Differential activation mechanisms of serum C5a in lupus nephritis and neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: To explore the role of C5a in the pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) and lupus nephritis (LN). METHODS: Sera were obtained from 29 patients with NPSLE, 25 with LN, 26 without NPSLE or LN [SLE alone], and 21 healthy donors. Cerebrospinal fluid (CSF) was obtained from 29 NPSLE patients. C5a and C5 were measured by ELISA. Blood-brain barrier (BBB) function was evaluated by Q albumin ([CSF albumin/serum albumin] × 103). RESULTS: Serum C5a, but not C5, was significantly increased in SLE compared with healthy control. Serum C5a, but not C5, was significantly higher in NPSLE and in LN than in SLE alone. Serum C4, but not C3, was lower in LN than in NPSLE. Q albumin was significantly higher in diffuse NPSLE than in focal NPSLE, whereas there were no significant differences in CSF or serum C5a between both groups. Notably, CSF C5 and C5a were significantly correlated with Q albumin, whereas serum C5a, but not C5, appeared to be inversely correlated with Q albumin. CONCLUSION: These results disclosed that serum C5a was elevated not only in NPSLE but also in LN through different mechanisms. Moreover, it is suggested that C5a might be consumed during BBB damages.

Reactivation of hepatitis B virus in rheumatoid arthritis patients treated with biological disease-modifying antirheumatic drugs.

OBJECTIVE: To examine the incidence of hepatitis B virus (HBV) reactivation in patients with rheumatoid arthritis (RA) receiving biological disease-modifying antirheumatic drugs (DMARDs). METHODS: We retrospectively reviewed RA patients treated with biological DMARDs at our institution from July 2010 to December 2012. Patients with antibodies for hepatitis B core antigen and/or hepatitis B surface antigen were regarded as having prior HBV infection. Clinical data on these patients, including HBV-DNA levels, were retrieved from the medical records. RESULTS: During the study period, 251 patients were administered various biological DMARDs. Six patients with a history of HBV vaccination and one patient with positive HBV surface antigen were excluded from the study. Fifty-seven of the remaining 244 patients (23.4%) had prior HBV infection. These patients were followed for a median of 18 months (range: 2-27 months) and HBV-DNA was examined a median of seven times (range: 2-27). HBV-DNA was detected in three patients (5.3%), comprising two receiving tocilizumab and one receiving etanercept. However, HBV-DNA levels were below the quantitation limit (<2.1 log copies mL(-1) ) in all three patients. HBV-DNA became negative again within several months in all three patients, while biological DMARDs were continued and liver function tests remained normal throughout. CONCLUSION: HBV-DNA reactivation occurred in 5.3% of RA patients with prior HBV infection during treatment with biological DMARDs, but there were no associated clinical manifestations. Accordingly, it seems that biological DMARDs can be used safely in patients with RA.

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.

Reversible basal ganglia lesions in neuropsychiatric lupus: a report of three pediatric cases.

AIM: Central nervous system involvement represents a serious and common complication of systemiclupus erythematosus (SLE). We describe the characteristics of patients with neuropsychiatric (NP) SLE complicated with reversible basal ganglia lesions. METHODS: We describe the cases of three NPSLE patients. RESULTS: They presented with NP manifestations such as headache, cognitive dysfunction, tremors, seizures, and mood disorder. The levels of autoantibodies to NMDA (N-methyl-d-aspartate) receptor antibodies and antiribosomal-P antibodies were elevated, indicating the presence of an acute phase. Marked elevation of interleukin-6 in cerebrospinal fluid was noted when these patients showed NP symptoms. Moreover, the patients presented with high-intensity lesions in the basal ganglia on T2-weighted images, fluid-attenuated inversion recovery (FLAIR) images, diffusion-weighted images (DWI) and apparent diffusion coefficient (ADC) maps. Following immunosuppressive treatment, almost complete improvement of the lesions was noted. CONCLUSION: The reported cases indicate that reversible vasculopathies represent vasogenic edema localized in basal ganglia lesions and that activation of the autoimmune system and inflammation could lead to NP manifestations in SLE.

IgG anti-NR2 glutamate receptor autoantibodies from patients with systemic lupus erythematosus activate endothelial cells.

