Impact of anti-interleukin-6 receptor blockade on circulating T and B cell subsets in patients with systemic lupus erythematosus.

BACKGROUND: Circulating plasmablasts/plasma cells and activated B and T cells are increased in systemic lupus erythematosus (SLE). Interleukin (IL)-6 induces differentiation of B cells into antibody-forming cells and of T cells into effector cells. OBJECTIVE: To examine the hypothesis that blocking IL-6 would reverse some of the immune abnormalities present in SLE. METHODS: Fifteen patients with SLE with mild-to moderate disease activity were treated with biweekly infusions of tocilizumab, a humanised anti-IL-6 receptor monoclonal antibody for 12 weeks. Lymphocyte subsets (analysed by flow cytometry) and serum immunoglobulin levels were compared at baseline and at weeks 6 and 12. RESULTS: Tocilizumab decreased activated T and B cells, the frequency of CD27(high)CD38(high)IgD- plasmablasts/plasma cells and IgD-CD27+ post-switched memory B cells as well as IgG+ memory B cell, whereas it increased the frequency of IgD+CD27- antigen-inexperienced B cells. Among antigen-inexperienced IgD+CD27- B cells, CD38(low) mature naïve B cells increased significantly and CD38(Intermediate)CD5+ pre-naïve B cells showed a decreasing trend, whereas CD38(high)CD5+ transitional type 1 B cells did not change. Most of the changes occurred in patients who had abnormal values at baseline. IgG, IgA, IgG1 and IgG3 serum levels decreased albeit within the normal range. The frequency of CD4+CD45RA+CCR7+ naïve T cells increased. CONCLUSIONS: In vivo blockade of the IL-6 receptor decreases lymphocyte activation and restores B and T cell homoeostasis by either blocking differentiation and/or trafficking in patients with SLE and leads to normalisation of the abnormal B and T cell subsets seen at baseline.

Efficacy of etanercept in the tumor necrosis factor receptor-associated periodic syndrome: a prospective, open-label, dose-escalation study.

OBJECTIVE: To investigate the efficacy of etanercept in improving the symptoms and underlying inflammation in patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS). METHODS: Fifteen patients with TRAPS were enrolled in a prospective, open-label, dose-escalation study. Patients recorded attacks, symptom severity, and use of ancillary medications in a daily diary. Blood samples were collected during each period and measured for levels of acute-phase reactants. Between 7 years and 9 years after the conclusion of the initial study, patients completed a followup survey and were evaluated to determine the long-term outcome of etanercept treatment. RESULTS: Etanercept treatment significantly attenuated the total symptom score and reduced the frequency of symptoms. Etanercept also reduced levels of acute-phase reactants, particularly during asymptomatic periods. During a 10-year followup period, patients continued to receive etanercept for a median of 3.3 years, with a number of patients switching to anti-interleukin-1ß receptor therapy or not receiving biologic agents, most frequently citing injection site reactions and lack of efficacy as reasons for discontinuation. However, patients continuing to receive etanercept had reduced symptoms at followup. CONCLUSION: Etanercept reduces symptoms and serum levels of inflammatory markers of TRAPS in a dose-dependent manner, but does not completely normalize symptoms or acute-phase reactant levels. Although long-term adherence to etanercept is poor, continuing to receive etanercept may provide continued symptomatic benefit.

Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions.

To determine the role of CD154-CD40 interactions in the B cell overactivity exhibited by patients with active systemic lupus erythematosus (SLE), CD19+ peripheral B cells were examined before and after treatment with humanized anti-CD154 mAb (BG9588, 5c8). Before treatment, SLE patients manifested activated B cells that expressed CD154, CD69, CD38, CD5, and CD27. Cells expressing CD38, CD5, or CD27 disappeared from the periphery during treatment with anti-CD154 mAb, and cells expressing CD69 and CD154 disappeared from the periphery during the post-treatment period. Before treatment, active-SLE patients had circulating CD38 (bright) Ig-secreting cells that were not found in normal individuals. Disappearance of this plasma cell subset during treatment was associated with decreases in anti-double-stranded DNA (anti-dsDNA) Ab levels, proteinuria, and SLE disease activity index. Consistent with this finding, peripheral B cells cultured in vitro spontaneously proliferated and secreted Ig in a manner that was inhibited by anti-CD154 mAb. Finally, the CD38(+/++)IgD(+), CD38(+++), and CD38(+)IgD(-) B cell subsets present in the peripheral blood also disappeared following treatment with humanized anti-CD154. Together, these results indicate that patients with active lupus nephritis exhibit abnormalities in the peripheral B cell compartment that are consistent with intensive germinal center activity, are driven via CD154-CD40 interactions, and may reflect or contribute to the propensity of these patients to produce autoantibodies.

