Immunogenicity and loss of response to TNF inhibitors: implications for rheumatoid arthritis treatment.

The availability of monoclonal antibodies has revolutionized the treatment of an increasingly broad spectrum of diseases. Inflammatory diseases are among those most widely treated with protein-based therapeutics, termed biologics. Following the first large-scale clinical trials with monoclonal antibodies performed in the 1990s by rheumatologists and clinical immunologists, the approval of these agents for use in daily clinical practice led to substantial progress in the treatment of rheumatic diseases. Despite this progress, however, only a proportion of patients achieve a long-term clinical response. Data on the use of agents blocking TNF, which were among the first biologics introduced into clinical practice, provide ample evidence of primary and secondary treatment inefficacy in patients with rheumatoid arthritis (RA). Important issues relevant to primary and secondary failure of these agents in RA include immunogenicity, methodological problems for the detection of antidrug antibodies and trough drug levels, and the implications for treatment strategies. Although there is no strong evidence to support the routine estimation of antidrug antibodies or serum trough levels during anti-TNF therapy, these assessments might be helpful in a few clinical situations; in particular, they might guide decisions on switching the therapeutic biologic in certain instances of secondary clinical failure.

Mucin production determines sensitivity to bortezomib and gemcitabine in pancreatic cancer cells.

The prognosis of pancreatic cancer remains disappointing due to a high intrinsic resistance against chemotherapeutic agents. Standard gemcitabine therapies have improved overall survival only marginally and recently, inhibition of the proteasome by the boronic acid derivative bortezomib has been introduced as a novel therapeutic strategy for solid and hematological malignancies including pancreatic cancer. The mucus-producing pancreatic cancer cell line Capan-1 was cultured under standard conditions and treated with different concentrations of gemcitabine or bortezomib. Mucus production was suppressed by siRNA-mediated silencing of apomucin genes. Cell proliferation was determined by 3H-thymidine incorporation and apoptosis was quantified after propidium iodide staining by flow cytometry. Apoptotic cell death was confirmed by TUNEL staining, determination of mitochondrial transmembrane potential and assessment of caspase 3/7 activity. NFκB-activity was determined by EMSA. The unfolded protein response (UPR) was further investigated by PCR, Western blotting and caspase 12 activity assays. Silencing of MUC4 significantly reduced expression of mucins for up to 5 days after transfection. While native cells showed an increased sensitivity to bortezomib treatment, silenced cells were more sensitive to gemcitabine treatment. Bortezomib induces mitochondrial damage in native cells and also activates the UPR by splicing of Xbp-1 and induction of CHOP, which is significantly reduced by silencing of MUC4. Our data suggest that mucinous pancreatic cancers are more sensitive towards proteasome inhibition by bortezomib and that alternative pathways of apoptosis are involved in cell death induction, while tumor cells with a low secretory activity show a better response to gemcitabine.

Advances in rheumatology: new targeted therapeutics.

Treatment of inflammatory arthritides - including rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis - has seen much progress in recent years, partially due to increased understanding of the pathogenesis of these diseases at the cellular and molecular levels. These conditions share some common mechanisms. Biologic therapies have provided a clear advance in the treatment of rheumatological conditions. Currently available TNF-targeting biologic agents that are licensed for at east one of the above-named diseases are etanercept, infliximab, adalimumab, golimumab, and certolizumab. Biologic agents with a different mechanism of action have also been approved in rheumatoid arthritis (rituximab, abatacept, and tocilizumab). Although these biologic agents are highly effective, there is a need for improved management strategies. There is also a need for education of family physicians and other healthcare professionals in the identification of early symptoms of inflammatory arthritides and the importance of early referral to rheumatologists for diagnosis and treatment. Also, researchers are developing molecules - for example, the Janus kinase inhibitor CP-690550 (tofacitinib) and the spleen tyrosine kinase inhibitor R788 (fostamatinib) - to target other aspects of the inflammatory cascade. Initial trial results with new agents are promising, and, in time, head-to-head trials will establish the best treatment options for patients. The key challenge is identifying how best to integrate these new, advanced therapies into daily practice.