OBJECTIVE: To investigate the possibility that IgG anti-NR2 glutamate receptor antibodies (anti-NR2) derived from patients with systemic lupus erythematosus (SLE) cause an immunologic interaction with endothelial cells (ECs) in the blood-brain barrier, resulting in inflammation of the blood-brain barrier, allowing the entrance of these autoantibodies into the cerebrospinal fluid. METHODS: Purified IgG anti-NR2 antibodies from 14 patients with SLE were tested for their ability to bind to double-stranded DNA (dsDNA) and ECs, to modulate endothelial adhesion molecule expression and cytokine production by ECs, and to activate the NF-κB pathways in the ECs. Purified IgG from 5 normal subjects was used as a negative control. RESULTS: Purified IgG anti-NR2 antibodies bound to dsDNA in an IgG-dose-dependent manner. This interaction up-regulated the expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on the EC surface and increased the production of interleukin-6 (IL-6) and IL-8, but not tumor necrosis factor α or IL-1ß, by ECs. Purified IgG anti-NR2 also activated the degradation of cytoplasmic IκB, indicating the activation of NF-κB in the ECs. CONCLUSION: EC activation through the NF-κB signaling pathway induced by IgG anti-NR2 antibodies in the central nervous system of SLE patients may lead to inflammation of the blood-brain barrier, initiating the pathogenesis of neuropsychiatric SLE.

No increased mortality in patients with rheumatoid arthritis treated with biologics: results from the biologics register of six rheumatology institutes in Japan.

OBJECTIVE: To investigate the influence of biologics on mortality and risk factors for death in rheumatoid arthritis (RA) patients. METHODS: RA patients treated with at least one dose of biologics in daily practice in six large rheumatology institutes ("biologics cohort") were observed until 15 May 2010 or death, whichever occurred first. Mortality of the biologics cohort and the "comparator cohort" (comprising patients among the IORRA cohort who had never been treated with biologics) was compared to that of the Japanese general population. Factors associated with mortality were assessed by a Cox model. RESULTS: Among 2683 patients with 6913.0 patient-years of observation, 38 deaths were identified in the biologics cohort. The probability of death in patients lost to follow-up, calculated using the weighted standardized mortality ratio (SMR), was 1.08 [95 % confidence interval (CI) 0.77-1.47] in the biologics cohort and 1.28 (95 % CI 1.17-1.41) in the comparator cohort. Pulmonary involvement was the main cause of death (47.4 %), and the disease-specific SMR of pneumonia was 4.19 (95 % CI 1.81-8.25). Risk factors for death included male gender [hazard ratio (HR) 2.78 (95 % CI 1.24-6.22)], advanced age (HR 1.07, 95 % CI 1.03-1.11), and corticosteroid dose (HR 1.08, 95 % CI 1.01-1.17). CONCLUSION: Mortality in RA patients exposed to biologics did not exceed that in patients not exposed to biologics, but death from pulmonary manifestations was proportionally increased in RA patients exposed to biologics.

Sustained elevation of interleukin-33 in sera and synovial fluids from patients with rheumatoid arthritis non-responsive to anti-tumor necrosis factor: possible association with persistent IL-1ß signaling and a poor clinical response.

Although TNF inhibitors have dramatically improved the outcome of patients with rheumatoid arthritis, 30-40% of patients do not respond well to them and treatment needs to be changed. In an effort to discriminate good and poor responders, we focused on the change in serum and synovial fluid levels of interleukin (IL-) 33 before and after treatment with TNF inhibitors. They were also measured in synovial fluids from 17 TNF inhibitor-naïve patients, and fibroblast-like synoviocytes (FLS) in-culture from 6 patients and correlated with various pro-inflammatory cytokines. Serum levels of IL-33 at 6 months after treatment decreased significantly in responders, while they did not change in non-responders. Synovial fluid levels of IL-33 in 6 patients under treatment with TNF inhibitors stayed high in 3 who were refractory and slightly elevated in 2 moderate responders, while they were undetectable in one patient under remission. Among inflammatory cytokines measured in 17 synovial fluids from TNF inhibitor-naïve patients, levels of IL-33 showed a significant positive correlation only to those of IL-1ß. IL-1ß increased IL-33 expression markedly in FLS in vitro, compared to TNF-α. IL-1ß might be inducing RA inflammation through producing pro-inflammatory IL-33 in TNF inhibitor-hypo-responders. Sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors, although how TNF inhibitors affect the level of IL-33 remains to be elucidated.