Elevated interleukin-10: a new cause of dyslipidemia leading to severe HDL deficiency.

BACKGROUND: Low high-density lipoprotein cholesterol (HDL-C) is a risk factor for coronary artery disease. Investigating mechanisms underlying acquired severe HDL deficiency in noncritically ill patients ("disappearing HDL syndrome") could provide new insights into HDL metabolism. OBJECTIVE: To determine the cause of low HDL-C in patients with severe acquired HDL deficiency. METHODS AND RESULTS: Patients with intravascular large B-cell lymphoma (n = 2), diffuse large B-cell lymphoma (n = 1), and autoimmune lymphoproliferative syndrome (n = 1) presenting with markedly decreased HDL-C, low low-density lipoprotein cholesterol (LDL-C), and elevated triglycerides were identified. The abnormal lipoprotein profile returned to normal after therapy in all 4 patients. All patients were found to have markedly elevated serum interleukin-10 (IL-10) levels that also normalized after therapy. In a cohort of autoimmune lymphoproliferative syndrome patients (n = 93), IL-10 showed a strong inverse correlation with HDL-C (R(2) = 0.3720, P < .0001). A direct causal role for increased serum IL-10 in inducing the observed changes in lipoproteins was established in a randomized, placebo-controlled clinical trial of recombinant human IL-10 in psoriatic arthritis patients (n = 18). Within a week of initiating subcutaneous recombinant human IL-10 injections, HDL-C precipitously decreased to near-undetectable levels. LDL-C also decreased by more than 50% (P < .0001) and triglycerides increased by approximately 2-fold (P < .005). All values returned to baseline after discontinuing IL-10 therapy. CONCLUSION: Increased IL-10 causes severe HDL-C deficiency, low LDL-C, and elevated triglycerides. IL-10 is thus a potent modulator of lipoprotein levels, a potential new biomarker for B-cell disorders, and a novel cause of disappearing HDL syndrome.

Tocilizumab in systemic lupus erythematosus: data on safety, preliminary efficacy, and impact on circulating plasma cells from an open-label phase I dosage-escalation study.

OBJECTIVE: To assess the safety of interleukin-6 receptor inhibition and to collect preliminary data on the clinical and immunologic efficacy of tocilizumab in patients with systemic lupus erythematosus (SLE). METHODS: In an open-label phase I dosage-escalation study, 16 patients with mild-to-moderate disease activity were assigned to receive 1 of 3 doses of tocilizumab given intravenously every other week for 12 weeks (total of 7 infusions): 2 mg/kg in 4 patients, 4 mg/kg in 6 patients, or 8 mg/kg in 6 patients. Patients were then monitored for an additional 8 weeks. RESULTS: The infusions were well tolerated. Tocilizumab treatment led to dosage-related decreases in the absolute neutrophil count, with a median decrease of 38% in the 4 mg/kg dosage group and 56% in the 8 mg/kg dosage group. Neutrophil counts returned to normal after cessation of treatment. One patient was withdrawn from the study because of neutropenia. Infections occurred in 11 patients; none was associated with neutropenia. Disease activity showed significant improvement, with a decrease of > or =4 points in the modified Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index score in 8 of the 15 evaluable patients. Arthritis improved in all 7 patients who had arthritis at baseline and resolved in 4 of them. Levels of anti-double-stranded DNA antibodies decreased by a median of 47% in patients in the 4 mg/kg and 8 mg/kg dosage groups, with a 7.8% decrease in their IgG levels. These changes, together with a significant decrease in the frequency of circulating plasma cells, suggest a specific effect of tocilizumab on autoantibody-producing cells. CONCLUSION: Although neutropenia may limit the maximum dosage of tocilizumab in patients with SLE, the observed clinical and serologic responses are promising and warrant further studies to establish the optimal dosing regimen and efficacy.

Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus.

CD4(+)CD25(+) T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4(+)CD25(high) Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4(+)CD25(high) Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4(+)CD25(high) Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4(+) effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4(+) effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4(+)CD25(high) Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4(+)CD25(high) Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.

Abnormal differentiation of memory T cells in systemic lupus erythematosus.