Patient global assessment in psoriatic arthritis: a multicenter GRAPPA and OMERACT study.

OBJECTIVE: During OMERACT 8, delegates selected patient global assessment (PGA) of disease as a domain to be evaluated in randomized controlled trials in psoriatic arthritis (PsA). This study assessed the reliability of the PGA, measured by means of 0-100 mm visual analog scale (VAS), and the additional utility of separate VAS scales for joints (PJA) and skin (PSA). METHODS: In total, 319 consecutive patients with PsA (186 men, 133 women, mean age 51 ± 13 yrs) were enrolled. PGA, PJA, and PSA were administered at enrolment (W0) and after 1 week (W1). Detailed clinical data, including ACR joint count, Psoriasis Area and Severity Index (PASI), and Hospital Anxiety and Depression Scale, were recorded. RESULTS: Comparison of W0 and W1 scores showed no significant variations (intraclass correlation coefficients for PGA 0.87, PJA 0.86, PSA 0.78), demonstrating the reliability of the instrument. PGA scores were not influenced by patient anxiety or depression, but were dependent on PJA and PSA (p = 0.00001). PJA was dependent on the number of swollen and tender joints (p < 0.00001). PSA scores were influenced by the extent of skin psoriasis and by hand skin involvement (p = 0.00001). Joint and skin disease were found not to correlate in terms of disease activity as evidenced by the swollen joint count compared to PASI (r = 0.11) and by the PJA compared to PSA (r = 0.38). CONCLUSION: PGA assessed by means of VAS is a reliable tool related to joint and skin disease activity. Because joint and skin disease often diverge it is suggested that in some circumstances both PJA and PSA are also assessed.

Inhibition of TNF-alpha reduces transplant arteriosclerosis in a murine aortic transplant model.

Experimental and clinical data provide evidence that TNF-alpha contributes to acute and chronic allograft rejection. In this study, we explored the effect of TNF-alpha blockade using the chimeric monoclonal antibody infliximab on the development of transplant arterisoclerosis in a fully mismatched aortic allograft model. Post-transplant treatment of CBA (H2(k)) recipients with 250 mug infliximab (cumulative dose 1.25 mg) reduced luminal occlusion of C57Bl/6 (H2(b)) aortic grafts on day 30 from 77 +/- 5% in untreated controls to 52 +/- 6%. Increasing the dose of anti-TNF-alpha antibody had no further beneficial effect. Treatment with human control immunoglobulin had no effect on intima proliferation. Under TNF-alpha blockade, ICAM-1 and PDGF mRNA expression within the grafts was strongly reduced, whereas iNOS expression was enhanced. The data show that TNF-alpha blockade using infliximab can reduce the development of transplant arteriosclerosis in fully mismatched murine aortic grafts.

Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE.

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1-nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1beta, IL-6, IL-10, and tumor necrosis factor (TNF) alpha and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2-dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1-nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis.

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

Increased cortisol relative to adrenocorticotropic hormone predicts improvement during anti-tumor necrosis factor therapy in rheumatoid arthritis.

OBJECTIVE: Some patients with chronic inflammatory diseases such as rheumatoid arthritis (RA) improve rapidly from anti-tumor necrosis factor (anti-TNF) therapy. No sensitive markers are available that might predict outcome of anti-TNF therapy. We undertook this study to investigate the predictive value of hypothalamic-pituitary-adrenal (HPA) axis hormones for clinical improvement during anti-TNF therapy. METHODS: An observational study in 23 RA patients was followed by a validation study in 38 RA patients. The patients receiving anti-TNF antibodies had no glucocorticoid treatment, and we measured baseline serum levels of adrenocorticotropic hormone (ACTH) and cortisol. Improvement during anti-TNF antibody treatment was judged by the Disease Activity Score in 28 joints (DAS28), and serum levels of cortisol were measured at followup. RESULTS: The observational study demonstrated that improvement in the DAS28 correlated negatively with baseline serum levels of cortisol (R=-0.520, P=0.011) and the cortisol:ACTH ratio (R=-0.700, P=0.0002). In the longitudinal part of the study at followup, those patients with good improvement and initially low serum levels of cortisol demonstrated an increase of serum cortisol, in contrast to patients with little or no improvement. Findings in the observational study were supported by those in the validation study in a group of RA patients with less inflammation (correlation of improvement in the DAS28 with cortisol:ACTH ratio: R=-0.320, P=0.025). CONCLUSION: This is the first study in a human chronic inflammatory disease to demonstrate that inflammation-induced TNF interferes with HPA axis integrity, which is linked to the disease outcome. These findings position the HPA axis centrally in the vicious circle of perpetuation of chronic inflammation.

