The role of neutrophil apoptosis in juvenile‐onset systemic lupus erythematosus

Objective

Accumulation of apoptotic cells may lead to the development of systemic lupus erythematosus (SLE) through a breakdown in immune tolerance. Altered neutrophil apoptosis may contribute to nuclear autoantigen exposure, ultimately leading to autoantibody generation. This study aimed to determine whether neutrophil apoptosis is altered in patients with juvenile‐onset SLE as compared with controls.

Methods

Apoptosis was measured in neutrophils from patients with juvenile‐onset SLE (n = 12), adult‐onset SLE (n = 6), and pediatric patients with inflammatory (n = 12) and noninflammatory (n = 12) conditions. Annexin V staining and flow cytometry were used to determine neutrophil apoptosis. Proapoptotic and antiapoptotic proteins were measured in sera and in neutrophil cell lysates.

Results

Neutrophil apoptosis was significantly increased in patients with juvenile‐onset SLE as compared with the noninflammatory controls at time 0. Incubation of neutrophils with sera from patients with juvenile‐onset SLE further increased neutrophil apoptosis as compared with incubation with sera from pediatric controls. Concentrations of TRAIL and FasL were significantly increased in sera from patients with juvenile‐onset SLE, whereas interleukin‐6, tumor necrosis factor α, and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) were significantly decreased. Addition of GM‐CSF to sera from patients with juvenile‐onset SLE significantly decreased neutrophil apoptosis as compared with juvenile‐onset SLE sera alone. The expression of proapoptotic proteins (caspase 3, Fas, and FADD) was elevated in juvenile‐onset SLE neutrophils, whereas the expression of antiapoptotic proteins (cellular inhibitor of apoptosis 1 and 2 and X‐linked inhibitor of apoptosis) was decreased. Neutrophil apoptosis correlated with biomarkers of disease activity (erythrocyte sedimentation rate and double‐stranded DNA concentration) and the British Isles Lupus Assessment Group disease activity score.

Conclusion

Our data demonstrate an imbalance in proapoptotic and antiapoptotic factors in both neutrophils and sera from patients with juvenile‐onset SLE. This imbalance results in increased neutrophil apoptosis in these patients. Correlations with markers of disease activity indicate that altered neutrophil apoptosis in juvenile‐onset SLE patients may play a pathogenic role in this condition.

     

系统性红斑狼疮患者外周血TB淋巴细胞Fas FasL表达及与凋亡的关系研究

目的探讨系统性红斑狼疮(SLE)患者外周血T、B淋巴细胞Fas、FasL表达及与凋亡的关系.方法采用流式细胞术(FCM)检测了30例SLE活动期患者、30例SLE非活动期患者和30名正常人外周血T、B淋巴细胞的凋亡率及Fas、FasL表达率.结果T、B淋巴细胞的Fas表达率依次为:活动期组＞非活动期组＞正常对照组(P＜0.01);FasL的表达率除B淋巴细胞在SLE活动期显著高于正常对照组外(P＜0.01),其余各组间差异无统计学意义;细胞的凋亡率在活动期显著高于非活动期(P＜0.01),而非活动期与正常对照组差异无统计学意义(P＞0.05):SLE的活动性与T或B细胞的Fas、FasL表达率间无相关关系,而与T或B细胞的凋亡率呈正相关(P＜0.01);无论是T或B细胞,Fas、FasL的表达率与凋亡率间无相关性.结论SLE患者体内外周血淋巴细胞的凋亡受到多种因素影响,但最终仍表现为凋亡率的异常增高,特别是活动期SLE,推测外周血淋巴细胞凋亡率的增高在SLE发病机制中起着重要作用.     