Influence of antibodies against infliximab and etanercept on the treatment effectiveness of these agents in Japanese patients with rheumatoid arthritis.

We investigated the influence of antibodies against infliximab and etanercept on the serum trough levels of these agents and the influence of these antibodies on the effectiveness of treatment in patients with rheumatoid arthritis treated with these agents. Forty patients treated with infliximab for 54 weeks and 40 patients treated with etanercept for 32 weeks were enrolled. They were divided into responder and non-responder groups. Serum trough levels of and antibodies against these agents were measured by enzyme-linked immunosorbent assay or radioimmunoassay. Of the 40 patients treated with infliximab, 14 (35%) had anti-infliximab antibodies. Serum trough levels were significantly lower in the non-responder group (14 patients) than in the responder group (26 patients) 6 weeks after initiation of infliximab (p < 0.05). Conversely, titers of anti-infliximab antibody were significantly higher in the non-responder group than in the responder group between 6 and 38 weeks after initiation of infliximab (p < 0.05). Anti-etanercept antibodies were not detected in any patients on etanercept. Serum trough levels of etanercept were not significantly different between the responder (31 patients) and non-responder groups (9 patients). It seems that the appearance of anti-infliximab antibodies might decrease infliximab serum concentrations and, thereby, reduce the agent's effectiveness. The clinical efficacy of etanercept does not appear to be affected by the serum concentrations if it is administered at standard doses.

Increased levels of interleukin 33 in sera and synovial fluid from patients with active rheumatoid arthritis.

OBJECTIVE: To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33. METHODS: Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1beta and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting. RESULTS: Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS. CONCLUSION: IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.

Inhibition of NF-kappaB signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and is characterized mainly by symmetric polyarticular joint disorders. The pathologic processes are mediated by a number of cytokines, chemokines, cell adhesion molecules, and matrix metalloproteinases. The expression of most of these molecules is controlled at the transcriptional level. In addition, activation of NF-kappaB is involved in RA pathogenesis. This study was performed to explore the role of a novel serine/threonine kinase inhibitor, fasudil, in the control of the NF-kappaB activation pathway and to investigate the therapeutic effects of fasudil on arthritis development in a rat model of RA. METHODS: Fibroblast-like synoviocytes (FLS) from RA patients and human endothelial cells (ECs) were established and maintained. To study the role of fasudil on cytokine expression, various cytokines expressed in the RA FLS and human ECs were measured by enzyme-linked immunosorbent assay following stimulation of the cells with interleukin-1beta (IL-1beta) in the presence of various concentrations of fasudil. The role of fasudil on NF-kappaB activation was studied using a reporter gene assay, Western blotting of IkappaBalpha, immunofluorescence analysis of the p65 subunit of NF-kappaB, and electrophoretic mobility shift assay. The in vivo effects of fasudil on arthritis were studied in a rat adjuvant-induced arthritis (AIA) model. RESULTS: Fasudil inhibited cytokine expression in RA FLS and human ECs and also inhibited the activation of ECs, in a dose-dependent manner. Fasudil inhibited IL-1beta-induced activation of NF-kappaB independent of the inhibition of IkappaBalpha degradation and nuclear translocation of NF-kappaB, and inhibited IL-1beta-induced DNA binding of NF-kappaB. Finally, in vivo, fasudil ameliorated arthritis in rats with AIA, without any adverse effects. CONCLUSION: Serine/threonine kinase inhibitor fasudil inhibits the development of arthritis in a rat model of RA, and also inhibits the NF-kappaB signaling required for binding of NF-kappaB to specific DNA sequences through, for example, the phosphorylation of p65, suggesting that a specific target of fasudil might be a novel NF-kappaB kinase. Thus, fasudil serves as a novel strategy for the treatment of RA.