OBJECTIVE: The chemokine receptor CCR7 and the tumor necrosis factor receptor family member CD27 define 3 distinct, progressively more differentiated maturational stages of CD4 memory subpopulations in healthy individuals: the CCR7+, CD27+, the CCR7-, CD27+, and the CCR7-, CD27- populations. The goal of this study was to examine maturational disturbances in CD4 T cell differentiation in systemic lupus erythematosus (SLE), using these phenotypic markers. METHODS: Phenotypic analysis by flow cytometry, in vitro stimulation experiments, telomere length measurement, and determination of inducible telomerase were carried out. RESULTS. In SLE patients, significant increases of CCR7-, CD27- and CCR7-, CD27+ and a reduction of CCR7+, CD27+ CD4 memory T cells were found. In vitro stimulation of SLE T cells showed a stepwise differentiation from naive to CCR7+, CD27+ to CCR7-, CD27+ to CCR7-, CD27-; telomere length and inducible telomerase decreased in these subsets in the same progressive sequence. The in vitro proliferative response of these populations progressively declined as their susceptibility to apoptosis increased. Interestingly, a significant reduction in inducible telomerase was noted in SLE naive and CCR7+, CD27+ CD4+ memory T cells. Additionally, SLE CCR7-, CD27+ and CCR7-, CD27- CD4 memory T cells proliferated poorly in response to in vitro stimulation and underwent significantly more apoptosis than their normal counterparts. Finally, expression of CXCR4 was significantly reduced in all SLE subsets compared with normal. CONCLUSION: Together these data indicate an increased degree of in vivo T cell stimulation in SLE, resulting in the accumulation of terminally differentiated memory T cells with a decreased proliferative capacity and an increased tendency to undergo apoptosis upon stimulation.

Use of computerized assessment to predict neuropsychological functioning and emotional distress in patients with systemic lupus erythematosus.

OBJECTIVE: Cognitive dysfunction and neuropsychiatric disturbance are common in systemic lupus erythematosus (SLE). This study addressed the ability of the Automated Neuropsychological Assessment Metrics (ANAM), a computerized cognitive testing battery consisting of cognitive subtests, a sleepiness rating scale, and a mood scale, to predict neuropsychological status in patients with SLE. METHODS: Sixty individuals with SLE and no overt neuropsychiatric symptoms were administered ANAM to determine its validity as a screening measure of cognitive dysfunction and emotional distress in SLE. RESULTS: Performance on ANAM was compared with results of a consecutively administered, 2-hour battery of traditional neuropsychological tests and the Beck Depression Inventory II (BDI-II). Individual ANAM cognitive test scores were significantly correlated with most neuropsychological tests, particularly those measuring psychomotor processing speed and executive functioning. Using logistic regression, ANAM cognitive subtests successfully predicted individuals with SLE who had probable versus no impairment after controlling for premorbid levels of cognitive ability. Sensitivity of group classification was 76.2% and specificity was 82.8%, with 80% correct classification overall. ANAM's ability to predict neuropsychological functioning remained even after controlling for subjective reports of depressed mood and current sleepiness. Further, the ANAM mood scale was significantly correlated with the BDI-II (r = 0.67, P < 0.001), indicating its potential future use as a screening tool for emotional distress. CONCLUSION: ANAM shows promise as a time- and cost-efficient tool for screening and monitoring cognitive and emotional functioning in SLE, and can indicate when a more thorough neuropsychological investigation is warranted.

Anti-N-methyl-D-aspartate receptor antibodies, cognitive dysfunction, and depression in systemic lupus erythematosus.

OBJECTIVE: To assess the association of cognitive dysfunction and depression with serum antibodies to N-methyl-D-aspartate (NMDA) receptor (anti-NR2) and analyze clinical and neuroimaging correlates in patients with systemic lupus erythematosus (SLE). METHODS: Sixty patients underwent neurocognitive assessment, evaluation for depression with the Beck Depression Inventory II (BDI-II) and psychiatric interview (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition [DSM-IV] criteria), brain magnetic resonance imaging, and proton magnetic resonance spectroscopy imaging (1H-MRSI). Cognition was assessed in 5 domains: memory, attention/executive, visuospatial, motor, and psychomotor, and adjusted to each individual's best level of prior cognitive functioning estimated from the reading subtest of the Wide Range Achievement Test-3 (WRAT-3). Serum anti-NR2 antibodies were measured by enzyme-linked immunosorbent assay using a pentapeptide from the human NMDA receptor. RESULTS: Cognitive dysfunction was found in 28 of 60 patients (mild in 8, moderate in 20) before adjustment for WRAT-3 and in 35 of 60 patients (mild in 15, moderate in 11, and severe in 9) after adjustment for WRAT-3. The changes were most pronounced in the memory and visuospatial domains. There was no significant association between anti-NR2 antibody levels and cognition. On 1H-MRSI, patients with moderate or severe cognitive dysfunction had significantly higher choline:creatine ratios in the dorsolateral prefrontal cortex and the white matter, compared with patients with mild or absent cognitive dysfunction. Anti-NR2 antibodies were significantly correlated with BDI scores; patients with BDI-II scores of > or =14 had higher serum levels of anti-NR2 antibodies (P = 0.005, 95% confidence interval 0.83, 4.31), and there was a trend toward higher anti-NR2 antibody levels among patients who fulfilled the DSM-IV criteria for major depression. CONCLUSION: Serum anti-NR2 antibodies are associated with depressive mood but not with cognitive dysfunction in SLE at a given time point. Larger longitudinal studies are needed to address the possible association between anti-NR2 antibodies and depression in SLE.