Increased Fas ligand expression of peripheral B‐1 cells correlated with CD4+ T‐cell apoptosis in filarial‐infected patients

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen‐presenting cells (APC) dysfunction, increased interleukin‐10(IL‐10), regulatory T cells and induction of CD4⁺ T (Th)‐cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B‐1 cells and induction of apoptosis of Th cells by FasL‐expressing B‐1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th‐cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th‐cell population and FasL‐expressing B‐1 cells in different clinical categories of filariasis. The peripheral apoptotic T‐helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B‐1 cells increased in filarial patients and positively correlated with peripheral apoptotic T‐helper cells indicating FasL‐expressing B‐1 cells may be one of the important mediators of Th‐cell apoptosis and immune anergy during filarial pathology.     

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‐[¹⁸F]‐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.

     

Copy number variations of interleukin‐17F,interleukin‐21, and interleukin‐22 are associated with systemic lupus erythematosus

Objective

Systemic lupus erythematosus (SLE) represents the classic prototype of systemic autoimmune disease. The identification of the Th17 cell subset has provided new understanding regarding the underlying mechanisms of autoimmunity. Copy number variations (CNVS ) have been discovered to have phenotypic consequences and are associated with various types of diseases. We undertook this study to explore a possible association between CNVS of Th17 cell–related genes and the risk of SLE.

Methods

We extracted genomic DNA and RNA from 938 SLE patients and 1,017 healthy controls. We examined CNVS of Th17 cell–related genes, including retinoic acid receptor–related orphan nuclear receptor γt, STAT‐3, interleukin‐6 (IL‐6), transforming growth factor β, tumor necrosis factor α, IL‐17A, IL‐17F, IL‐21, IL‐22, IL‐23A, CCL20, and CCR6, and levels of messenger RNA (mRNA) for IL‐17F, IL‐21, and IL‐22.

Results

Genotype and allele frequencies for copy number amplifications of IL‐17F, IL‐21, and IL‐22 were found to be significantly higher in SLE patients than in healthy controls. CNVS of IL‐17F, IL‐21, and IL‐22 had no synergistic contribution to SLE. The mRNA expression of IL‐17F, IL‐21, and IL‐22 in the samples with >2 copies of DNA was significantly higher than that in those with 2 copies of DNA.

Conclusion

Our findings indicate that CNVS of IL‐17F, IL‐21, and IL‐22 are associated with the risk of SLE.

     

Rapid responses to anakinra in patients with refractory adult‐onset Still's disease

Objective

To assess the efficacy of anakinra treatment in patients with adult‐onset Still's disease (AOSD) that is refractory to corticosteroids, methotrexate (MTX), and etanercept.

Methods

Four patients with AOSD were treated with prednisone and MTX and 2 patients were also treated with etanercept for worsening symptoms and indicators of systemic inflammation. White blood cells (WBCs), C‐reactive protein (CRP) levels and/or erythrocyte sedimentation rate, and ferritin levels were measured and, in 1 patient, serum creatinine levels were determined. Treatment with anakinra at 100 mg/day was initiated.

Results

The index patient's disease was refractory to treatment with prednisone (30 mg/day) and MTX, with spiking fevers, rash, synovitis, a serum ferritin level of 8,400 ng/ml (normal ≤200), and a CRP level of 86 mg/liter (normal <8). Levels of interleukin‐1β (IL‐1β), IL‐1α, IL‐6, IL‐1 receptor antagonist, and IL‐18 were elevated. Just prior to anakinra treatment, the WBC count was 14,600/mm³, the CRP level was 86 mg/liter, and the ferritin level was 573 ng/ml, with daily spiking fevers to 104°F, rash, and swollen joints. Within hours of the first injection, the patient was afebrile and asymptomatic; within days, the WBC count, ferritin level, and CRP level decreased into the normal range. On 2 occasions, anakinra was withheld. Within a few days, the WBC count rose to >20,000/mm³ with prominent neutrophilia, the CRP level rose to >200 mg/liter, and the ferritin level rose to >3,000 ng/ml. Upon restarting anakinra, the patient became afebrile, the WBC count fell to 8,000/mm³, the CRP level fell to <3 mg/liter, and the ferritin level fell to <300 ng/ml. Three additional patients with refractory AOSD who experienced rapid reductions in fever, symptoms, and markers of inflammation when treated with anakinra are reported.

