J. Langner

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.

Stimulation of the expression and the enzyme activity of aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 on human renal cell carcinoma cells and renal tubular epithelial cells by T cell-derived cytokines, such as IL-4 and IL-13

Aminopeptidase N (APN) and dipeptidylpeptidase TV (DPIV) are transmembrane type II molecules widely distributed in mammalian tissues. In recent years, the interest in cell surface peptidases has increased considerably because, among other things, several reports indicate roles of ectopeptidases in tumour cell metastasis. Investigations into the regulation of APN and DPIV on tumour cells are rare. We report, for the first time, that IL‐4 and IL‐13 can up‐regulate protein expression as well as enzymatic activity of both the peptidases on renal carcinoma cells and renal tubular epithelial cells in culture. The analysis of mRNA by competitive polymerase chain reaction (PCR) confirmed our results with respect to the APN increase at the level of gene expression. IL‐1β and tumour necrosis factor‐alpha (TNF‐a) augmented the IL‐4‐induced effect with respect to APN but not to DPIV, A 5‐day incubation with interferon‐gamma (IFN‐γ) increased protein expression, especially of APN and, to a lesser extent, also of DPIV, whereas no significant increase in enzymatic activity could be observed. Small concentrations of transforming growth factor‐beta I (TGF‐β1) inhibit the expression and enzyme activity of DPIV. IL‐6, IL‐7. IL‐10 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) have been found to be without any effect on APN and DPIV. For a prospective therapeutic regimen with T cell‐derived cytokines it has to be considered that—besides their effect on tumour cell growth—cytokines might affect surface ectopeptidases involved in tumour cell adhesion processes. The inhibition of APN and DPIV could be a new approach to suppression of cancer spread.

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.