Identification and characterization of circulating human transitional B cells.

Murine B-cell development begins in bone marrow and results in the generation of immature transitional B cells that transit to the spleen to complete their maturation. It remains unclear whether the same developmental pathway takes place in humans. Using markers characteristic of human bone marrow immature B cells, we have identified a population of circulating human B cells with a phenotype most similar to mouse transitional type I (T1) B cells, although these human counterparts express CD5. These cells die rapidly in culture, and B-cell activation factor member of the tumor necrosis factor (TNF) family (BAFF) does not effect their survival regardless of B-cell receptor (BCR) stimulation. In contrast, bone marrow stromal cells or interleukin-4 (IL-4) significantly enhanced their survival. In the presence of T-cell signals provided by IL-4 or CD40 ligation, BCR stimulation can induce progression into cell cycle. Interestingly, circulating B cells that phenotypically and functionally resemble murine T2 B cells are found in cord blood and adult peripheral blood, suggesting that B-cell maturation may not be restricted to the spleen. Notably, increased proportions of T1 B cells were found in blood of patients with systemic lupus erythematosus (SLE), although bone marrow production and selection appeared to be normal.

Cytochrome P450 pharmacogenetics as a predictor of toxicity and clinical response to pulse cyclophosphamide in lupus nephritis.

OBJECTIVE: Pulse cyclophosphamide is the treatment of choice for severe lupus nephritis. However, not all patients respond to this therapy, and gonadal toxicity is of particular concern. Cyclophosphamide is a prodrug that requires activation by cytochrome P450 (CYP) enzymes. We conducted a retrospective cohort study to test whether genetic polymorphisms of these enzymes are associated with the toxicity of, and clinical response to, cyclophosphamide in patients with lupus nephritis. METHODS: Sixty-two patients with proliferative lupus nephritis treated with cyclophosphamide were genotyped for common variant alleles of CYP2B6, 2C19, 2C9, and 3A5. We examined the association between these genotypes and the following clinical end points: development of premature ovarian failure, end-stage renal disease (ESRD), doubling of serum creatinine level, and achievement of complete renal response. RESULTS: The observed frequencies of the variant alleles CYP2B6*5, CYP2C19*2, CYP2C9*2, and CYP3A5*3 were 12.1%, 25.0%, 4.0%, and 75.8%, respectively. Patients who were either heterozygous or homozygous for CYP2C19*2 had a significantly lower risk of developing premature ovarian failure (relative risk 0.10; 95% confidence interval 0.02-0.52), after adjustment for age and total number of cyclophosphamide pulses received. In a survival analysis, patients homozygous for CYP2B6*5 (n = 3) or CYP2C19*2 (n = 4) had a higher probability of reaching ESRD (P = 0.0005) and of doubling the creatinine level (P = 0.0005) as well as a trend toward a lower probability of achieving a complete renal response (P = 0.051). CONCLUSION: Determination of selected cytochrome P450 enzyme genotypes may be valuable for predicting the risk of premature ovarian failure in lupus nephritis patients treated with cyclophosphamide. The association of these genotypes with renal response needs further validation.

Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis.

