Elke Kunisch

Detection of Oncofetal H19 RNA in Rheumatoid Arthritis Synovial Tissue

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

IL-33 regulates TNF-α dependent effects in synovial fibroblasts.

The recently described IL-33 acts as a pro-inflammatory cytokine, inducing the expression of multiple responses in the target cells. Although a nuclear localization of IL-33 has been described, its exact functional relevance is presently unknown. The present study was conducted to analyze the effects of IL-33 on the TNF-α induced synthesis of the pro-inflammatory mediators IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the pro-destructive molecules matrix metalloproteinase-1 (MMP-1), MMP-3, and TIMP-1 of rheumatoid arthritis synovial fibroblast (RA-SFs) using RNA overexpression and silencing. TNF-α significantly induced IL-33 mRNA expression and protein synthesis in RA-SFs. TNF-α-induced IL-33 protein expression was mediated via p38 signaling. Immunohistochemistry for IL-33 clearly showed that nuclear translocation of IL-33 was induced in TNF-α stimulated RA-SFs. IL-33 overexpression enhanced TNF-α-induced pro-inflammatory and pro-destructive functions in RA-SFs. IL-33 silencing significantly downregulated TNF-α-induced pro-inflammatory functions, whereas TNF-α-induced pro-destructive functions were less influenced by IL-33 silencing. This study identifies IL-33 as a critical regulator/enhancer of TNF-α-induced functions in RA-SFs, pointing to a central role of this cytokine in the perpetuation of pro-inflammatory and pro-destructive processes in rheumatoid arthritis (RA) and other inflammatory and degenerative diseases.

Prostaglandin E2 Differentially Modulates Proinflammatory/Prodestructive Effects of TNF-α on Synovial Fibroblasts via Specific E Prostanoid Receptors/cAMP

The present study investigated the influence of PGE2, E prostanoid (EP) receptors, and their signaling pathways on matrix metalloproteinase (MMP)-1 and IL-6 expression in synovial fibroblasts (SFs) from rheumatoid arthritis (RA) patients. RASFs expressed all four EP receptors, with selective induction of EP2 by TNF-α. TNF-α time-dependently increased intracellular cAMP/protein kinase A signaling (maximum, 6–12 h) and PGE2 secretion (maximum, 24 h). PGE2 and the EP2 agonists butaprost or ONO-AE1-259 ((16)-9-deoxy-9β-chloro-15-deoxy-16-hydroxy-17,17-trimethylene-19,20-didehydro PGE1), in turn, induced a rapid, time-dependent (maximum, 15–30 min) increase of cAMP. Additionally, cyclooxygenase-2 inhibition by NS-398 (N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide) reduced the TNF-α-induced increase in IL-6 mRNA/protein, which was restored by stimulation with PGE2 or EP2, EP3, and EP4 agonists. In contrast, TNF-α-induced MMP-1 secretion was not influenced by NS-398 and diminished by PGE2 via EP2. Finally, 3-isobutyl-1-methylxanthine enhanced the effects of PGE2 on MMP-1, but not on IL-6 mRNA. In conclusion, PGE2 differentially affects TNF-α-induced mRNA expression of proinflammatory IL-6 and prodestructive MMP-1 regarding the usage of EP receptors and the dependency on cAMP. Although specific blockade of EP2 receptors is considered a promising therapeutic strategy in RA, opposite regulation of proinflammatory IL-6 and prodestructive MMP-1 by PGE2 via EP2 may require more complex approaches to successfully inhibit the cyclooxygenase-1/2 cAMP axis.

Predominant activation of MAP kinases and pro-destructive/pro-inflammatory features by TNF α in early-passage synovial fibroblasts via TNF receptor-1: failure of p38 inhibition to suppress matrix metalloproteinase-1 in rheumatoid arthritis

