Humoral responses after influenza vaccination are severely reduced in patients with rheumatoid arthritis treated with rituximab

Objective

For patients with rheumatoid arthritis (RA), yearly influenza vaccination is recommended. However, its efficacy in patients treated with rituximab is unknown. The objectives of this study were to investigate the efficacy of influenza vaccination in RA patients treated with rituximab and to investigate the duration of the possible suppression of the humoral immune response following rituximab treatment. We also undertook to assess the safety of influenza vaccination and the effects of previous influenza vaccination.

Methods

Trivalent influenza subunit vaccine was administered to 23 RA patients who had received rituximab (4–8 weeks after rituximab for 11 patients [the early rituximab subgroup] and 6–10 months after rituximab for 12 patients [the late rituximab subgroup]), 20 RA patients receiving methotrexate (MTX), and 29 healthy controls. Levels of antibodies against the 3 vaccine strains were measured before and 28 days after vaccination using hemagglutination inhibition assay. The Disease Activity Score in 28 joints (DAS28) was used to assess RA activity.

Results

Following vaccination, geometric mean titers (GMTs) of antiinfluenza antibodies significantly increased for all influenza strains in the MTX‐treated group and in healthy controls, but for no strains in the rituximab‐treated group. However, in the late rituximab subgroup, a rise in GMT for the A/H3N2 and A/H1N1 strains was demonstrated, in the absence of a repopulation of CD19+ cells at the time of vaccination. Seroconversion and seroprotection occurred less often in the rituximab‐treated group than in the MTX‐treated group for the A/H3N2 and A/H1N1 strains, while seroprotection occurred less often in the rituximab‐treated group than in the healthy controls for the A/H1N1 strain. Compared with unvaccinated patients in the rituximab‐treated group, previously vaccinated patients in the rituximab‐treated group had higher pre‐ and postvaccination GMTs for the A/H1N1 strain. The DAS28 did not change after vaccination.

Conclusion

Rituximab reduces humoral responses following influenza vaccination in RA patients, with a modestly restored response 6–10 months after rituximab administration. Previous influenza vaccination in rituximab‐treated patients increases pre‐ and postvaccination titers. RA activity was not influenced.

     

Interferon‐α priming promotes lipid uptake and macrophage‐derived foam cell formation: A novel link between interferon‐α and atherosclerosis in lupus

Objective

An increased risk of premature atherosclerosis has been associated with systemic lupus erythematosus (SLE), and type I interferon (IFN) has been shown to play a pathogenic role in human SLE. The aim of this study was to determine whether IFNα is involved in the development of atherosclerosis in patients with SLE by promoting lipid uptake and macrophage‐derived foam cell formation, which is a crucial step in early atherosclerosis.

Methods

The effects of IFNα on lipid uptake and foam cell formation were determined by flow cytometry and oil red O staining. Messenger RNA and protein expression of scavenger receptors (SRs) was examined. Promoter activity was detected by luciferase reporter assay. Expression of macrophage SR class A (SR‐A) and IFN‐inducible genes (IFIGs) was measured in peripheral blood mononuclear cells obtained from 42 patients with SLE and 42 healthy donors.

Results

IFNα priming increased the uptake of oxidized low‐density lipoprotein and hence enhanced foam cell formation by up‐regulating SR‐A expression. IFNα increased SR‐A expression via enhancing its promoter activities. Examination using signaling inhibitors revealed that a phosphatidylinositol 3‐kinase/Akt signaling pathway appeared to be involved in this process. Notably, SR‐A messenger RNA was significantly increased in patients with SLE compared to normal subjects and positively correlated with IFIG expression.

Conclusion

Our data suggest that IFNα priming up‐regulated the expression of SR‐A in human monocyte/macrophages, leading to increased lipid uptake and foam cell formation. Activation of the IFN signaling pathway may be linked to the risk of atherosclerosis by affecting plaque formation in patients with SLE. These findings provide novel insights into the mechanisms of and potential therapeutic approaches to premature atherosclerosis in patients with SLE.