Two-year efficacy and safety of infliximab treatment in patients with active psoriatic arthritis: findings of the Infliximab Multinational Psoriatic Arthritis Controlled Trial (IMPACT).

OBJECTIVE: To investigate longterm efficacy/safety of infliximab over 2 years in patients with active psoriatic arthritis (PsA). METHODS: Initially, 104 patients were randomized to receive blinded infusions of infliximab 5 mg/kg or placebo at Weeks 0, 2, 6, and 14. At Week 16, all patients received infliximab 5 mg/kg every 8 weeks through Week 46. Seventy-eight of the 87 patients completing the first year continued into the open-label longterm extension and received infliximab 5 mg/kg at Weeks 54, 62, 70, 78, 86, and 94. The primary efficacy endpoint for the study extension was the proportion of patients with at least 20% improvement in the American College of Rheumatology response criteria (ACR20) at Week 98. Radiographic progression was assessed by the PsA-modified van der Heijde-Sharp score in patients with radiographs available at baseline and Week 98 (n = 43). RESULTS: At Week 98, 62% (48/78) of infliximab-treated patients achieved an ACR20 response; 45% (35/78) and 35% (27/78) of patients achieved ACR50 and ACR70 responses, respectively. Among patients with baseline Psoriasis Area and Severity Index scores >or= 2.5, 64% (16/25) achieved > 75% improvement from baseline to Week 98. The average estimated annual radiographic progression with infliximab treatment was significantly reduced versus the estimated baseline rate of progression. No new safety issues were observed during the second year of the study. CONCLUSION: Therapy with infliximab 5 mg/kg through Week 94 produced sustained improvement in joint and skin symptoms, inhibited radiographic progression, and continued to exhibit a favorable benefit-risk ratio in this population with treatment-refractory PsA.

An open-label pilot study of the effectiveness of adalimumab in patients with rheumatoid arthritis and previous infliximab treatment: relationship to reasons for failure and anti-infliximab antibody status.

This prospective open-label pilot study evaluated the effectiveness and safety of adalimumab and the relationship to antibodies against infliximab (IFX) in adult patients with active rheumatoid arthritis (RA) who had been treated previously with IFX and experienced treatment failure owing to lack or loss of response or intolerance. Patients self-administered adalimumab 40 mg subcutaneously every other week for 16 weeks, followed by maintenance therapy for up to Week 56. Measures of effectiveness included American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) response criteria, 28-joint Disease Activity Score, and the Health Assessment Questionnaire Disability Index. Serum IFX concentrations, human antichimeric antibody against IFX (HACA), adalimumab serum concentrations, antiadalimumab antibody, and safety also were assessed. Of the 41 enrolled patients, 37 completed 16 weeks and 30 completed 56 weeks of treatment. Patients experienced clinically meaningful improvements in all measures of RA activity, with greater response rates observed for patients who had experienced loss of initial response to or intolerance of IFX. At Week 16, 46% of patients achieved an ACR20 and 28% achieved an ACR50; 61% achieved an at least moderate and 17% achieved a good EULAR response. Clinical benefit was maintained through Week 56 in all effectiveness parameters. Baseline HACA status did not significantly impact effectiveness. No new safety signals were observed; neither former IFX intolerance status nor baseline HACA status had a clinically relevant impact on adverse event frequency or severity. Adalimumab was effective and well-tolerated in patients with RA who previously failed IFX therapy, irrespective of reason for discontinuation and of HACA status.

High mobility group box 1 in the pathogenesis of inflammatory and autoimmune diseases.

