Airway-specific recruitment of T cells is reduced in a CD26-deficient F344 rat substrain.

Asthma is a chronic inflammatory disease affecting the airways. Increased levels of T cells are found in the lungs after the induction of an allergic-like inflammation in rats, and flow cytometry studies have shown that these levels are reduced in CD26-deficient rats. However, the precise anatomical sites where these newly recruited T cells appear primarily are unknown. Therefore, we quantified the distribution of T cells in lung parenchyma as well as in large, medium and small airways using immunohistochemical stainings combined with morphometric analyses. The number of T cells increased after the induction of an allergic-like inflammation. However, the differences between CD26-deficient and wild-type rats were not attributable to different cell numbers in the lung parenchyma, but the medium- and large-sized bronchi revealed significantly fewer T cells in CD26-deficient rats. These sites of T cell recruitment were screened further using immunohistochemistry and quantitative real-time polymerase chain reaction with regard to two hypotheses: (i) involvement of the nervous system or (ii) expression of chemokines with properties of a T cell attractor. No topographical association was found between nerves and T cells, but a differential transcription of chemokines was revealed in bronchi and parenchyma. Thus, the site-specific recruitment of T cells appears to be a process mediated by chemokines rather than nerve-T cell interactions. In conclusion, this is the first report showing a differential site-specific recruitment of T cells to the bronchi in a CD26-deficient rat substrain during an asthma-like inflammation.

Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients.

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma.

Self-renewal is considered as a common property of stem cells. Dysregulation of stem cell self-renewal is likely a requirement for the development of cancer. Hiwi, the human Piwi gene, encodes a protein responsible for stem cell self-renewal. In this study, we investigated the expression of Hiwi at the RNA level by real-time quantitative PCR in 65 primary soft-tissue sarcomas (STS) and ascertained its impact on prognosis for STS patients. In a multivariate Cox's proportional hazards regression model, we found that an increased expression of Hiwi mRNA is a significant negative prognostic factor for patients with STS (P=0.017; relative risk 4.6, 95% confidence interval (CI) 1.3-16.1) compared to medium expression of Hiwi transcript. However, a low expression of Hiwi transcript is correlated with a 2.4-fold (CI 0.7-8.0) increased risk, but this effect was not significant (P=0.17). Altogether, high-level expression of Hiwi mRNA identifies STS patients at high risk of tumour-related death. This is the first report showing a correlation between expression of a gene involved in stem cell self-renewal and prognosis of cancer patients.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells.

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen-fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

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.

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-beta, 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-1beta and tumour necrosis factor-alpha 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.

Expression of cathepsin B, D and L protein in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common childhood autoimmune rheumatic disease and like rheumatoid arthritis (RA), it is characterized by inflammation and the progressive destruction of joints. In RA, cathepsins as proteinases play a major role in destroying synovial tissue and cartilage matrix. So far no data on cathepsin expression in pannus tissue of HA patients exist. The aim of this study was to characterize the expression levels of cathepsins B, D, H, and L in HA and to compare them with those in RA. Synovectomy tissue from 16 HA and 12 RA patients was investigated for cathepsin expression levels by Western blot analysis. Expression of cathepsins B, D and L was on comparable levels in the synovectomy tissue of HA and RA patients. The following graduation of expression was determined: cathepsin D > cathepsin L > cathepsin B. Cathepsin H was neither found to be expressed in HA nor in RA patients. The expression levels of cathepsins in pannus tissue showed no clear difference between patients with systemic JIA and patients with monoarticular JIA. In summary, the comparable expression of cathepsins B, D and L in RA and JIA synovectomy tissue suggests that they may play a similarly important role in destroying synovial tissue and cartilage matrix in the course of HA and RA.

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.

Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: interleukin-8 is resistant to degradation by aminopeptidase N/CD13.

In rheumatic joints, high concentrations of interleukin-8 (IL-8) have been measured in synovial fluid and in pannus tissue. In both locations aminopeptidase N (APN)/CD13, an exopeptidase with reported activity towards IL-8 is also present. The surprising stability of IL-8 in the presence of an alleged IL-8-degrading peptidase prompted us to undertake the present study. Cocultivation of fibroblast-like synoviocytes (SFC) with T cells or with T lymphocytic cell membranes, or of T cells with SFC cell membranes, all resulted in increased IL-8 mRNA expression and IL-8 secretion into the medium, and an increase of APN expression on lymphocytes. IL-8 degradation was monitored by Western blots and HPLC. IL-8(72), as a partially processed form, was used throughout this study since it is abundant in tissues and has increased biological activity in comparison to IL-8(77). Thus its degradation/inactivation is considered of high biological significance. Whereas trypsin as a positive control rapidly degraded IL-8, we did not see any IL-8 degradation, either by a variety of soluble APNs, by leucine aminopeptidase or by APN expressed on the surface of SFC, or on ECV304 cells transfected with an APN expression vector. The much more sensitive HPLC technique resulted in negative results as well.

IL-1 beta- 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/microg total RNA) was five-fold higher than the expression in synoviocytes from non-RA patients (80 pg/microg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/microg 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-1beta or IL-4. IL-1beta 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.

Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains.

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and 'rosettes' made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.

Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell-cell contact.

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell-cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast-like synoviocytes (SFCs) results in an increase of steady-state APN/CD13 mRNA and a rapid expression of cell-surface protein on the lymphocytes. In this study using the Dual-Luciferase reporter assay, we demonstrate that interaction of the T-cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell-cell contact-induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell-cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides -48 to -35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase-promoter activity via cell-cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction.