     

Intravenous immunoglobulin abrogates dendritic cell differentiation induced by interferon‐α present in serum from patients with systemic lupus erythematosus

Objective

Alterations in the function of dendritic cells (DCs) may explain the systemic autoimmune responses that characterize systemic lupus erythematosus (SLE). Even though several reports have documented the beneficial effect of intravenous immunoglobulin (IVIG) in SLE, the underlying mechanisms of action remain poorly understood. Considering the effect of serum factors, including interferon‐α (IFNα), on the activity of DCs, we investigated the effects of IVIG on the differentiation of DCs mediated by serum from SLE patients.

Methods

DCs were differentiated from peripheral blood monocytes obtained from SLE patients and from healthy blood donors, in the presence of SLE serum. IVIG was used at a concentration of 0.15 mM. A functional assay was performed to assess the inhibitory effect of IVIG on the uptake of nucleosomes by DCs.

Results

IVIG interfered with the differentiation of DCs from SLE patients and healthy donors cultured in the presence of SLE serum. Treatment of DCs with IVIG inhibited the ingestion of nucleosomes by immature DCs, by up to 36%.

Conclusion

The present findings indicate that IVIG, by down‐regulating the IFNα‐mediated differentiation of DCs and by inhibiting uptake of nucleosomes, may exert an essential immunoregulatory effect in SLE patients at the onset of the immune response, at the DC level. Given the critical role of HLA molecules and the costimulatory signals delivered by CD80 and CD86 in optimal antigen presentation and T cell activation, inhibition of expression of HLA and CD80/CD86 on DCs by IVIG offers a plausible explanation for the efficacy of IVIG in SLE and other immune‐mediated inflammatory conditions.

     

Epstein‐Barr virus promotes interferon‐α production by plasmacytoid dendritic cells

Objective

Epstein‐Barr virus (EBV) infection has been linked to systemic lupus erythematosus (SLE), as demonstrated by the presence of increased seroprevalence and elevated viral loads, but the mechanism of this linkage has not been elucidated. Increased interferon‐α (IFNα) levels and signatures, which are associated with innate immune responses, have been found in patients with SLE. Plasmacytoid dendritic cells (PDCs) are innate immune cells that mediate viral immunity by producing large quantities of IFNα, but the role they play during infection with EBV remains unclear. To address this issue, we investigated the ability of EBV to promote IFNα production by PDCs in healthy subjects.

Methods

Human PDCs were sorted and cultured in the presence of EBV, EBV‐encoded RNA, and EBV double‐stranded DNA. IFNα production by PDCs was measured by enzyme‐linked immunosorbent assay, with the activation of these cells measured by flow cytometry.

Results

We found that EBV DNA and RNA promoted IFNα production by human PDCs through engagement of Toll‐like receptor 9 (TLR‐9) and TLR‐7, respectively, with the initial viral recognition by PDCs mediated by binding to class II major histocompatibility complex (MHC) molecules.

Conclusion

These data demonstrate that class II MHC–specific engagement by virus, with subsequent viral nucleic acid recognition, mediates IFNα production by PDCs. Our results suggest that elevated levels of IFNα found in SLE patients may be a result of aberrantly controlled chronic viral infection.

     

Coordinate overexpression of interferon‐α–induced genes in systemic lupus erythematosus

Objective

To study the contribution of interferon‐α (IFNα) and IFNγ to the IFN gene expression signature that has been observed in microarray screens of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE).

Methods

Quantitative real‐time polymerase chain reaction analysis of healthy control PBMCs was used to determine the relative induction of a panel of IFN‐inducible genes (IFIGs) by IFNα and IFNγ. PBMCs from 77 SLE patients were compared with those from 22 disease controls and 28 healthy donors for expression of IFIGs.

Results

Expression of IFNα‐inducible genes was significantly higher in SLE PBMCs than in those from disease controls or healthy donors. The level of expression of all IFIGs in PBMCs from SLE patients with IFNα pathway activation correlated highly with the inherent responsiveness of those genes to IFNα, suggesting coordinate activation of that cytokine pathway. Expression of genes preferentially induced by IFNγ was not significantly increased in SLE PBMCs compared with control PBMCs. IFNα‐regulated gene‐inducing activity was detected in some SLE plasma samples.

Conclusion

The coordinate activation of IFNα‐induced genes is a characteristic of PBMCs from many SLE patients, supporting the hypothesis that IFNα is the predominant stimulus for IFIG expression in lupus.

