Untersuchungen zur differentiellen Wirkung von verschiedenen Anti-CD4 monoklonalen Antikörpern auf T-Zellen

CD4+-T-Helferzellen sind in großer Zahl in der entzündeten Synovialmembran bei rheumatoider Arthritis (RA) sowie in Arthritismodellen vorhanden und spielen mit großer Wahrscheinlichkeit eine bedeutende Rolle in der Pathogenese von Arthritiden. Bei der präventiven Behandlung mit drei verschiedenen Anti-CD4 monoklonalen Antikörpern (mAk) im Modell der Adjuvansarthritis der Ratte (AA) wurden abhängig von dem jeweils eingesetzten mAk unterschiedliche klinische Verbesserungen beobachtet. Im Mittelpunkt der Untersuchungen stand deshalb die Suche nach Parallelen zwischen der unterschiedlichen klinischen Effizienz der Anti-CD4 mAk W3/25, OX35 (klinisch effizient) und RIB5/2 (klinisch ineffizient) bei der präventiven Therapie der AA und ihren in vitro Effekten auf TZell-Funktionen als Erklärung für die unterschiedlichen Therapieeffekte. Keine klaren Parallelen zur differentiellen klinischen Effizienz ergaben sich bei den folgenden Untersuchungen: 1.) Bestimmung der Affinitäten der mAk zum CD4-Molekül. 2.) Inhibition der Proliferation in der primären gemischten Lymphozytenkultur (1° MLC) mit CD4+-T-Zellen und CD8+-T-Zellen durch die drei mAk 3.) Beeinflussung der Produktion der Zytokine IL-2, IFNg, IL-10 und IL-4 in verschiedenen experimentellen Ansätzen (sekundäre MLC nach Anwesenheit der mAk in der 1° MLC, Kreuzvernetzung des CD4-Moleküls mittels der mAk nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR). 4.) Einfluss der drei Anti-CD4 mAk auf die TZR-vermittelte Apoptose. 5.) Mobilisierung von intrazellulärem Kalzium durch CD4-Kreuzvernetzung mittels der mAk. 6.) Aktivität der Tyrosinkinasen p56lck und p59fyn nach CD4-Kreuzvernetzung mittels der mAk. 7) Phosphorylierung des Shc-Adaptermoleküls durch CD4-Kreuzvernetzung mittels der drei mAk. 8.) Effekte der drei mAk auf die Aktivität der Transkriptionsfaktoren NF-AT und AP-1. Dagegen ergaben sich bei den Untersuchungen zur Produktion von TNFa und zur Aktivität des Transkriptionsfaktors NF-kB eindeutige Parallelen zur differentiellen klinischen Effizienz: 1.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR induzierte eine signifikant höhere Sekretion von TNFa als mit den mAk W3/25 und OX35. 2.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 vor einer Stimulation von CD4+-T-Zellen über den TZR führte zu einer signifikant stärkeren Erhöhung der Aktivität von NF-kB als mit den mAk W3/25 und OX35. Beide differentiellen Effekte könnten daher die Erklärung für die unterschiedliche klinische Effizienz der drei Anti-CD4 mAk darstellen

Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-β pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-β1, its receptor TGFBR1, the TGF-β binding proteins LTBP1/2, the TGF-β-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-β2 was downregulated. Upregulation of TGF-β1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-β2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-β1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-β-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-β pathway in RA SFBs, stimulation with TGF-β1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-β pathway. The abundance of TGF-β, in conjunction with an augmented mRNA and/or protein expression of TGF-β-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-β-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling

Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies

Expression of cytokine mRNA and protein in joints and lymphoid organs during the course of rat antigen-induced arthritis

Cytokine expression was assessed during antigen-induced arthritis (AIA) in synovial membrane (SM), inguinal lymph node (LN), and spleen using competitive RT-PCR and sandwich ELISA. In the SM, early elevations of IL-1β and IL-6 mRNA (by 6 hours; 450- and 200-fold, respectively) correlated with the joint swelling; a 6-fold increase in tumor necrosis factor α (TNFα) was not significant. Not only IL-2 and IFN-γ (which increased 10,000-fold and 200-fold, respectively), but also IL-5 and IL-10, increased acutely (6 hours – day 1; 3-fold and 35-fold, respectively) in the SM. In general, the protein levels in the SM for IL-1β, IL-6, TNFα, IFN-γ, IL-4, and IL-10 (increase from 4-fold to 15-fold) matched the course of mRNA expression. In the inguinal LN, there were early mRNA elevations of IL-6 (a 2.5-fold increase by 6 hours, which correlated positively with the joint swelling) and IL-2 (4-fold by 6 hours), as well as later rises of IL-4 and IL-5 (2.5- and 4-fold, respectively, by day 3). No significant elevations of the corresponding proteins in this tissue were observed, except for IL-1β (by day 6) and IL-10 (by day 1). In the spleen, there were significant mRNA elevations at 6 hours of IL-1β (1.5-fold), IL-6 (4-fold; positively correlated with the joint swelling), IFN-γ (3-fold), and IL-2 (7- to 10-fold). IL-5 and IL-10 (2- and 3-fold, respectively) peaked from 6 hours to day 3 in the spleen. Increases of the corresponding proteins were significant in comparison with day 0 only in the case of IL-2 (day 6). By day 6 (transition to the chronic phase), the mRNA for cytokines declined to or below prearthritis levels in all the tissues studied except for IL-1β in the SM and IL-6 in the spleen. AIA is thus characterized by four phenomena: early synovial activation of macrophages, T helper (Th)1-like, and Th2-like cells; late, well-segregated Th2-like responses in the inguinal LN; late, overlapping Th1-like/Th2-like peaks in the spleen; and chronic elevation of synovial IL-1β mRNA and spleen IL-6 mRNA

Association of Human FOS Promoter Variants with the Occurrence of Knee-Osteoarthritis in a Case Control Association Study

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2–3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2–2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA

Association of Human FOS Promoter Variants with the Occurrence of Knee-Osteoarthritis in a Case Control Association Study

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2-3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2-2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.Peer reviewe

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

Introduction The objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes. Methods Efficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry. Results Liposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug. Conclusions This new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P