K. Thiele

Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis

Interleukin‐17 (IL‐17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL‐17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared to patients with osteoarthritis. In contrast to the restricted expression of IL‐17, the IL‐17 receptor (IL‐17R/CDw217) is expressed ubiquitously. Using a real‐time RT‐PCR assay, we detected similar absolute levels of IL‐17R mRNA expression in fibroblast‐like synoviocytes (SFC) from patients with RA (mean 9 pg/μg total RNA; ranged from 0·1 pg to 96 pg IL‐17R mRNA/μg total RNA) compared to synoviocytes of non‐RA patients. Analysis of the IL‐17R surface expression confirmed the results obtained for IL‐17R mRNA expression. Exposure of SFC to IL‐17 led to a mRNA induction of CXC chemokines IL‐8, GRO‐α and GRO‐β. An anti‐IL‐17 antibody blocked these effects of IL‐17. The MAPK p38 appears necessary for the regulation of IL‐8, GRO‐α and GRO‐β expression as shown by inhibition with SB203580. The inhibitors genistein (tyrosine kinase inhibitor) and calphostin C (inhibitor of protein kinase C) reduced significantly the IL‐17‐stimulated mRNA expression of IL‐8, GRO‐α and GRO‐β in SFC, whereas PD98059 (inhibitor of MEK‐1/2) was without effect. Pharmacological drugs used in therapy of RA, such as cyclosporin and methotrexate, induced a fourfold increase of IL‐17R mRNA expression and augmented the IL‐17‐stimulated IL‐8 expression. Our results support the hypothesis that IL‐17/IL‐17R may play a significant role in the pathogenesis of RA contributing to an unbalanced production of cytokines as well as participating in connective tissue remodelling.

Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-β, and bone morphogenetic protein-6 with an expression 3.6–10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1β and tumour necrosis factor-α on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.

Interleukin-17 stimulates the expression of IκBα mRNA and the secretion of IL-6 and IL-8 in glioblastoma cell lines

Abstract Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4 + activated memory T cells. In an effort to elucidate the biological effects of IL-17 in glial cells, we investigated the ability of this cytokine in order to activate nuclear factor (NF)-κB, which is being discussed as one of the most important transcription factors in the regulation of neuronal and glial cell function. Activation of NF-κB involves the degradation of its cytoplasmatic inhibitor IκB-α, which allows the nuclear translocation of NF-κB, and ensures transcriptional activation of genes including IκB-α itself. Using a competitive RT-PCR, we examined the IL-17-induced IκB-α mRNA expression in glioblastoma cells, and we examined IL-17 up-regulated IκB-α mRNA expression in a dose- and time-dependent fashion with a maximum time between 1 and 3 h. This induction could be inhibited by Calphostin C (proteinkinase C inhibitor) and genistein (tyrosine kinase inhibitor). After 60 min of IL-17 stimulation, a degradation of the IκB-α protein was detectable. Furthermore, IL-17 stimulated the secretion of IL-6 and IL-8 in glial cells, and IL-17 and IL-1β in combination showed a superadditive effect. We suggest IL-17 to play a role as an immune factor, possibly involved in complex pathophysiological interactions of neurodegenerative diseases.

IL-1β- and IL-4-induced down-regulation of autotaxin mRNA and PC-1 in fibroblast-like synoviocytes of patients with rheumatoid arthritis (RA)

Autotaxin (ATX) is a 125‐kD ectonucleotide pyrophosphate/phosphodiesterase, which was initially isolated and cloned from human melanoma cells as a potent stimulator of tumour cell motility. ATX shows 44% identity to the plasma cell membrane marker PC‐1. Recently, we described the decreased expression of ATX mRNA in cultured fibroblast‐like synoviocytes (SFC) of patients with RA by interferon‐gamma. In this study using a competitive reverse transcriptase‐polymerase chain reaction, we show an increased ATX mRNA expression in SFC from patients with RA in comparison with synoviocytes from non‐RA patients. The median ATX mRNA amount in SFC of RA patients (440 pg/μg total RNA) was five‐fold higher than the expression in synoviocytes from non‐RA patients (80 pg/μg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/μg total RNA). In contrast to the elevated ATX mRNA expression in SFC of patients with RA, we did not measure increased mRNA amounts of PC‐1 in these cells. Both the ATX mRNA amount and the 5′‐nucleotide phosphodiesterase (PDE) activity of SFC lysate were reduced after treatment of SFC with the cytokines IL‐1β or IL‐4. IL‐1β and IL‐4 induced a down‐regulation of PC‐1 mRNA and protein expression in SFC. In SFC treated with transforming growth factor‐beta the expression of PC‐1 mRNA and protein was increased, whereas no significant effect on ATX mRNA expression was detectable. Pharmacological drugs used in therapy for RA, such as dexamethasone, cyclosporin, methotrexate and indomethacin, did not show a statistically significant effect on either ATX mRNA or PC‐1 mRNA expression. Only pentoxifylline suppressed ATX mRNA as well as PC‐1 mRNA expression. In conclusion, we show a tight regulation of ATX and PC‐1 gene expression by cytokines detectable in the inflamed tissue of RA. Further investigations will deal with the regulation of ATX protein expression as well as with the function of ATX in RA.