     

Impact of synthetic and biologic disease‐modifying antirheumatic drugs on antibody responses to the AS03‐adjuvanted pandemic influenza vaccine: A prospective,open‐label,parallel‐cohort,single‐center study

Objective

To identify the determinants of antibody responses to adjuvanted split influenza A (H1N1) vaccines in patients with inflammatory rheumatic diseases.

Methods

One hundred seventy‐three patients (82 with rheumatoid arthritis, 45 with spondylarthritis, and 46 with other inflammatory rheumatic diseases) and 138 control subjects were enrolled in this prospective single‐center study. Controls received 1 dose of adjuvanted influenza A/09/H1N1 vaccine, and patients received 2 doses of the vaccine. Antibody responses were measured by hemagglutination inhibition assay before and 3–4 weeks after each dose. Geometric mean titers (GMTs) and rates of seroprotection (GMT ≥40) were calculated. A comprehensive medical questionnaire was used to identify the determinants of vaccine responses and adverse events.

Results

Baseline influenza A/09/H1N1 antibody levels were low in patients and controls (seroprotection rates 14.8% and 14.2%, respectively). A significant response to dose 1 was observed in both groups. However, the GMT and the seroprotection rate remained significantly lower in patients (GMT 146 versus 340, seroprotection rate 74.6% versus 87%; both P < 0.001). The second dose markedly increased antibody titers in patients, with achievement of a similar GMT and seroprotection rate as elicited with a single dose in healthy controls. By multivariate regression analysis, increasing age, use of disease‐modifying antirheumatic drugs (DMARDs) (except hydroxychloroquine and sulfasalazine), and recent (within 3 months) B cell depletion treatment were identified as the main determinants of vaccine responses; tumor necrosis factor α antagonist treatment was not identified as a major determinant. Immunization was well tolerated, without any adverse effect on disease activity.

Conclusion

DMARDs exert distinct influences on influenza vaccine responses in patients with inflammatory rheumatic diseases. Two doses of adjuvanted vaccine were necessary and sufficient to elicit responses in patients similar to those achieved with 1 dose in healthy controls.

     

Association of endogenous anti–interferon‐α autoantibodies with decreased interferon‐pathway and disease activity in patients with systemic lupus erythematosus

Objective

Numerous observations implicate interferon‐α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti‐IFNα autoantibodies (AIAAs) on IFN‐pathway and disease activity is unclear. The aim of this study was to characterize IFN‐pathway activity and the serologic and clinical profiles of AIAA‐positive patients with SLE.

Methods

Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA‐positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN‐regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN‐inducible chemokines, and other autoantibodies were measured using immunoassays.

Results

AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN^low and IFN^high, that differed in the levels of serum type I IFN bioactivity, IFN‐regulated gene expression, BAFF, anti–ribosomal P, and anti‐chromatin autoantibodies, and in AIAA status. The majority of AIAA‐positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN‐pathway activity, and lower disease activity compared with the IFN^high patients. AIAA‐positive sera were able to effectively neutralize type I IFN activity in vitro.

Conclusion

Patients with SLE commonly harbor AIAAs. AIAA‐positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN‐pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

     

Interferon‐α mediates suppression of C‐reactive protein: Explanation for muted C‐reactive protein response in lupus flares?

Objective

C‐reactive protein (CRP) is synthesized by hepatocytes in response to interleukin‐6 (IL‐6) during inflammation. Despite raised IL‐6 levels and extensive systemic inflammation, serum CRP levels remain low during most viral infections and disease flares of systemic lupus erythematosus (SLE). Because both viral infections and SLE are characterized by high levels of interferon‐α (IFNα), the aim of this study was to determine whether this cytokine can inhibit the induction of CRP.

Methods

The interference of all 12 IFNα subtypes with CRP promoter activity induced by IL‐6 and IL‐1β was studied in a CRP promoter– and luciferase reporter–transfected human hepatoma cell line, Hep‐G2. CRP secretion by primary human hepatocytes was analyzed by enzyme‐linked immunosorbent assay.

Results

CRP promoter activity was inhibited by all single IFNα subtypes, as well as by 2 different mixtures of biologically relevant IFNα subtypes. The most prominent effect was seen using a leukocyte‐produced mixture of IFNα (56% inhibition at 1,000 IU/ml). The inhibitory effect of IFNα was confirmed in primary human hepatocytes. CRP promoter inhibition was dose dependent and mediated via the type I IFN receptor. Transferrin production and Hep‐G2 proliferation/viability were not affected by IFNα.