High mobility group box 1 is a nuclear protein participating in chromatin architecture and transcriptional regulation. When released from cells, HMGB1 can also act as a pro-inflammatory mediator or alarmin. Upon stimulation with lipopolysaccharides or tumor necrosis factor-alpha, HMGB1 is secreted from certain cells such as monocytes/macrophages and fosters inflammatory responses. In addition, HMGB1 is passively released from necrotic cells and mediates inflammation and immune activation. In contrast, during apoptotic cell death, nuclear HMGB1 becomes tightly attached to hypo-acetylated chromatin and is not released into the extracellular milieu, thereby preventing an inflammatory response. There is accumulating evidence that extracellular HMGB1 contributes to the pathogenesis of many inflammatory diseases, including autoimmune diseases. Increased concentrations of HMGB1 have been detected in the synovial fluid of patients with rheumatoid arthritis. In animal models of RA, HMGB1 appears to be crucially involved in the pathogenesis of arthritis since neutralization of HMGB1 significantly ameliorates the disease. Also, in the serum and plasma of patients with systemic lupus erythematosus we detected substantial amounts of HMGB1, which may contribute to the disease process. However, investigations of blood concentrations of HMGB1 and its relevance in human diseases are hindered by the lack of reliable routine test systems.

Hypothesis: human serum-borne albumin bound lipids promote cellular survival after apoptosis induction by a variety of stimuli.

A tight control of proliferation and cell death is required to maintain homeostasis in multicellular organisms. Several specific pro- and anti-apoptotic regulators and pathways have been deciphered being responsible for these complex tasks. Here we describe a human serum-borne activity promoting cellular fitness and inhibiting apoptosis after a plethora of different cell death stimuli. The factor(s) do not inhibit a specific death pathway, instead it/they can be considered as general pro-survival factor(s) for cultured cells. The activity is heat stable (30 min, 96 degrees C), co-migrates with albumin in size exclusion chromatography, and is sensitive to chemical delipidation. A similar activity is observed in native, non-delipidated preparations of human albumin, while delipidated albumin is not effective. These properties point to heat stable factors that exert anti-apoptotic activities, most likely albumin bound bioactive lipids. The activity prevented Akt dephosphorylation and degradation, after apoptosis induction by staurosporine and the production of reactive oxygen species after UV-B irradiation. In conclusion human serum-enriched bioactive lipids promote survival of cultured cells overriding the pro-apoptotic effects of a variety of apoptosis inducing agents.

Expression and regulation of CCL18 in synovial fluid neutrophils of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor alpha, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.

Palifermin as treatment in dose-intense conventional polychemotherapy induced mucositis.

Polychemotherapy or radiation frequently causes oral mucositis. Until now, only supportive options are available. Palifermin, a recombinant form of the human keratinocyte growth factor can avoid mucositis in patients treated with myeloablative polychemotherapy for allogeneic stem cell transplantation. Here, we present a patient treated with dose-intense conventional polychemotherapy suffering from severe mucositis leading to additional hospitalization. By using palifermin, the mucositis as well as further admissions were totally prevented. Furthermore, application in subsequent chemotherapy cycles demonstrated a safe and efficacious use. Hence, treatment with palifermin can prevent severe mucositis in a patient treated with dose-intense conventional polychemotherapy.

Interleukin-4 supports interleukin-12-induced proliferation and interferon-gamma secretion in human activated lymphoblasts and T helper type 1 cells.