Potent induction of IFN-alpha and chemokines by autoantibodies in the cerebrospinal fluid of patients with neuropsychiatric lupus.

Neuropsychiatric disease in systemic lupus erythematosus (NPSLE) is a poorly understood, but potentially fatal, disease manifestation. A pathogenetic role for autoantibodies is suspected, but the mechanism is unclear. Since immune complexes in SLE can stimulate IFN-alpha and there is strong evidence in humans and in mice that IFN-alpha can cause neuropsychiatric manifestations, we asked whether NPSLE patient serum and/or cerebrospinal fluid (CSF) contain abnormally high IFN-alpha-inducing activity. In a bioassay containing plasmacytoid dendritic cells and a source of Ag, NPSLE CSF induced significantly higher IFN-alpha compared with CSF from patients with multiple sclerosis or other autoimmune disease controls. When normalized for IgG concentration, NPSLE CSF was 800-fold more potent at inducing IFN-alpha compared with paired serum due to inhibitors present in serum. Analysis of Ig-deficient patient serum, depletion of IgG from normal serum, as well as addition of purified IgG to NPSLE CSF and serum in the bioassays revealed that one inhibitor was contained within the IgG fraction itself. In addition to IFN-alpha, immune complexes formed by CSF autoantibodies produced significantly increased levels of IFN-gamma-inducible protein 10 (IP-10/CXCL), IL-8, and MCP-1, all of which have been reported to be elevated in CSF from NPSLE patients. Taken together, these findings are consistent with a two-step model of NPSLE whereby CSF autoantibodies bind to Ags released by neurocytotoxic Abs or other brain cell injury, and the resulting immune complexes stimulate IFN-alpha and proinflammatory cytokines and chemokines.

Prognostic indicators related to death in patients with Pneumocystis pneumonia associated with collagen vascular diseases.

The objective of this study is to investigate the clinical markers of life-threatening Pneumocystis pneumonia (PCP) in patients with collagen vascular diseases (CVD). The patients who contracted Pneumocystis jeroveccii were retrospectively selected from our medical charts and conditions related to the patients' death were reviewed. The findings indicated that lower levels of serum albumin and cholinesterase, increased alveolar-arterial oxygen gradient, intratracheal intubation, and necessity to treat in the intensive care unit were significantly related to deaths associated with PCP in CVD. A special attention should be paid to decreased serum albumin and cholinesterase as ominous predictors in PCP occurred in patients with CVD.

Anti-beta 1-adrenoreceptor autoantibodies and myocardial sympathetic nerve activity in chronic heart failure.

BACKGROUND: The autoantibodies stimulate the beta1-adrenoreceptors on cardiac myocytes similar to norepinephrine, and are associated with reduced cardiac function. Iodine-123 metaiodobenzylguanidine ((123)I-MIBG) is metabolized similarly to norepinephrine. This study was undertaken to investigate the relationship between cardiac stimulation by anti-beta1-adrenoreceptor autoantibodies and myocardial sympathetic nervous activity in patients with chronic heart failure. METHODS: We screened for the anti-beta1-adrenoreceptor autoantibodies in 52 patients with chronic heart failure by conducting an enzyme-linked immunosorbent assay, and underwent (123)I-MIBG scintigraphy in 27 of the patients. Anterior planar images of (123)I-MIBG were obtained 15 min and 3 h after the injection. We determined the heart to mediastinum radioactivity ratio (H/M), and calculated the rate of washout of (123)I-MIBG from the heart. RESULTS: Patients with New York Heart Association functional class III or IV had higher levels of anti-beta1-adrenoreceptor autoantibodies than those with class I or II (p<0.01). The autoantibody level was significantly correlated with delayed H/M (r=-0.65, p<0.001) and washout rate (r=0.65, p<0.001). Sixteen patients with a cardiac event showed higher levels of the autoantibodies (p<0.05). Cardiac event-free survival was poorer in patients with the autoantibody levels >10 U/ml than that <10 U/ml (log-rank=12.1, p<0.001). CONCLUSION: The anti-beta1-adrenoreceptor autoantibodies are closely associated with cardiac sympathetic nervous activity assessed by (123)I-MIBG and cardiac event in patients with chronic heart failure.