Conclusion

The current study demonstrates that IFNα is an inhibitor of CRP promoter activity and CRP secretion. This finding concords with previous observations of up‐regulated IFNα and a muted CRP response during SLE disease flares. Given the fundamental role of both IFNα and CRP in the immune response, our results are of importance for understanding the pathogenesis of SLE and may also contribute to understanding the differences in the CRP response between viral and bacterial infections.

     

Lupus‐like disease and high interferon levels corresponding to trisomy of the type I interferon cluster on chromosome 9p

Objective

Systemic lupus erythematosus (SLE) is associated with type I interferons (IFNs) and can be induced by IFNα treatment. This study looked for evidence of autoimmunity in a pedigree consisting of 4 family members with a balanced translocation 9;21 and 2 members with an unbalanced translocation resulting in trisomy of the short (p) arm and part of the long (q) arm of chromosome 9. These latter 2 subjects had 3 copies of the IFN gene cluster.

Methods

Subjects were evaluated clinically and serologically for autoimmune disease. Expression levels of IFNα4, IFNβ, the type I IFN–inducible gene Mx1, the type I IFN receptor, interleukin‐6, and tumor necrosis factor α were determined by real‐time polymerase chain reaction. Circulating plasmacytoid dendritic cells, the main IFN‐producing cells, were quantified by flow cytometry.

Results

Both subjects with trisomy of chromosome 9p had a lupus‐like syndrome with joint manifestations and antinuclear antibodies: one had anti‐RNP and antiphospholipid autoantibodies, and the other had anti–Ro 60. The 3 family members with a balanced translocation 9;21 had no clinical or serologic evidence of autoimmunity, similar to that in relatives who were unaffected by the chromosomal translocation. In the 2 subjects with trisomy of 9p, high levels of IFNα/β (comparable with those found in patients with SLE), increased signaling through the IFN receptor (as indicated by high Mx1 expression), and low levels of circulating plasmacytoid dendritic cells (as observed in patients with SLE) were evident. These abnormalities were not seen in individuals with a balanced translocation.

Conclusion

Trisomy of the type I IFN cluster of chromosome 9p was associated with lupus‐like autoimmunity and increased IFNα/β and IFN receptor signaling. The data support the idea that abnormal regulation of type I IFN production is involved in the pathogenesis of SLE.

     

Genetic variation at the IRF7/PHRF1 locus is associated with autoantibody profile and serum interferon‐α activity in lupus patients

Objective

Interferon‐α (IFNα) is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variation near IRF7 is implicated in SLE susceptibility. SLE‐associated autoantibodies can stimulate IFNα production through the Toll‐like receptor/IRF7 pathway. This study was undertaken to determine whether variants of IRF7 act as risk factors for SLE by increasing IFNα production and whether autoantibodies are important to this phenomenon.

Methods

We studied 492 patients with SLE (236 African American, 162 European American, and 94 Hispanic American subjects). Serum levels of IFNα were measured using a reporter cell assay, and single‐nucleotide polymorphisms (SNPs) in the IRF7/PHRF1 locus were genotyped.

Results

In a joint analysis of European American and Hispanic American subjects, the rs702966 C allele was associated with the presence of anti–double‐stranded DNA (anti‐dsDNA) antibodies (odds ratio [OR] 1.83, P = 0.0069). The rs702966 CC genotype was only associated with higher serum levels of IFNα in European American and Hispanic American patients with anti‐dsDNA antibodies (joint analysis P = 4.1 × 10⁻⁵ in anti‐dsDNA–positive patients and P = 0.99 in anti‐dsDNA–negative patients). In African American subjects, anti‐Sm antibodies were associated with the rs4963128 SNP near IRF7 (OR 1.95, P = 0.0017). The rs4963128 CT and TT genotypes were associated with higher serum levels of IFNα only in African American patients with anti‐Sm antibodies (P = 0.0012). In African American patients lacking anti‐Sm antibodies, an effect of anti‐dsDNA–rs702966 C allele interaction on serum levels of IFNα was observed, similar to the other patient groups (overall joint analysis P = 1.0 × 10⁻⁶). In European American and Hispanic American patients, the IRF5 SLE risk haplotype showed an additive effect with the rs702966 C allele on IFNα level in anti‐dsDNA–positive patients.