Aminopeptidase N-Mediated Signal Transduction and Inhibition of Proliferation of Human Myeloid Cells

Cell surface peptidases are involved in degradative processes of various peptides, in cell-cell communcation, in lymphocyte activation and in cell growth1,2,3,4. Aminopeptidase N (APN, CD 13), a ubiquitously occurring surface molecule of many tissues, among them human liver, kidneys, intestine, placenta, bone marrow or brain microvessels, acts with a relatively broad substrate specifity on peptides with N-terminal neutral amino acids 5. Within the haematopoietic system CD13 is localized on monocytes, granulocytes and activated T lymphocytes5,6,7,8.

Constitutive expression of HLA class II mRNA in synovial fibroblast-like cells from patients with rheumatoid arthritis

We describe the quantification of the absolute amounts of HLA class II mRNA and class II transactivator (CIITA) mRNA by competitive reverse transcription polymerase chain reaction in cultured synovial fibroblast-like cells (SFC) of patients with rheumatoid arthritis. High basal levels of transcription of class II mRNA (10(7)-10(9) molecules/microgram total RNA) and CIITA mRNA were detected in cultured SFC, with DPB < DRB = DQB, although SFC only express small amounts of MHC class II proteins. In contrast to SFC, we did not detect class II mRNA nor CIITA mRNA in skin fibroblasts. After treatment with IFN-gamma, we observed a 3- to 28-fold increase in class II mRNA in SFC and an increase of DRB and DPB in skin fibroblasts from undetectable levels to 10(8)-10(9) molecules/microgram total RNA.

Co-Incubation of Lymphocytes with Fibroblast-Like Synoviocytes and other Cell Types Can Induce Lymphocytic Surface Expression of Aminopeptidase N/CD13

Aminopeptidase N (APN, EC 3.4.11.2) is an ubiquitously occurring transmembrane ectoenzyme. Sequence comparisons of the cloned cDNA showed that APN is identical to CD13 antigen1. The coding part of the CD13 gene is encoded by 20 exons2. CD13 has been considered to be a myeloid-specific marker because lymphocytes of peripheral blood and tonsills always are CD13 negative. However, we have previously demonstrated the expression of CD l3 on synovial T cells from patients with different forms of arthritis3 or on tumor-infiltrating lymphocytes from renal cell carcinoma4. To learn more about conditions in tissues resulting in the expression of CD13 on lymphocytes, we incubated tonsillar T and B cells as well as peripheral blood lympho-cytes (PBL) with various mediators in the absence and presence of different stromal cells.

Two Transfected Endothelial Cell Lines Expressing High Levels of Membrane Bound or Soluble Aminopeptidase N

The CD13 molecule, found to be identical with aminopeptidase N (APN/EC 3.4.11.2)1 has been considered as a specific marker for the myeloid lineage within the hematopoietic system, however the occurrence of APN on lymphoid cells was recently described e.g. on tumor-infiltrating lymphocytes2, on T cells in the synovial fluid from patients with rheumatoid arthritis3 and some kinds of leukemia or lymphoma4,5,6. Our group very recently obtained evidence that the coculture of lymphocytic cells with various adherent APN positive cells such as fibroblast-like synoviocytes, HUVEC, kidney tubular epithelial cells or some tumor cell lines can induce the APN molecule on lymphocytes7. In need of a coculture system to further investigate the influence of the CD13/APN molecule we focused our interest on transfecting a APN negative adherent cell line with an expression vector for APN. To this end we used the spontaneously transformed immortal endothelial cell line ECV304 (ATCC CRL-1998) established from the vein of an apparently normal human umbilical cord8. Some reports describe the existence of a solubilized form of aminopeptidase N in serum, originating from liver and increased in hepatobiliary disease9, 10, 11. Therefore it seemed to us of further interest, wether the soluble form of this molecule is also able to induce the lymphocytic expression and if direct cell contact is necessary for induction. For that reason we additionally transformed the ECV304 cell line with an expression vector for the secretory form of aminopeptidase N.