Interleukin-12 (IL-12) and IL-4 are known to differentially promote T helper (Th) cell differentiation. While IL-12 induces interferon-gamma (IFN-gamma) production and maturation of Th1 cells, IL-4 is thought to antagonize IL-12 and to favour Th2 development. Here we studied the combined action of various concentrations of common gamma-chain (gamma(c)-chain) cytokines, including IL-4 and the Th1 cytokine IL-12, in human activated lymphoblasts and Th1 cells. IL-4 and IL-7 potentiated IL-12-induced proliferation at every concentration tested (1-10 ng/ml) without increasing rescue from apoptosis, indicating that proliferation was directly affected by these cytokine combinations. With regards to cytokine secretion, IL-2 together with IL-12 initiated tumour necrosis factor-alpha synthesis, enhanced IFN-gamma production, and shedding of soluble IL-2 receptor alpha as expected. Importantly, combining IL-4 with IL-12 also enhanced IFN-gamma secretion in lymphoblasts and a Th1 cell line. Investigating signal transduction in lymphoblasts induced by these cytokines, we found that not only IL-2 but also IL-4 enhances signal transducer and activator of transcription 3 (STAT3) tyrosine phosphorylation by IL-12. Tyrosine phosphorylations of janus kinase 2 (JAK-2), tyrosine kinase 2 (TYK2), extracellular signal-regulated kinase (ERK) and STAT4, STAT5 and STAT6 were not potentiated by combinations of these cytokines, suggesting specificity for increased STAT3 phosphorylation. In conclusion, two otherwise antagonizing cytokines co-operate in activated human lymphoblasts and Th1 cells, possibly via STAT3 as a converging signal. These data demonstrate that IL-4 can directly enhance human Th1 cell function independently of its known actions on antigen-presenting cells. These findings should be of importance for the design of cytokine-targeted therapies of human Th-cell-driven diseases.

Prevention of gonadal toxicity and preservation of gonadal function and fertility in young women with systemic lupus erythematosus treated by cyclophosphamide: the PREGO-Study.

BACKGROUND: With dramatically improved survival rates of SLE patients, comorbidity and long-term damage such as premature ovarian failure (POF) gain increasing importance. In the Erlangen cohort, 14% of cyclophosphamide treated patients younger than 41 years have POF, which is a common consequence of cyclophosphamide treatment. PATIENTS AND METHODS: We tested the concentrations of FSH and LH, before, during and after cyclophosphamide treatment in 63 premenopausal women with SLE without ovarian protection and initiated the PREGO-Study (Prospective randomized study on protection against gonadal toxicity) in patients with SLE. RESULTS: In lupus patients treated with cyclophosphamide, 60% suffered from POF and hypergonadotropic amenorrhea. Whereas the POF rate was <50% in women below 30 years, it was 60% between 30 and 40 years. The cumulative dosage of cyclophosphamide also strongly influenced POF rate. CONCLUSIONS: Our present results, with a high POF rate in Cyclophosphamide treated SLE patients demonstrate the urgent need for ovarian protection in this patient group. Besides POF these women are at high risk for premature atherosclerosis which is the major cause of death in lupus. Following preliminary encouraging experience in women with lymphoma, in whom the temporary induction of a prepubertal hormonal milieu during chemotherapy, has significantly decreased the risk of POF, we have initiated the PREGO-Study, comparing randomised monthly injection versus no injection of gonadotropin-releasing hormone analogue (GnRH-a) to young SLE patients during cyclophosphamide therapy.

Involvement of phosphatidylserine, alphavbeta3, CD14, CD36, and complement C1q in the phagocytosis of primary necrotic lymphocytes by macrophages.

OBJECTIVE: Uningested dead cells may be an important source of autoantigens and may trigger autoimmune diseases such as systemic lupus erythematosus (SLE). Multiple receptors involved in the clearance of apoptotic cells have been described; however, little is known about the receptors and ligands involved in uptake of necrotic cells that release autoantigens as well. METHODS: The uptake of autologous necrotic peripheral blood lymphocytes into human monocyte-derived macrophages was qualitatively and quantitatively monitored by confocal microscopy and 2-color flow cytometry, respectively. Blocking experiments were performed to examine the receptors and molecules involved in the phagocytosis of necrotic cells. Cytokine secretion by lipopolysaccharide-activated monocytes and macrophages was determined by enzyme-linked immunosorbent assay. RESULTS: Phosphatidylserine, which was exposed on necrotic as well as apoptotic cells, promoted the recognition and removal of primary necrotic lymphocytes. Several macrophage receptor systems, including the thrombospondin-CD36-alphavbeta3 complex, CD14, and the complement component C1q, contributed to the engulfment of necrotic cells. Necrotic peripheral blood lymphocytes slightly increased the lipopolysaccharide-induced secretion of interleukin-10 and reduced the secretion of tumor necrosis factor alpha in monocytes and macrophages. CONCLUSION: Our results indicate that at least some of the receptors and adaptors mediating the uptake of apoptotic cells are also involved in the clearance of necrotic cells. Hence, necrotic cells engage phagocyte receptors such as CD36, which mediate antiinflammatory signals from apoptotic cells. Necrotic cells consequently also have the potency to provide antiinflammatory signals to phagocytes; however, these signals may be overridden by proinflammatory factors released during necrosis. These findings have implications regarding the etiopathogenesis of autoimmune diseases such as SLE, in which impaired clearance of dead cells may foster autoimmunity by the release of potential autoantigens.