Conclusion

Our findings indicate that IRF7/PHRF1 variants in combination with SLE‐associated autoantibodies result in higher serum levels of IFNα, providing a biologic relevance for this locus at the protein level in human SLE in vivo.

     

Expression of the markers BDCA‐2 and BDCA‐4 and production of interferon‐α by plasmacytoid dendritic cells in systemic lupus erythematosus

Objective

To study the expression of blood dendritic cell antigen 2 (BDCA‐2) and BDCA‐4 molecules by plasmacytoid dendritic cells (PDCs) in the blood of patients with systemic lupus erythematosus (SLE), and to study PDC production of interferon‐α (IFNα) and its inhibition by anti–BDCA‐2 and anti–BDCA‐4 antibodies.

Methods

Peripheral blood mononuclear cells (PBMCs) from SLE patients (SLE PBMCs) and from healthy controls were induced to produce IFNα in vitro by SLE serum containing an endogenous IFNα‐inducing factor (SLE‐IIF) or by herpes simplex virus type 1 (HSV‐1). The frequencies and numbers of BDCA‐2–, BDCA‐3–, and BDCA‐4–expressing cells were analyzed by flow cytometry, and the effects of anti–BDCA‐2 and anti–BDCA‐4 monoclonal antibodies (mAb) on IFNα production were investigated.

Results

IFNα production by SLE PBMCs induced by SLE‐IIF or HSV‐1 was decreased compared with that of healthy control PBMCs (P = 0.002 and P = 0.0007, respectively). The proportions of BDCA‐2– and BDCA‐3–expressing cells in SLE PBMCs were reduced compared with those in PBMCs from healthy controls (P = 0.01 and P = 0.004, respectively). IFNα producers in culture, especially among SLE PBMCs, displayed reduced BDCA‐2 expression and constituted only a minority of the BDCA‐2–positive cells, at least in healthy control PBMCs (median 18%). IFNα production by both SLE and healthy control PBMCs stimulated by SLE‐IIF or HSV‐1 was markedly reduced by anti–BDCA‐2 mAb (median 81–98% inhibition). Anti–BDCA‐4 mAb only partially inhibited SLE‐IIF–induced IFNα production.

Conclusion

SLE patients had a reduced number of BDCA‐2–expressing PDCs, also termed natural IFNα‐producing cells, and their IFNα production could be inhibited by anti–BDCA‐2/4 mAb. Such mAb may be a therapeutic option for inhibiting the ongoing IFNα production in SLE patients.

     

Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti–RNA binding protein autoantibodies

Objective

Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) have increased expression of genes typically induced by type I interferon (IFN). However, it has been difficult to identify and quantify the factors responsible for activation of the IFN pathway in SLE. To characterize these mediators, we developed an assay that measures the functional effects of plasma or serum components on the gene expression of cultured target cells.

Methods

WISH epithelial cell line cells were cultured with medium, with recombinant IFNα, IFNβ, or IFNγ, or with 50% plasma from SLE patients (n = 73), rheumatoid arthritis (RA) patients (n = 19), or healthy donors (n = 30). Real‐time quantitative polymerase chain reaction was used to determine WISH cell expression of IFN target genes, including PRKR, IFIT1, IFI44, MX1, and C1orf29 (preferentially induced by IFNα) and CXCL9 (Mig) (preferentially induced by IFNγ).

Results

IFNα‐regulated genes were induced by SLE plasma samples, but not by most of the RA or healthy control plasma samples. The activity in SLE plasma was inhibited >90% by anti‐IFNα antibody, but not by anti‐IFNβ or anti‐IFNγ antibodies. The expression of each IFNα target gene induced by SLE plasma correlated with the expression of that gene studied ex vivo in PBMCs from the same patients and with the titer of anti–RNA binding protein (anti‐RBP)–specific autoantibodies. Plasma activity paralleled PBMC expression of IFNα‐inducible genes over time.

Conclusion

IFNα in SLE plasma is a major stimulus of IFN target gene expression and is related to expression of those genes in PBMCs from SLE patients and to the titer of anti‐RBP autoantibodies. These data provide additional support for the view that IFNα mediates immune system activation and dysregulation in SLE.