Conclusion

Refractory AOSD appears to be IL‐1–mediated since anakinra decreases hematologic, biochemical, and cytokine markers and also produces rapid reductions in systemic and local inflammation. Reported efficacy of tumor necrosis factor–blocking therapies in AOSD may be due to a reduction in IL‐1.

     

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.

     

Apoptosis‐induced acetylation of histones is pathogenic in systemic lupus erythematosus

Objective

In systemic lupus erythematosus (SLE), inadequate removal of apoptotic cells may lead to challenge of the immune system with immunogenic self antigens that have been modified during apoptosis. We undertook this study to evaluate whether apoptosis‐induced histone modifications are targets for the immune system in SLE.

Methods

The epitope of KM‐2, a monoclonal antihistone autoantibody derived from a lupus mouse, was mapped by random peptide phage display. The reactivity of KM‐2 and plasma with (acetylated) histone H4 (H4) peptides and with nonapoptotic, apoptotic, and hyperacetylated histones was determined by immunofluorescence staining, enzyme‐linked immunosorbent assay, and Western blotting.

Results

KM‐2 recognized apoptosis‐induced acetylation of H4 at lysines 8, 12, and 16. The majority of plasma samples from SLE patients and lupus mice showed higher reactivity with triacetylated H4 peptide (residues 1–22) and with hyperacetylated and apoptotic histones than with nonacetylated H4 peptide and normal histones. Importantly, administration of triacetylated H4 peptide to lupus‐prone mice before disease onset accelerated the disease by enhancing mortality and aggravating proteinuria, skin lesions, and glomerular IgG deposition, while the nonacetylated H4 peptide had no pathogenic effect. The delayed‐type hypersensitivity response in lupus mice against the triacetylated H4 peptide was higher than that against the nonacetylated H4 peptide. Bone marrow–derived dendritic cells (DCs) cultured in the presence of hyperacetylated nucleosomes showed increased expression/production of CD40, CD86, interleukin‐6 (IL‐6), and tumor necrosis factor α compared with DCs cultured in the presence of normal nucleosomes. Finally, DCs cultured in the presence of hyperacetylated nucleosomes were able to activate syngeneic T cells, because IL‐2 production increased.

Conclusion

Apoptosis‐induced acetylation of nucleosomes may represent an important driving force in the development of lupus.

     

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.

     

Extracellular calreticulin is present in the joints of patients with rheumatoid arthritis and inhibits FasL (CD95L)–mediated apoptosis of T cells

Objective

The binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL‐induced apoptosis in vivo but are susceptible to FasL‐induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL–Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system.

Methods

Human Jurkat T cells rendered apoptotic by FasL exposure were analyzed by flow cytometry. Necrosis was determined according to measurement of lactate dehydrogenase release. Quantification of calreticulin in plasma and synovial fluid and of calreticulin–FasL binding was performed by enzyme‐linked immunosorbent assay. Measurement of nitrite/nitrate in the plasma and synovial fluid was carried out by chemiluminescence assay.

Results

Extracellular calreticulin was present at a significantly higher concentration in the plasma (median 10.3 ng/ml, interquartile range [IQR] 14.8 ng/ml) and synovial fluid (median 10.3 ng/ml, IQR 12.0 ng/ml) of RA patients (each P < 0.05) compared with the plasma (median 3.1 ng/ml, IQR 1.3 ng/ml) and synovial fluid (median 2.9 ng/ml, IQR 0.9 ng/ml) of patients with psoriatic arthritis and the plasma of healthy control subjects (median 2.9 ng/ml, IQR 0.9 ng/ml). Calreticulin concentrations in the synovial fluid correlated with the tender and swollen joint counts and the activity scores on the 28‐joint Disease Activity Score assessment. Calreticulin also bound directly to FasL. In vitro, calreticulin (2–16 ng/ml) inhibited FasL‐induced apoptosis of Jurkat T cells.

