, as determined by microscopy. The expression of vascular endothelial growth factor receptor 2/kinase insert domain receptor (KDR) messenger RNA (mRNA) in CD34+ cells was examined by quantitative reverse transcription-polymerase chain reaction. RESULTS: The generation of CD14+ cells from bone marrow-derived CD34+ cells from RA patients was comparable to that from control subjects. However, the generation of von Willebrand factor (vWF)-positive cells and CD31+/vWF+ cells from RA bone marrow-derived CD34+ cells was significantly higher than that from control subjects (P = 0.004 and P = 0.030, respectively). The generation of vWF+ cells from bone marrow CD34+ cells correlated significantly with the microvessel densities in the synovial tissues (r = 0.569, P = 0.021). Finally, RA bone marrow CD34+ cells expressed KDR mRNA at higher levels than OA bone marrow CD34+ cells. CONCLUSION: These results indicate that RA bone marrow CD34+ cells have enhanced capacities to differentiate into endothelial cells in relation to synovial vascularization. The data therefore suggest that bone marrow CD34+ cells might contribute to synovial neovascularization by supplying endothelial precursor cells and, thus, play an important role in the pathogenesis of RA.

Bone marrow CD34+ progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD- B cells through direct cellular interaction.

Recent studies have suggested the involvement of bone marrow in the pathogenesis of rheumatoid arthritis (RA), in which proliferation of monocyte-lineage cells (MLC) as well as local B cell activation in the synovium play an important role. Here, we show that bone marrow-derived MLC have the capacity to activate human peripheral blood IgD- B cells. Bone marrow CD34+ cells from RA patients that had been stimulated with stem cell factor and GM-CSF for 3-4 weeks (>90% CD14+ HLA-DR+ cells, <0.5% CD19+ B cells, and <0.5% CD3+ T cells; MLC) induced the production of IgG much more effectively than that of IgM by highly purified B cells from healthy donors in the presence of IL-2 and IL-10. CD34+ cells from cord blood or from bone marrow of osteoarthritis patients also displayed the capacity to induce IgG production. The induction of IgG production by the bone marrow-derived MLC was markedly decreased when they were separated from B cells by a membrane filter. The bone marrow-derived MLC interacted preferentially with IgD- B cells to induce IgG production. These results indicate that upon stimulation with stem cell factor and GM-CSF, CD34+ progenitor cells differentiate into MLC that activate preferentially IgD- B cells through direct cellular interactions to produce IgG. Therefore, the data suggest that the accelerated recruitment of MLC from the bone marrow to the synovium might play a role in the local B cell activation in RA.

Regulation of human B cell function by sulfasalazine and its metabolites.

Although sulfasalazine is a well-known disease-modifying antirheumatic drug (DMARD), the mechanisms of its action remain unclear. Indeed, it remains uncertain whether sulfasalazine itself or one of its metabolites is responsible for the antirheumatic effects of sulfasalazine. Since one of the characteristic features of rheumatoid arthritis (RA) is chronic stimulation of B cells, we compared the effects of sulfasalazine and its metabolites on the in vitro function of human B cells. Ig production was induced from highly purified B cells from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus IL-2. Sulfasalazine suppressed the production of IgM and IgG at its pharmacologically attainable concentrations (1-10 microg/ml). Of the metabolites of sulfasalazine, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), but not 4-acethyl SP, also significantly suppressed the production of IgM and IgG at their pharmacologically relevant concentrations. By contrast, any of sulfasalazine, SP, 5-ASA and 4-acethyl SP did not suppress the IFN-gamma production of immobilized anti-CD3 stimulated CD4+ T cells. These results indicate that sulfasalazine and its metabolites preferentially suppress the function of B cells, but not that of T cells, at their pharmacologically attainable concentrations. The data therefore suggest that not only sulfasalazine, but its metabolites, might contribute to the beneficial effects of sulfasalazine.