Advanced glycation end-product (AGE)-damaged IgG occurs as a result of hyperglycemia and/or oxidative stress. Autoantibodies to IgG-AGE were previously demonstrated in patients with severe, longstanding rheumatoid arthritis (RA). We investigated whether IgG-AGE and anti-IgG-AGE antibodies were present early in the course of RA and other inflammatory arthropathies. We prospectively followed a cohort of 238 patients with inflammatory arthritis of duration less than 1 year. Patients were evaluated clinically and serologically, and radiographs were obtained at initial and 1-year visits. Sera were assayed for IgG-AGE and anti-IgG-AGE antibodies by enzyme-linked immunosorbent assay (ELISA). Rheumatoid factor (RF) was determined by nephelometry and ELISA. Of all patients, 29% had RF-positive RA, 15% had RF-negative RA, 18% had spondyloarthropathy, and 38% had undifferentiated arthritis. IgG-AGE was present in 19% of patients, and was similar in amount and frequency in all groups. Patients with elevated IgG-AGE levels had significantly higher levels of the inflammatory markers C-reactive protein and erythrocyte sedimentation rate, but there was no correlation with blood glucose levels. Overall, 27% of the patients had IgM anti-IgG-AGE antibodies. These antibodies were highly significantly associated with RFs (P < 0.0001) and with swollen joint count (P < 0.01). In early onset arthritis, IgG damaged by AGE was detected in all patient groups. The ability to make IgM anti-IgG-AGE antibodies, however, was restricted to a subset of RF-positive RA patients with more active disease. The persistence of the anti-IgG-AGE response was more specific to RA, and was transient in the patients with spondyloarthropathy and with undifferentiated arthritis who were initially found to be positive for anti-IgG-AGE antibodies.

Pilot clinical trial of intravenous doxycycline versus placebo for rheumatoid arthritis.

OBJECTIVE: To screen for potential efficacy and assess the feasibility of intravenous (IV) doxycycline as a treatment for rheumatoid arthritis (RA). METHODS: The study was a (stratified, block) randomized, double blind, 12 week, pilot trial of IV doxycycline 300 mg/day versus identical appearing IV placebo given over 2 h for 14 days. The primary comparison was to a hypothesized placebo rate of 20% as described by Paulus. If a total of 14 consecutive subjects receiving doxycycline treatment did not respond, it would be considered futile to proceed to a Phase III trial. We planned a placebo group of 14 subjects to verify the placebo response rate and estimate sample size required for a definitive Phase III trial, if such a trial was warranted based on the pilot study. American College of Rheumatology (ACR) RA response criteria were used. After 23 subjects entered, the study was closed due to recruitment difficulties. RESULTS: At baseline, mean (SD) tender joint count was 37 (11.9), swollen joint count 30 (9.6), morning stiffness 317 (319) min, and erythrocyte sedimentation rate 72 mm/h (27.5). Randomization resulted in 10 subjects receiving doxycycline and 13 receiving placebo. Treatment was stopped in 8 subjects: in 6, treatment was ineffective (one taking doxycycline, 5 placebo), and in 2, rashes occurred (one taking doxycycline, one placebo). Only one subject met ACR response criteria in the doxycycline group and none in the placebo group. Having no responders in the placebo group was consistent with placebo response rate of 20% or less. Several patients required peripherally inserted central catheters for venous access. CONCLUSION: The efficacy of IV doxycycline as a treatment for RA could not be ruled out. However, as the proportion of responders was small, it is unlikely that potential efficacy of IV doxycycline would outweigh potential disadvantages of IV administration.

A pilot study of the use of 2-[18F]-fluoro-2-deoxy-D-glucose-positron emission tomography to assess the distribution of activated lymphocytes in patients with systemic lupus erythematosus.

OBJECTIVE: The 2-[(18)F]-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) technique provides information on uptake and metabolism of glucose in various tissues. Compared with resting cells, activated lymphocytes take up radioactively labeled glucose analog at a higher rate, which makes it possible to identify lymphoid organs with higher concentrations of activated lymphocytes. This study was undertaken to compare the pattern of PET images and quantitative FDG uptake in lymphoid organs of patients with active systemic lupus erythematosus (SLE) versus patients with inactive SLE and to correlate these findings with peripheral blood lymphocyte phenotypes. METHODS: Ten patients with active SLE and 9 patients with inactive SLE were studied. FDG-PET images were obtained from the inguinal region to above the ear, starting at 60 minutes after injection of FDG. Standardized uptake values using lean body mass were determined over areas of interest. RESULTS: Both patients with active lupus and those with inactive lupus had increased FDG uptake in lymph nodes when compared with healthy volunteers, and there was no statistically significant difference between the 2 groups of lupus patients. Thymic uptake was demonstrated in 5 of 10 patients with active lupus compared with 0 of 9 patients with inactive disease. Three of the 5 patients with active SLE who were over 29 years of age had thymic uptake. Of the activation markers tested, only the CD3/CD71 population of cells was significantly different between the patient groups, with an increased percentage in the active disease group (P = 0.0247). CONCLUSION: Increased FDG uptake in lymph nodes of both patients with active SLE and patients with inactive SLE suggests that metabolic, and probably immunologic, activity is enhanced not only in active, but also in clinically quiescent, disease. The increased thymic uptake observed only in patients with active disease suggests that the thymus plays an important role during periods of disease activity.