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 IV (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-1 beta and tumour necrosis factor-alpha (TNF-alpha) augmented the IL-4-induced effect with respect to APN but not to DPIV. A 5-day incubation with interferon-gamma (IFN-gamma) 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 1 (TGF-beta 1) 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.     

Regulation of the expression of aminopeptidase A,aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-β1) and tumour necrosis factor-alpha (TNF-α) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-α), IFN-β and IFN-γ increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.     

Fetal microchimerism in Hashimoto's thyroiditis: a quantitative approach

OBJECTIVE: Fetal microchimerism (MCH) has been implicated in the etiology of autoimmune diseases such as autoimmune thyroiditis. The goal of the study was to reliably estimate the number of fetal engrafted cells and to further investigate factors influencing the development of MCH. METHODS: Quantitative real-time PCR amplification using Y-chromosome specific (DYS14) and autosomal (beta-globin) loci was performed on thyroid gland specimens. Furthermore, we compared the distribution of ABO and rhesus systems in mothers with and without blood MCH in relation to the blood groups of the children. RESULTS: MCH was detected in eight of 21 Hashimoto patients in a frequency range of 15 to 4900 male cells per 100,000 total cells (median 97 cells), but in none of 17 healthy thyroid glands. In a third group, consisting of 18 nodular goiters, only one sample was positive (182 male cells/100,000 total cells). No woman who had not had a prior pregnancy with a male fetus showed MCH. Mothers both with and without MCH showed the same rate of mother/child incompatibilities for the ABO and rhesus systems. CONCLUSIONS: The percentage of microchimeric cells varies to a great extent in Hashimoto's thyroiditis, and this phenomenon can occur in nodular goiter in rare instances, but it appears to be absent from normal thyroid glands. Nevertheless, the biological significance of MCH remains unclear. Moreover, we have concluded that the tested blood group systems (as opposed to their role in graft vs host disease after transplantations) have no effect on fetal MCH.     

Inhibition of Glutaminyl Cyclases alleviates CCL2‐mediated inflammation of non‐alcoholic fatty liver disease in mice

Inflammation is an integral part of non‐alcoholic fatty liver disease (NAFLD), the most prevalent form of hepatic pathology found in the general population. In this context, recently we have examined the potential role of Glutaminyl Cyclases (QC and isoQC), and their inhibitors, in the maturation of chemokines, for example, monocyte chemoattractant protein 1 (MCP‐1, CCL2), to generate their bioactive conformation. Catalysis by isoQC leads to the formation of an N‐terminal pyroglutamate residue protecting CCL2 against degradation by aminopeptidases. This is of importance because truncated forms possess a reduced potential to attract immune cells. Since liver inflammation is characterized by the up‐regulation of different chemokine pathways, and within this CCL2 is known to be a prominent example, we hypothesised that application of QC/isoQC inhibitors may alleviate liver inflammation by destabilizing CCL2. Therefore, we investigated the role of QC/isoQC inhibition, in comparison with the angiotensin receptor blocker Telmisartan, during development of pathology in a mouse model of non‐alcoholic fatty liver disease. Application of a QC/isoQC inhibitor led to a significant reduction in circulating alanine aminotransferase and NAFLD activity score accompanied by an inhibitory effect on hepatocyte ballooning. Further analysis revealed a specific reduction of inflammation by decreasing the number of F4/80‐positive macrophages, which is in agreement with the proposed CCL2‐related mechanism of action of QC/isoQC inhibitors. Finally, QC/isoQC inhibitor application attenuated liver fibrosis as characterized by reduced collagen deposition in the liver parenchyma. Thus in conclusion, QC/isoQC inhibitors are a promising novel class of anti‐non‐alcoholic steatohepatitis drugs which have a comparable disease‐modifying effect to that of Telmisartan, which is probably mediated via specific interference with a comparable monocyte/macrophage infiltration that occurs under inflammatory conditions.     

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/microg total RNA; ranged from 0.1 pg to 96 pg IL-17R mRNA/microg 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-alpha and GRO-beta. An anti-IL-17 antibody blocked these effects of IL-17. The MAPK p38 appears necessary for the regulation of IL-8, GRO-alpha and GRO-beta 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-alpha and GRO-beta 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.     

IL-10 and TGF-beta differ in their regulation of aminopeptidase N/CD13 expression in monocytes

Human monocytic cells express considerable amounts of aminopeptidase N (APN)/CD13, a transmembrane protein proposed to play a role in the modulation of kinins, neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Previous studies have shown that APN/CD13 participates in antigen processing and presentation, trimming peptides protruding out of MHC class II molecules. In several inflammatory processes, macrophages have been shown to express especially high amounts of MHC class II molecules and of this peptidase. To learn more about the regulation of APN/CD13 on monocytes we investigated its expression under the influence of cytokines. Here, we report a dose- and time-dependent up-regulation of APN/CD13 mRNA and protein expression by transforming growth factor (TGF)-beta on human monocytes. To the contrary, we found IL-10 down-regulating the expression of APN/CD13 mRNA and protein. Both the regulation of the APN/CD13 protein assessed by immunofluorescence and the gene expression assessed by real-time PCR were highly correlated. Using the Dual-Luciferase reporter assay, we demonstrate that TGF-beta treatment of monocytes results in a higher activity of the APN/CD13 myeloid promoter. Our results implicate differences in the expression of the membrane peptidase APN/CD13 and therefore in the peptide-modulating ability of monocytes after exposure to these two immunosuppressive cytokines, TGF-beta and IL-10.