Conclusion

Calreticulin was present at higher concentrations in the plasma and synovial fluid of RA patients. Calreticulin had the capacity to bind directly to FasL and to inhibit FasL‐mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA.

     

Expansion of toll‐like receptor 9–expressing B cells in active systemic lupus erythematosus: Implications for the induction and maintenance of the autoimmune process

Objective

Toll‐like receptors (TLRs) are pattern‐associated receptors in innate immunity that may be involved in the recognition of self antigens and the production of pathogenic autoantibodies. This study was undertaken to examine the expression and function of various TLRs in subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE).

Methods

The expression of TLRs in PBMCs from 50 SLE patients with active disease (SLE Disease Activity Index [SLEDAI] score ≥8; n = 26) or inactive disease (SLEDAI score <8; n = 24) and 20 healthy controls was studied by flow cytometry. TLR expression was assessed on various subpopulations of PBMCs (TLR‐2 and TLR‐4 by membrane staining; TLR‐3 and TLR‐9 by intracellular staining). TLR function was accessed by stimulating PBMCs with specific ligands.

Results

The proportion of B cells and monocytes expressing TLR‐9 was higher among patients with active SLE (mean ± SD 49.5 ± 24.4% and 30.7 ± 24.1%, respectively) than among patients with inactive disease (22.8 ± 19.6% and 14.3 ± 8.4%, respectively; P = 0.02 and P = 0.03). Among B cells, the proportion of plasma cells and memory B cells expressing TLR‐9 was increased in patients with active SLE. Increased percentages of TLR‐9–expressing B cells correlated with the presence of anti–double‐stranded DNA antibodies (P = 0.007). Treatment with serum from patients with active disease increased the percentage of TLR‐9–expressing plasma cells in serum from healthy controls. Enhanced induction of HLA–DR after TLR‐9 stimulation was documented in B cells from patients with active disease.

Conclusion

In patients with active SLE, the proportion of peripheral blood memory B cells and plasma cells expressing TLR‐9 is increased. Endogenous nucleic acids released during apoptotic cell death may stimulate B cells via TLR‐9 and contribute to SLE pathogenesis.

     

Outside‐to‐inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin‐1β production and deficient apoptosis of rheumatoid arthritis monocytes

Objective

Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and inhibitors of monocytic cytokines are highly efficient agents for treatment of the disease. The aim of this study was to analyze the effects of a therapeutic anti–tumor necrosis factor α (anti‐TNFα) antibody on monocytes from patients with RA and healthy control subjects.

Methods

Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti‐TNFα antibody or IgG. Annexin V staining, caspase activation, poly(ADP‐ribose) polymerase cleavage, and DNA staining with propidium iodide were used to analyze apoptosis. The signaling events elicited in monocytes by infliximab were analyzed by Western blotting and electromobility shift assay.

Results

Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNFα, spontaneous in vitro production of interleukin‐1β (IL‐1β), and a decreased rate of spontaneous ex vivo apoptosis. Incubation with infliximab induced significantly increased apoptosis in monocytes from patients with RA but not in monocytes from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after ligation by infliximab and was independent of caspase activation. Instead, transmembrane TNF reverse signaling inhibited the constitutive NF‐κB activation in RA monocytes, suppressed IL‐1β secretion, and normalized spontaneous in vitro apoptosis. This normalization was reversible by the addition of exogenous IL‐1β.

Conclusion

This study demonstrates that outside‐to‐inside signaling through transmembrane TNF after ligation by infliximab inhibits constitutive NF‐κB activation and suppresses spontaneous IL‐1β production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects observed during treatment with TNFα inhibitors.