Objective: To examine the relative importance of tumour necrosis factor-receptor 1 (TNF-R1) and TNF-R2 and their signalling pathways for pro-inflammatory and pro-destructive features of early-passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA). Methods: Cells were stimulated with tumour necrosis factor (TNF)α or agonistic anti-TNF-R1/TNF-R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-1 secretion by ELISA; and MMP-3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125). Results: In RA- and OA-SFB, TNFα-induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF-R1. Reduction of proliferation and induction of IL6, IL8 and MMP-1 were solely mediated by TNF-R1, whereas PGE2 and MMP-3 secretion was mediated by both TNF-Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFα influence on these effector molecules. In contrast, inhibition of p38 reversed TNFα effects on proliferation and IL6/PGE2 secretion (but not on IL8 and MMP-3 secretion). The above effects were comparable in RA- and OA-SFB, except that TNFα-induced MMP-1 secretion was reversed by p38 inhibition only in OA-SFB. Conclusion: In early-passage RA/OA-SFB, activation of MAPK cascades and pro-inflammatory/pro-destructive features by TNFα is predominantly mediated by TNF-R1 and, for proliferation and IL6/PGE2 secretion, exclusively regulated by p38. Strikingly, RA-SFB are insensitive to p38 inhibition of MMP-1 secretion. This indicates a resistance of RA-SFB to the inhibition of pro-destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.

Enhanced mechanical properties of a novel, injectable, fiber-reinforced brushite cement.

Injectable, brushite-forming calcium phosphate cements (CPCs) have great potential as bone replacement materials due to enhanced degradability and long-term inclusion in bone remodeling. However, the use of such brushite-forming CPCs in load-bearing areas is limited by their low mechanical strength. One approach to overcome this limitation is the use of reinforcing fibers. Thus, an injectable, biodegradable, brushite-forming CPC based on beta-tricalcium phosphate/phosphoric acid with fiber reinforcement was developed for minimally invasive surgery. The fibers (diameter 25 µm; length 0.25, 1 or 2mm) were extruded from poly(l-lactide-co-glycolide) acid (PLGA) and added to the CPC (2.5, 5 or 7.5% (w/w)). Independent of the fiber content, injectability of the CPC was retained up to a fiber length of 1mm. The addition of all PLGA fiber types increased diametral tensile strength, biaxial flexural strength, and flexural strength by up to 25% (p ≤ 0.05 for the diametral tensile strength for the CPC with 5% (w/w) 1mm fibers and the biaxial flexural strength of the CPC with 5% (w/w) 0.25 mm fibers). In contrast, the work of fracture strongly and significantly increased (p<0.01) by up to 12.5-fold. At constant fiber content, the mechanical properties of the fiber-reinforced CPC were mostly augmented with increasing fiber length. Also, the addition of PLGA fibers to the brushite-forming CPC (up to 7.5% (w/w)) only transiently delayed cell growth and did not decrease cell viability. Fiber reinforcement of CPCs thus augments their mechanical strength while preserving the injectability and biocompatibility required for their application in modern surgery.

Pro-inflammatory IL-1beta and/or TNF-alpha up-regulate matrix metalloproteases-1 and -3 mRNA in chondrocyte subpopulations potentially pathogenic in osteoarthritis: in situ hybridization studies on a single cell level.

In osteoarthritis chondrocytes, matrix metalloproteases (MMPs) and their inhibitors are induced by interleukin (IL)‐1beta or tumor necrosis factor (TNF)‐alpha and balanced by inhibitors, but their messenger RNA (mRNA) expression has not been studied in individual cells.

Synovial fibroblasts and synovial macrophages from patients with rheumatoid arthritis and other inflammatory joint diseases show chromosomal aberrations

Chromosomal aberrations were investigated in nuclei extracted from synovial tissue and first‐passage synovial fibroblasts (P‐1 SFB, 98% enrichment) or macrophages (P‐1 Mϕ) from patients with rheumatoid arthritis (n = 10). The findings were compared with those in other rheumatic diseases (osteoarthritis, n = 14; reactive arthritis, n = 1), as well as with those in chronic obstructive pulmonary disease (n = 8). Controls were paired peripheral blood lymphocytes from arthritic patients, synovial tissue or SFB/Mϕ from joint trauma/normals (n = 9), and peripheral blood monocytes from normal donors (n = 10). GTG banding of metaphase chromosomes and interphase fluorescence in situ hybridization with centromere‐specific probes were used. Comparable chromosomal aberrations were observed in synovial tissue and P‐1 SFB of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis (polysomy 7 and aneusomies of chromosomes 4, 8, 9, 12, and 18). Notably, aneusomies of chromosomes 4, 6, 7, 8, 9, 11, 12, and/or X were also detected in P‐1 synovial Mϕ from rheumatoid arthritis (90% of the cases), osteoarthritis (93%), and reactive arthritis (1/1), as well as bronchial Mϕ from chronic obstructive pulmonary disease (25%). No aberrations were detected in paired peripheral blood lymphocytes (except for one osteoarthritis case with a karyotype 45,X[10]/46,XX[17]), or in peripheral blood monocytes and synovial tissue of normals/joint trauma. Because Mϕ aberrations were common to chronic joint and pulmonary disease, chronic inflammatory stress may induce chromosomal aberrations with potential functional relevance in local mesenchymal cells and infiltrating leukocytes in an organ‐independent fashion.

