Bernard Lauwerys

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18.

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3−), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3−) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-γ, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-γ, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-γ and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-γR knockout mice demonstrated that IFN-γ was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.

The anti-CD20 antibody rituximab reduces the Th17 cell response

OBJECTIVE Rituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA. METHODS Twelve patients with active RA were treated with rituximab. Disease activity was evaluated using the 28-joint Disease Activity Score. Synovial biopsy samples obtained at baseline and 12 weeks after treatment initiation were analyzed by microarray, quantitative polymerase chain reaction, and immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and from 4 patients with X-linked agammaglobulinemia were stimulated with the Th17-inducing stimulus Candida albicans, and the response in the presence and absence of rituximab was examined. RESULTS In RA patients, rituximab reduced expression of retinoic acid-related orphan receptor γt and interleukin-22 (IL-22) and numbers of Th17-positive cells in synovial tissue, and this correlated with better clinical outcome. Rituximab did not affect tumor necrosis factor α (TNFα), Th1 cell, or Treg cell responses. Rituximab strongly reduced in vitro IL-17 and IL-22 production induced by C albicans. This effect was not observed in PBMCs from patients with X-linked agammaglobulinemia. CONCLUSION Rituximab reduced the local Th17 response in RA patients, whereas it did not influence Th1 cell, Treg cell, or TNFα responses. The decreased Th17 response was associated with reduced inflammation and better clinical outcome. Moreover, inhibition of the Th17 response by rituximab was lost in the absence of B cells, providing evidence that the effects of rituximab are due to B cell depletion. These data demonstrate an unexpected role of B cells in the development of Th17 responses, which could possibly lead to B cell-based strategies for the treatment of Th17-related autoimmune diseases.

Juvenile-onset systemic lupus erythematosus: different clinical and serological pattern than adult-onset systemic lupus erythematosus

Objective: To investigate differences in clinical signs and symptoms, and in antinuclear antibodies (ANA), between patients with juvenile-onset and adult-onset systemic lupus erythematosus (SLE). Methods: Clinical and serological data of 56 patients with juvenile-onset SLE were compared with data of 194 patients with adult-onset SLE. ANA were determined by line immunoassay and by indirect immunofluorescence on Crithidia luciliae. Results: Renal involvement, encephalopathy and haemolytic anaemia were seen, and anti-dsDNA, anti-ribosomal P and antihistone antibodies found, significantly more often in juvenile-onset SLE. Anti-dsDNA antibodies were directly associated, and anti-ribosomal P antibodies inversely associated, with renal involvement in juvenile-onset SLE. In juvenile patients with SLE and anti-dsDNA and without anti-ribosomal P antibodies the odds ratio for glomerulonephritis was 9.00; no patients with anti-ribosomal P but without anti-dsDNA had renal involvement. Conclusion: Patients with juvenile-onset SLE more often have renal involvement and encephalopathy than patients with adult-onset SLE. Anti-ribosomal P, anti-dsDNA and antihistone antibodies are more often found in patients with juvenile-onset SLE.

Down-regulation of interferon signature in systemic lupus erythematosus patients by active immunization with interferon α–kinoid†

OBJECTIVE We developed interferon-α-kinoid (IFN-K), a drug composed of inactivated IFNα coupled to a carrier protein, keyhole limpet hemocyanin. In human IFNα-transgenic mice, IFN-K induces polyclonal antibodies that neutralize all 13 subtypes of human IFNα. We also previously demonstrated that IFN-K slows disease progression in a mouse model of systemic lupus erythematosus (SLE). This study was undertaken to examine the safety, immunogenicity, and biologic effects of active immunization with IFN-K in patients with SLE. METHODS We performed a randomized, double-blind, placebo-controlled, phase I/II dose-escalation study comparing 3 or 4 doses of 30 μg, 60 μg, 120 μg, or 240 μg of IFN-K or placebo in 28 women with mild to moderate SLE. RESULTS IFN-K was well tolerated. Two SLE flares were reported as serious adverse events, one in the placebo group and the other in a patient who concomitantly stopped corticosteroids 2 days after the first IFN-K dose, due to mild fever not related to infection. Transcriptome analysis was used to separate patients at baseline into IFN signature-positive and -negative groups, based on the spontaneous expression of IFN-induced genes. IFN-K induced anti-IFNα antibodies in all immunized patients. Notably, significantly higher anti-IFNα titers were found in signature-positive patients than in signature-negative patients. In IFN signature-positive patients, IFN-K significantly reduced the expression of IFN-induced genes. The decrease in IFN score correlated with the anti-IFNα antibody titer. Serum complement C3 levels were significantly increased in patients with high anti-IFNα antibody titers. CONCLUSION These results show that IFN-K is well tolerated, immunogenic, and significantly improves disease biomarkers in SLE patients, indicating that further studies of its clinical efficacy are warranted.

Genetic association of miRNA-146a with systemic lupus erythematosus in Europeans through decreased expression of the gene.

A recent genome-wide association study revealed a variant (rs2431697) in an intergenic region, between the pituitary tumor-transforming 1 (PTTG1) and microRNA (miR-146a) genes, associated with systemic lupus erythematosus (SLE) susceptibility. Here, we analyzed with a case–control design this variant and other candidate polymorphisms in this region together with expression analysis in order to clarify to which gene this association is related. The single-nucleotide polymorphisms (SNPs) rs2431697, rs2910164 and rs2277920 were genotyped by TaqMan assays in 1324 SLE patients and 1453 healthy controls of European ancestry. Genetic association was statistically analyzed using Unphased. Gene expression of PTTG1, the miRNAs miR-3142 and primary and mature forms of miR-146a in peripheral blood mononuclear cells (PBMCs) were assessed by quantitative real-time PCR. Of the three variants analyzed, only rs2431697 was genetically associated with SLE in Europeans. Gene expression analysis revealed that this SNP was not associated with PTTG1 expression levels, but with the microRNA-146a, where the risk allele correlates with lower expression of the miRNA. We replicated the genetic association of rs2341697 with SLE in a case–control study in Europeans and demonstrated that the risk allele of this SNP correlates with a downregulation of the miRNA 146a, potentially important in SLE etiology.