     

Targeting interleukin‐15 in patients with rheumatoid arthritis: A proof‐of‐concept study

Objective

Interleukin‐15 (IL‐15) is a proinflammatory, innate response cytokine that mediates pleiotropic effector function in rheumatoid arthritis (RA) inflammatory synovitis. Our objective was to study the ability of HuMax‐IL15, a human IgG1 anti–IL‐15 monoclonal antibody, to neutralize exogenous and endogenous IL‐15 activity in vitro and to perform a phase I–II dose‐escalation trial with HuMax‐IL15 in patients with active RA.

Methods

Mononuclear cells from blood and synovial fluid (SF) of RA patients were isolated and cultured in vitro under experimental conditions involving the addition of HuMax‐IL15. HuMax‐IL15 was administered to 30 RA patients who received no other disease‐modifying antirheumatic drugs in a 12‐week, dose‐ascending, placebo‐controlled, double‐blind, phase I–II proof‐of‐concept study.

Results

In vitro studies showed that HuMax‐IL15 suppressed proliferation and induced apoptosis in an IL‐15–dependent cell line, BDB2, and was capable of suppressing the release of interferon‐γ by synovial fluid mononuclear cell (SFMC) cultures induced by exogenous IL‐15. Furthermore, HuMax‐IL15 F(ab′)₂ fragments suppressed exogenous IL‐15–induced CD69 expression in RA peripheral blood mononuclear cells and SFMCs, which indicates that HuMax‐IL15 can specifically neutralize several biologic effects of IL‐15 in synovial tissue in vitro. In a phase I–II clinical trial, HuMax‐IL15 was well tolerated clinically, with no significant effects on T lymphocyte subset and natural killer cell numbers. Substantial improvements in disease activity were observed according to the American College of Rheumatology criteria for 20% improvement (63% of patients), 50% improvement (38%), and 70% improvement (25%).

Conclusion

These clinical data suggest for the first time that IL‐15 could represent a novel therapeutic target in RA.

     

Interleukin‐18 enhances monocyte tumor necrosis factor α and interleukin‐1β production induced by direct contact with T lymphocytes: Implications in rheumatoid arthritis

Objective

At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up‐regulation of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL‐18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA).

Methods

Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL‐1β, IL‐10, and IL‐18 were measured by enzyme‐linked immunosorbent assay. Expression of adhesion molecules, IL‐18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF‐κB p65, phosphorylated IκBα, and phosphatidylinositol 3‐kinase (PI 3‐kinase) p110 was analyzed by Western blotting.

Results

IL‐18 dose‐dependently enhanced the production of IL‐1β and TNFα, but not IL‐10, by monocytes following contact with RA synovial T cells or PHA‐prestimulated T cells. NF‐κB inhibitors N‐acetyl‐L ‐cysteine and Bay 11‐7085 and PI 3‐kinase inhibitor LY294002 inhibited the enhancing effects of IL‐18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF‐κB in the nucleus, phosphorylated IκB, and PI 3‐kinase were confirmed in monocytes cocultured with PHA‐prestimulated T cells, and the levels were further increased by stimulation with IL‐18. Neutralizing antibody to IL‐18 inhibited monocyte activation induced by direct contact with PHA‐prestimulated T cells. Via cell–cell contact, PHA‐prestimulated T cells increased autocrine production of IL‐18 by monocytes, which was mediated by activation of the NF‐κB and PI 3‐kinase pathways, and up‐regulated the expression of the IL‐18 receptor in monocytes. IL‐18 up‐regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) on monocytes. Blocking the binding of the TNF receptors VCAM‐1 or ICAM‐1 on monocytes to their ligands on stimulated T cells suppressed the IL‐18–enhanced production of TNFα and IL‐1β in monocytes induced by contact with PHA‐prestimulated T cells.

Conclusion

IL‐18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF‐κB and PI 3‐kinase pathways. IL‐18 up‐regulates the expression of the TNF receptors VCAM‐1 and ICAM‐1 on monocytes, which mediate the enhancing effects of IL‐18 on T cell–monocyte contact.