Hepatic Fibrosis in a Long-term Murine Model of Sepsis.

ABSTRACT Chronic sequelae of sepsis represent a major, yet underappreciated clinical problem, contributing to long-term mortality and quality-of-life impairment. In chronic liver disease, inflammation perpetuates fibrogenesis, but development of fibrosis in the post–acute phase of systemic inflammation has not been studied. Therefore, a mouse model of post–acute sequelae of sepsis was established based on polymicrobial peritonitis under antibiotic protection. Survival decreased to approximately 40% within 7 days and remained constant until day 28 (post–acute phase). In survivors, clinical recovery was observed within 1 week, whereas white blood cell and platelet count, as well as markers of liver injury, remained elevated until day 28. Macroscopically, inflammation and abscess formation were detected in the peritoneal space and on/in the liver. Microscopically, acute-chronic inflammation with ductular proliferation, focal granuloma formation in the parenchyma, and substantial hepatic fibrosis were observed. Increased numbers of potentially pathogenetic macrophages and &agr;-smooth muscle actin–positive cells, presumably activated hepatic stellate cells, were detected in the vicinity of fibrotic areas. Fibrosis was associated with the presence of elastin and an augmented production/deposition of collagen types I and III. Microarray analyses revealed early activation of canonical and noncanonical pathways of hepatic stellate cell transdifferentiation. Thus, chronic sequelae of experimental sepsis were characterized by abscess formation, persistent inflammation, and substantial liver injury and fibrosis, the latter associated with increased numbers of macrophages/&agr;-smooth muscle actin–positive cells and deposition of collagen types I and III. This suggests persistent activation of stellate cells, with consecutive fibrosis—a hallmark of chronic liver disease—as a result of acute life-threatening infection.

Functional role of the KCa3.1 potassium channel in synovial fibroblasts from rheumatoid arthritis patients.

Rheumatoid arthritis synovial fibroblasts (RA‐SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA‐SFs would therefore be of high therapeutic interest. The present study shows that the intermediate‐conductance, calcium‐activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA‐SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro‐inflammatory and pro‐destructive mediators. Whole‐cell patch‐clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor β1 (TGF‐β1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore‐blocking inhibitor TRAM‐34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro‐inflammatory factors (IL‐6, IL‐8, and MCP1) and the tissue‐destructive protease MMP3. These effects were observed in non‐stimulated and/or TGF‐β1‐stimulated RA‐SFs. Since small molecule‐based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease. J. Cell. Physiol. 230: 1677–1688, 2015.

Vergleich zwischen konventioneller und Real-Time RT-PCR zur Quantifizierung der jun-Protoonkogen-mRNA und Analyse der junB-mRNA-Expression in Synovialmembranen und isolierten synovialen Fibroblasten von Patienten mit rheumatoider Arthritis