     

Cytokine response in pediatric patients with pandemic influenza H1N1 2009 virus infection and pneumonia: comparison with pediatric pneumonia without H1N1 2009 infection

Objectives

We investigated serum cytokine levels in pediatric patients with pandemic influenza H1N1 2009 virus (H1N1) infection‐pneumonia and in pediatric patients with pneumonia but without H1N1 infection, and examined correlations between cytokine levels and clinical/laboratory findings.

Methods

Fifty‐seven cases of infection by H1N1 were confirmed by RT‐PCR and enrolled. Of these 57 cases, 26 had a severe H1N1 infection (group 1), and 31 had a mild H1N1 infection (group 2). Sera from 18 cases with pneumonia without H1N1 infection (group 3) were used as controls. The serum levels of 10 cytokines were determined by multiplex assay.

Results

The serum levels of IFN‐α, IL‐6, and IP‐10 were significantly higher in H1N1 infected cases than in group 3, and levels of IL‐6 and IP‐10 were significantly higher in group 1 than in group 2. The level of IL‐10 was significantly higher in groups 1 and 3 than in group 2. However, levels of IFN‐γ and IL‐17 were not significantly different between the three groups. IL‐1β, IL‐4, and MIP‐1α were not detectable in most patients. IP‐10 and IL‐6 levels were found to show negative correlations with lymphocyte count and oxygen saturation.

Conclusions

We found higher levels of cytokines (IFN‐α, IL‐6, IP‐10) of innate immunity than those of acquired immunity in pediatric H1N1 infection. Of the cytokines found to be increased in cases with H1N1 infection, IP‐10 and IL‐6 were found to be correlated with disease severity (lymphopenia and hypoxia). IP‐10 and IL‐6 may be important markers in pediatric H1N1 infection. Pediatr Pulmonol. 2011; 46: 1233–1239. © 2011 Wiley Periodicals, Inc.

     

Induction of costimulation of human CD4 T cells by tumor necrosis factor–related apoptosis‐inducing ligand: Possible role in T cell activation in systemic lupus erythematosus

Objective

Tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) has recently been shown to induce costimulation of mouse T cells in conjunction with signals from the T cell receptor. This study was undertaken to investigate TRAIL‐induced costimulation of human T cells in order to determine the role of TRAIL‐induced T cell activation in human systemic lupus erythematosus (SLE).

Methods

An in vitro T cell stimulation system with immobilized anti‐CD3 and recombinant TRAIL receptor DR4‐Fc proteins was used to activate human T cells purified from healthy individuals and from patients with SLE. The T cells were stimulated in vitro to assay their proliferation response by ³H‐thymidine incorporation, and their cytokine production by enzyme‐linked immunosorbent assay. Activation of p38 MAPK after TRAIL stimulation was detected with specific anti–phospho‐p38 MAPK monoclonal antibodies in Western blots.

Results

Enhanced T cell proliferation and increased interleukin‐2 and interferon‐γ (IFNγ) production were demonstrated in human T cells after stimulation with immobilized DR4‐Fc and anti‐CD3 in vitro. TRAIL engagement selectively activated human CD4, rather than CD8, T cells and augmented IFNγ production. Activation of p38 MAPK was detected after TRAIL‐induced T cell activation. T cells isolated from patients with SLE demonstrated a stronger response to TRAIL‐induced costimulation, in terms of proliferation and increased up‐regulation of CD25 after activation, when compared with T cells from healthy subjects.

Conclusion

TRAIL engagement induces costimulation of human CD4 T cells via a p38 MAPK–dependent pathway. The results suggest that enhanced reactivity of T cells to autoantigens as a result of TRAIL‐induced costimulation may play a role in the development of human autoimmune diseases.

     

Phenotype and function of natural killer cells in systemic lupus erythematosus: Excess interferon‐γ production in patients with active disease

Objective

To determine the phenotype and the functionality of natural killer (NK) cells in patients with systemic lupus erythematosus (SLE).

Methods

A total of 94 patients with SLE (91 women and 3 men) were compared with 26 healthy controls. Active SLE was defined by an SLE Disease Activity Index score ≥4. Immunologic tests were performed using nonactivated and/or interleukin‐2 (IL‐2)–activated peripheral blood mononuclear cells. NK cell phenotype was determined by flow cytometry. NK cell natural cytotoxicity and antibody‐dependent cellular cytotoxicity (ADCC) were determined by ⁵¹Cr release and CD107a degranulation experiments. Intracellular interferon‐γ (IFNγ) production by NK cells was evaluated after overnight stimulation with IL‐12 and IL‐18. IFNα levels were assessed using an antiviral cytopathic bioassay.