Lack of evidence of stimulatory autoantibodies to platelet-derived growth factor receptor in patients with systemic sclerosis

OBJECTIVE Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal organs. Recent findings suggested that the disease is driven by stimulatory autoantibodies to platelet-derived growth factor receptor (PDGFR), which stimulate the production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues of research into the diagnosis and treatment of SSc. The present study was undertaken to confirm the presence of anti-PDGFR antibodies in patients with SSc. METHODS Immunoglobulins from 37 patients with SSc were purified by protein A/G chromatography. PDGFR activation was tested using 4 different sensitive bioassays, i.e., cell proliferation, ROS production, signal transduction, and receptor phosphorylation; the latter was also tested in a separate population of 7 patients with SSc from a different research center. RESULTS Purified IgG samples from patients with SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFRalpha or PDGFRbeta in any of the tests. Cell stimulation with PDGF itself consistently produced a strong signal. CONCLUSION The present results raise questions regarding the existence of agonistic autoantibodies to PDGFR in SSc.

Side-effects of intravenous cyclophosphamide pulse therapy.

We reviewed the side-effects of intravenous (IV) cyclophosphamide (CPM) pulse therapy in a group of 75 patients suffering from various autoimmune disorders (mostly systemic lupus erythematosus and vasculitis) who received a total of 451 IV CPM pulses, given on a monthly basis (mean ± s.d. CPM dose per pulse: 764 ± 217 mg; mean ± s.d. follow-up period: 26.7 ± 22.1 mon). Infection was the most common side-effect (30 episodes in 21 patients; 28% of the patients) but rarely required in-patient treatment (8 episodes in 7 patients; 9% of the patients). No relationship could be found between the occurrence of infection and the dose of CPM or of glucocorticoids. Other side-effects were rare. Only one patient suffered from neutropenia. Haemorragic cystitis was never observed nor did premature ovarian failure in the 25 female patients at risk. Four patients developed neoplasia and three died suddenly a few days after receiving a CPM pulse but the causal relationship between CPM therapy and these poor outcomes is speculative. Taken together, our data confirm in a large group of patients that IV CPM pulse therapy is relatively safe. In particular, the rate of severe infection requiring in-patient treatment is rare (1.8% of 451 pulses).

Gene expression profiling in the synovium identifies a predictive signature of absence of response to adalimumab therapy in rheumatoid arthritis

IntroductionTo identify markers and mechanisms of resistance to adalimumab therapy, we studied global gene expression profiles in synovial tissue specimens obtained from severe rheumatoid arthritis (RA) patients before and after initiation of treatment.MethodsPaired synovial biopsies were obtained from the affected knee of 25 DMARD (disease-modifying antirheumatic drug)-resistant RA patients at baseline (T0) and 12 weeks (T12) after initiation of adalimumab therapy. DAS28-CRP (disease activity score using 28 joint counts-C-reactive protein) scores were computed at the same time points, and patients were categorized as good, moderate, or poor responders according to European League Against Rheumatism criteria. Global gene expression profiles were performed in a subset of patients by means of GeneChip Human Genome U133 Plus 2.0 Arrays, and confirmatory immunohistochemistry experiments were performed on the entire cohort.ResultsGene expression studies performed at baseline identified 439 genes associated with poor response to therapy. The majority (n = 411) of these genes were upregulated in poor responders and clustered into two specific pathways: cell division and regulation of immune responses (in particular, cytokines, chemokines, and their receptors). Immunohistochemistry experiments confirmed that high baseline synovial expression of interleukin-7 receptor α chain (IL-7R), chemokine (C-X-C motif) ligand 11 (CXCL11), IL-18, IL-18 receptor accessory (IL-18rap), and MKI67 is associated with poor response to adalimumab therapy. In vitro experiments indicated that genes overexpressed in poor responders could be induced in fibroblast-like synoviocytes (FLS) cultures by the addition of tumor necrosis factor-alpha (TNF-α) alone, IL-1β alone, the combination of TNF-α and IL-17, and the combination of TNF-α and IL-1β.ConclusionsGene expression studies of the RA synovium may be useful in the identification of early markers of response to TNF blockade. Genes significantly overexpressed at baseline in poor responders are induced by several cytokines in FLSs, thereby suggesting a role for these cytokines in the resistance to TNF blockade in RA.

Type I interferon blockade in systemic lupus erythematosus: where do we stand?

SLE is an autoimmune condition characterized by loss of tolerance to chromatin constituents and the production of ANAs. The majority of SLE patients display spontaneous expression of type I IFN-induced genes in circulating mononuclear cells and peripheral tissues, and type I IFNs play a role in the pathogenesis of the disease via the sustained activation of autoreactive T and B cells necessary for the production of pathogenic autoantibodies. Several IFN-blocking strategies are currently being evaluated in clinical trials: monoclonal antibodies directed against IFN-α and type I IFN-α receptor (IFNAR), as well as active immunization against IFN-α. This review describes the rationale behind these trials and the results obtained, and discusses the perspectives for further development of these drugs.

Analysis of autosomal genes reveals gene-sex interactions and higher total genetic risk in men with systemic lupus erythematosus

Objectives Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci. Methods A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and non-parametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients. Results A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P=4.52x10-8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men. Conclusions The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.