CTLA-4 lacking the cytoplasmic domain costimulates IL-2 production in T-cell hybridomas.

Optimal T-cell activation depends on the antigen-specific signal mediated by the TCR and engagement of costimulatory receptors such as CD28. CTLA-4, a homologous counterpart of CD28, is considered to be a crucial inhibitory receptor. To test its function separately from CD28 in an antigen-driven and ligand-specific model, we stably transfected the T-cell hybridomas A1.1 and DO11.10, which lack significant endogenous CD28 or CTLA-4 expression, with wild-type CTLA-4 (CTLA-4 WT) and a construct lacking the cytoplasmic tail (tailless [TL]). Functional studies were carried out by co-incubation with APC expressing the B7 ligands for CTLA-4 and appropriate MHC molecules loaded with their cognate antigens. IL-2 production on costimulation of CTLA-4WT and TCR did not differ significantly from untransfected controls. However, coligation of TCR and CTLA-4TL resulted in a vigorous IL-2 response specific for the interaction of CTLA-4 with B7. Thus, lack of the cytoplasmic tail converted CTLA-4 into a costimulatory receptor. This indicates that the CTLA-4 inhibitory function may not be attributable to sequestration of the common B7 ligands when competing with CD28. Rather, ligation of B7 by the CTLA-4 extracellular domain can enhance TCR activation, whereas in the full-length receptor, inhibitory signals mediated by the cytoplasmic domain may override this activation.

Extravasation into synovial tissue induces CCL20 mRNA expression in polymorphonuclear neutrophils of patients with rheumatoid arthritis.

OBJECTIVE: Examination of expression of the chemokine macrophage inflammatory protein-3a (CCL20/Mip-3alpha) in blood polymorphonuclear neutrophils (PMN) and synovial fluid (SF) PMN of patients with rheumatoid arthritis (RA). METHODS: Paired samples of blood PMN and SF PMN were obtained from 11 patients with RA. In addition, SF was prepared from 9 patients with osteoarthritis (OA) and 10 patients with juvenile idiopathic arthritis (JIA). PMN were isolated via density centrifugation to a purity of 98%. Total RNA was isolated and the expression of CCL20 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In some experiments blood PMN obtained from healthy donors were stimulated with individual SF of patients with RA. For quantitative considerations, CXCL8, CCL20, interleukin 1, and tumor necrosis factor-alpha (TNF-alpha) levels were determined in SF by ELISA. RESULTS: In SF of RA patients CCL20 and CXCL8 levels were elevated, up to 7.5 ng/ml and 23.6 ng/ml, respectively. No significant level of either chemokine was found in SF of patients with JIA and OA. CCL20 mRNA was undetectable in blood PMN of all patients with RA. In SF PMN, CCL20 mRNA was found in 6/11 RA patients. Expression of CCL20 mRNA in 5/6 SF PMN samples was observed in the absence of detectable TNF-alpha levels in SF. Cell culture experiments, however, confirmed the ability of TNF-alpha in SF to induce CCL20 mRNA expression in blood PMN. Notably, expression of CCL20 was also found in PMN after stimulation with SF lacking TNF-alpha. CONCLUSION: Recruitment of PMN to the synovial microenvironment induces expression of CCL20 mRNA independent of the concentrations of TNF-alpha accumulating in SF of patients with RA.