Results

The absolute NK cell count was decreased in patients with active SLE, but the relative frequencies of total CD3−CD56^bright NK cells and CD3−CD56^dim NK cells were unaffected. The CD3−CD56^dim NK cells in patients with active SLE displayed unique phenotypic characteristics, including significant increases in CD69 and NKG2A and decreased expression of Fcγ receptor type IIIa/CD16, CD8α, and the killer cell immunoglobulin‐like receptor (KIR) KIR2DL1/KIR2DS1. Concomitant with these findings, NK cells from SLE patients had lower cytotoxicity but a normal level of ADCC compared with NK cells from healthy controls. There was a significant positive correlation between the increased level of IFNα in the serum and the enhanced frequency of IFNγ+ cells in patients with active SLE (r = 0.370, P = 0.04).

Conclusion

NK cells in patients with active SLE display phenotypic and functional features associated with activation. Furthermore, NK cells from patients with active SLE have the capacity to produce large amounts of IFNγ. This could contribute to the dysregulation of the link between innate and adaptive immunity seen in SLE.

     

Systemic lupus erythematosus monocytes are less responsive to interleukin‐10 in the presence of immune complexes

Objective

Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. The level of interleukin‐10 (IL‐10), predominantly an antiinflammatory cytokine, is paradoxically elevated in patients with SLE. The aim of this study was to examine the hypothesis that the antiinflammatory function of IL‐10 is impaired in monocytes from patients with SLE with long‐term exposure to immune complexes.

Methods

CD14+ monocytes were isolated from healthy donors and patients with SLE. Cultured CD14+ cells were treated with heat‐aggregated human IgG (325 μg/ml) in the presence or absence of IL‐10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL‐10 signaling, cell lysates were obtained from CD14+ cells treated with human IgG (325 μg/ml) for 1 hour followed by IL‐10 (20 ng/ml) treatment for 10 minutes. Western blot analysis was used to assess STAT‐3 phosphorylation. All experiments were performed in pairs.

Results

When stimulated with human IgG, SLE monocytes produced more tumor necrosis factor α (TNFα) and IL‐6 than did control cells. The suppressive effect of IL‐10 on human IgG–induced TNFα and IL‐6 production was lower in SLE monocytes compared with control monocytes, although IL‐10 receptor expression was similar in SLE and control monocytes. Human IgG suppressed IL‐10 receptor expression and altered IL‐10 signaling in control monocytes. Like SLE monocytes, interferon‐α (IFNα)–primed control monocytes stimulated with human IgG were also less responsive to IL‐10.

Conclusion

Human IgG and IFNα modulate IL‐10 function. In SLE monocytes, which are considered to be IFNα primed and persistently exposed to immune complexes, responses to IL‐10 are abnormal, limiting the antiinflammatory effect of this cytokine.

     

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

Objective

To investigate interferon‐γ (IFNγ) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNγ receptor (IFNγR) expression, STAT‐1 expression and phosphorylation, and the regulation of IFNγ‐inducible genes.

Methods

Fluorocytometry was used to investigate expression of STAT‐1, pSTAT‐1, CD95, HLA–DR, class I major histocompatibility complex (MHC), IFNγ‐inducible 10‐kd protein (IP‐10), monokine induced by IFNγ (Mig), and IFNγR in PBMCs from SLE patients and healthy individuals. STAT‐1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNα or IFNγ. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNγ‐inducible genes IP‐10 and Mig shortly after preparation or after stimulation with IFNγ in monocytes.

Results

STAT‐1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN‐inducible expression of CD95 and HLA–DR. STAT‐1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNγ‐inducible genes, such as IP‐10 or Mig, was increased in SLE monocytes. While STAT‐1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNα stimulation, incubation with IFNγ induced STAT‐1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT‐1 expression upon IFNγ stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNγ was also confirmed on the mRNA level, where expression of the IFN‐inducible, STAT‐1–dependent genes IP‐10 and Mig was more efficiently increased in SLE cells. However, IFNγR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.

Conclusion

In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNγ in this disease.