Tamiko Yanagida

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.

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

IntroductionAlthough 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.MethodsPaired 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.ResultsCSF 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).ConclusionsThese 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.

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.

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.

Induction of fibroblast-like cells from CD34(+) progenitor cells of the bone marrow in rheumatoid arthritis.

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34+ cells from bone marrow to generate fibroblast‐like type B synoviocytes. CD34+ cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast‐like morphology, which expressed prolyl 4‐hydroxylase, in the presence of stem cell factor (SCF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and tumor necrosis factor α (TNF‐α), much more effectively than CD34+ cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast‐like cells was not at all observed in cultures with SCF, GM‐CSF, and interleukin 4 (IL‐4) with or without TNF‐α. Generation of fibroblast‐like cells was correlated with matrix metalloproteinase (MMP)‐1 levels in culture supernatants. Thus, MMP‐1 levels were significantly higher in TNF‐α‐stimulated cultures of bone marrow CD34+ cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34+ cells from patients with RA have abnormal capacities to respond to TNF‐α and to differentiate into fibroblast‐like cells producing MMP‐1, suggesting that bone marrow CD34+ progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.

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

IntroductionNeuropsychiatric 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.MethodsPaired 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.ResultsAnti-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.ConclusionsThese 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.

Inhibition of cyclin A gene expression in human B cells by an immunosuppressant mizoribine

Mizoribine has been shown to have beneficial effects in the treatment of rheumatoid arthritis and lupus nephritis, in which abnormal B cell functions are involved. Previous studies demonstrated that mizoribine directly suppresses the function of human B cells. The current study explored in detail the mechanism of the suppression of human B cell responses by mizoribine at the molecular level. Highly purified peripheral blood B cells obtained from normal healthy individuals were stimulated with Staphylococcus aureus Cowan I (SAC) plus IL‐2 in the presence or absence of mizoribine or methotrexate for 48 h to 72 h. The expression of cyclin A mRNA was determined by semiquantitative reverse transcriptase‐polymerase chain reaction followed by Southern hybridization. Although at pharmacologically attainable concentrations both mizoribine and methotrexate suppressed the production of IgM of SAC‐activated B cells, mizoribine, but not methotrexate, decreased the expression of cyclin A protein as well as mRNA in B cells stimulated with SAC + IL‐2. Of note, mizoribine facilitated the degradation of cyclin A mRNA in the presence of actinomycin D, indicating that mizoribine shortens the stability of cyclin A mRNA. The results indicate that mizoribine suppresses the expression of cyclin A mRNA in human B cells by down‐regulating its stability, and thus down‐regulates their responses.

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.

Bone marrow CD34+ progenitor cells from rheumatoid arthritis patients support spontaneous transformation of peripheral blood B cells from healthy individuals.

Abstract We show that bone marrow (BM) CD34+ progenitor cells from rheumatoid arthritis (RA) patients have the capacity to support spontaneous transformation of peripheral blood B cells. CD34+ cells purified from BM blood from eight RA patients and eight osteoarthritis (OA) patients were expanded with granulocyte/macrophage colony stimulating factor (GM-CSF) for 4–6 weeks. GM-CSF-stimulated BM CD34+ cells from three of eight RA patients, but none from seven OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)- seronegative healthy donors. GM-CSF-stimulated BM CD34+ cells from four of six RA patients and from one of four OA patients also supported the spontaneous transformation of peripheral blood B cells from EBV-seropositive healthy donors. All the transformed B cell lines were positive for EBV-DNA as determined by PCR. Neither GM-CSF-stimulated BM CD34+ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines. These results suggest that the capacity of BM CD34+ cells to support survival of B cells might contribute to the pathogenesis of RA by sustaining abnormal B cell responses.

Polymorphonuclear neutrophils enhance suppressive activities of anti-CD3-induced CD4+ suppressor T cells

We investigated the effects of polymorphonuclear neutrophils (PMN) on the suppressive activities of CD4+ suppressor T cells induced by immobilized mAb to the CD3 molecular complex in order to explore the role of PMN in the regulation of humoral immune responses. CD4+ T cells that had been treated with mitomycin C induced the IgM production from highly purified B cells in cultures stimulated with immobilized anti-CD3. Addition of CD4+ T cells that had not been treated with mitomycin C (control T4 cells) suppressed the IgM production induced by immobilized anti-CD3-stimulated T4 mito. PMN enhanced the degree of suppression of the IgM production by anti-CD3-stimulated control T4 cells. The capacity of PMN to enhance the suppressive activity of anti-CD3-stimulated control T4 cells was restored when PMN were fixed with paraformaldehyde (PFA), suggesting that direct interactions between PMN and CD4+ T cells, but not soluble factors secreted by PMN, were involved in the enhancement of suppression. Fresh PMN as well as PFA-fixed PMN enhanced the endogenous IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells. Moreover, neither fresh PMN nor PFA-fixed PMN significantly augmented the suppressive activity of anti-CD3-stimulated control T4 cells in the presence of exogenous IL-2. These results indicate that PMN enhance the suppressive activity of anti-CD3-stimulated control T4 cells through direct interactions between PMN and CD4+ T cells. The enhancement of the suppressive activity of CD4+ suppressor T cells by PMN is accounted for by the enhancement of the endogenous IL-2 production by anti-CD3-stimulated CD4+ T cells. Thus, the data demonstrate that PMN influence the magnitude of humoral immune responses by regulating the production of IL-2 through direct interactions with T cells.