Summary. AP-1 dependent genes, e.g., matrix-metallo-proteinases, are involved in the pathogenesis of rheumatoid arthritis (RA). Therefore, the transcription factor AP-1 and its subunits, proteins of the Jun and Fos proto-oncogene families, are interesting targets for analysis in RA. In this study, we analyzed the mRNA expression of junB in synovial membrane (SM) samples and isolated synovial fibroblasts of patients with RA, osteoarthritis (OA), and normal, non-inflammatory controls.To address the suitability of real-time RT-PCR for the quantitation of Jun proto-oncogene family members, conventional RTPCR and real-time PCR were comparatively applied for junD, a gene representing a major challenge because of its high GC-content (70%, increasing the probability of secondary structures interfering with the PCR) and its sequence homology to other Jun proto-oncogenes. In addition, a comparison was performed concerning the precision, reproducibility, costs, as well as labor and time consumption of the two PCR methods.Real-time RT-PCR proved superior to conventional PCR in terms of precision (mean deviation of measured from employed concentration 58% for real-time PCR vs 225% for conventional PCR), reproducibility, as well as labor and time consumption (4 times less for real-time RT-PCR). Experimental cDNA normalization for equivalent cDNA concentrations by sample dilution was more reliable than mathematical cDNA normalization. However, real-time PCR was 3.6-fold more expensive.Applying the more reliable real-time RT-PCR for the exvivo analysis of junB mRNA-expression, no significantly different expression of junB was observed in SM or isolated synovial fibroblasts from RA as compared to OA. Interestingly, however, junBmRNA expression was significantly lower in RA SM and borderline significantly lower in OA SM than in normal/non-inflammatory SM, with potential effects on the functional properties of the resulting AP-1 complexes. Immunohistochemical staining of the SM with JunB-specific antibodies showed comparable JunB protein expression in SFB (collagen III mRNA-positive) of RA and OA samples.Thus, real-time RT-PCR appears suitable and time-saving for the quantitation of jun proto-oncogene mRNA-expression in tissue and cell samples with high precision and reproducibility. Zusammenfassung. AP-1-abhängige Gene, z. B. Matrix-Metallo- Proteinasen, sind an der Pathogenese der rheumatoiden Arthritis (RA) beteiligt. Deshalb sind der Transkriptionsfaktor AP-1 und seine Untereinheiten – die Proteine der Jun- und Fos-Proto-Onkogen-Familien – interessante Ziele für die Analyse in der RA. In dieser Studie wurde die mRNA-Expression von JunB in Synovialmembran-Gewebeproben (SM) und primären synovialen Fibroblasten von Patienten mit RA und Osteoarthritis (OA), sowie normalen, nicht entzündlichen Kontrollen analysiert.Um die Eignung der Real-time PCR für die Quantifizierung von Mitgliedern der Jun-Genfamilie zu überprüfen, wurde ein Vergleich der konventionellen RT-PCR mit der Real-time PCR für das Gen junD durchgeführt. Dieses stellt aufgrund seines hohen GC-Gehaltes (70%, wodurch die mögliche Bildung von mit der Polymerase- Reaktion interferierenden Sekundärstrukturen deutlich erhöht wird) und der Sequenzhomologien zu anderen Jun-Genen eine große methodische Herausforderung dar. Außerdem wurde in die Untersuchung ein Vergleich in Hinblick auf die Präzision, die Reproduzierbarkeit, die Kosten, sowie den Arbeits- und Zeitaufwand der beiden Methoden einbezogen.Die Real-time PCR erwies sich der konventionellen PCR in den Punkten Präzision (die mittlere Abweichung der gemessenen von der eingesetzten Konzentration betrug bei der Real-time PCR 58% gegenüber 225% bei der konventionellen PCR), Reproduzierbarkeit und Arbeits-/Zeitaufwand (4-fach geringer bei der Real-time RT-PCR) überlegen. Eine experimentelle Normalisierung durch Verdünnung der untersuchten cDNA-Proben auf äquivalente cDNA-Konzentrationen stellte sich gegenüber einer rein mathematischen Normalisierung als genauer heraus. Allerdings waren die Kosten der Realtime PCR 3,6-mal so hoch wie die der konventionellen PCR.Die zuverlässigere Real-time PCR wurde anschließend zur Ex-vivo- Analyse der junB mRNA-Expression eingesetzt. Dabei konnten keine signifikanten Unterschiede zwischen den Expressionsniveaus von junB in SM oder primären synovialen Fibroblasten von RA- und OA-Patienten nachgewiesen werden.Interessanterweise wurde allerdings eine signifikant niedrigere junB-Expression in der RA-SM und eine grenzwertig signifikant niedrigere junB-Expression in der OA-SM im Vergleich zu den Normalkontrollen beobachtet, was die Funktion der resultierenden AP-1 Komplexe beeinflussen könnte. Die immunhistologische Färbung der SM mit JunB-spezifischen Antikörpern zeigte eine vergleichbare JunB Proteinexpression in SFB (Kollagen III mRNA positiv) bei RA- und OA-Proben.Insgesamt erwies sich die Realtime RT-PCR in dieser Studie als eine geeignete und zeitsparende Methode für die Quantifizierung der mRNA-Expression von jun- Proto-Onkogenen in Gewebe und Zellproben mit hoher Präzision und Reproduzierbarkeit.