Transforming growth factor-beta increases the expression of aminopeptidase N/CD13 mRNA and protein in monocytes and monocytic cell lines.

Aminopeptidase N (APN)/CD13 is a membrane-bound surface ectopeptidase with a ubiquitous distribution. In hematopoiesis, APN/CD13 is expressed on stem cells and during most developmental stages of myeloid cells. Because APN/CD13 has been implicated in the trimming on the cell surface of peptides that protrude out of MHC class II molecules, we wanted to study the regulation of this membrane peptidase in antigen presenting cells by TGF-beta. TGF-beta is a potent inducer of the maturation of monocyte precursors towards a macrophage phenotype. Using competitive RT-PCR and cytofluorimetric analyses, we quantified the modulation of the APN/CD13 mRNA as well as protein expression by TGF-beta 1 and -2 and found a stimulation of the APN/CD13 expression in a time- and dose-dependent manner in monocytic cells. In U937 cells, the time course showed a maximum for APN/CD13 mRNA at 24 hours incubation with TGF-beta. In experiments with actinomycin D- treated cells was found a stabilization of APN/CD13 mRNA by TGF-beta 1. Contrary to the IL-4-induced expression of APN/CD13 as well as of MHC class II in monocytic cells, we could show that TGF-beta is able to augment the APN/CD13 expression but decreases the MHC class II expression.

Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor.

Using random arbitrarily primed-reverse transcribed-PCR and sequence analysis, we investigated changes in lymphocytic molecules after cell-cell contact with fibroblasts. An mRNA species which was upregulated in Jurkat T cells by cell-cell contact with MRHF cells (a human foreskin fibroblast line) was identified as coding for the human interleukin-17 receptor. This finding was confirmed by quantitative RT-PCR for the HUT78 and Jurkat T cell lines, for peripheral blood lymphocytes, and for tonsillar T cells. Furthermore, the interleukin-17 mRNA, coding for a proinflammatory cytokine, was also upregulated in peripheral blood lymphocytes and tonsillar T cells after cell-cell contact with fibroblasts. Supernatants obtained from cell-cell contact-stimulated peripheral blood lymphocytes enhance the production of interleukin-6 and interleukin-8 by fibroblast-like synoviocytes and this effect could be blocked by interleukin-17 antibodies. Changes in the mRNA levels of Jurkat T cells induced by cell-cell contact with adherent cells were also found for M-type pyruvate kinase, for tropomyosin TM30 and for the p54nrb gene product.

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

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)-kappaB, which is being discussed as one of the most important transcription factors in the regulation of neuronal and glial cell function. Activation of NF-kappaB involves the degradation of its cytoplasmatic inhibitor IkappaB-alpha, which allows the nuclear translocation of NF-kappaB, and ensures transcriptional activation of genes including IkappaB-alpha itself. Using a competitive RT-PCR, we examined the IL-17-induced IkappaB-alpha mRNA expression in glioblastoma cells, and we examined IL-17 up-regulated IkappaB-alpha 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 (protein kinase C inhibitor) and genistein (tyrosine kinase inhibitor). After 60 min of IL-17 stimulation, a degradation of the IkappaB-alpha protein was detectable. Furthermore, IL-17 stimulated the secretion of IL-6 and IL-8 in glial cells, and IL-17 and IL-1beta 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.

CD13--not just a marker in leukemia typing.

Membrane peptidases are a multifunctional group of ectoenzymes that have been implicated in the control of growth and differentiation of many cellular systems. Here, using aminopeptidase N/CD13 as an example, Dagmar Riemann and colleagues discuss the role of cell-cell contact in peptidase regulation and the influence of peptidases on cellular functions.

Regulation by transforming growth factor-beta1 of class II mRNA and protein expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Transforming growth factor (TGF)-beta1 is an immunosuppressive cytokine that modulates the expression of class II histocompatibility antigens on human cells. Aberrant HLA class II expression on synovial lining cells of rheumatoid arthritis synovial membrane has been described, and the extent and intensity of class II expression on the cells was claimed to be linked with the severity of the disease. In this study, the effects of TGF-beta1 on HLA class II antigen expression in fibroblast-like synoviocytes (SFC) from rheumatoid synovectomy tissues were determined by flow cytometric analysis and quantitative RT-PCR. We found that pre-incubation of cells with TGF-beta1 was able to down-regulate IFN-gamma-induced DR protein expression in SFC. TGF-beta1, additionally, down-regulated IFN-gamma-stimulated class II transactivator (CIITA) and DRB mRNA expression. The constitutive expression of CIITA mRNA was completely abolished and the constitutive expression of DRB mRNA was decreased after treatment of SFC with TGF-beta1 for 24 h. Addition of the TGF-beta inhibitor decorin to SFC for 24 h before TGF-beta1/IFN-gamma treatment was able to reduce the down-regulatory effect of TGF-beta1 on DR antigen expression induced by IFN-gamma. Using competitive RT-PCR, we found that SFC constitutively expressed decorin mRNA and that treatment of cells with TGF-beta1 for 24 h reduced the constitutive expression of decorin mRNA by 65%. Our results show that TGF-beta1 is able to reduce the expression of HLA class II mRNA and protein, and suggest a tight regulation between TGF-beta1 and decorin in SFC of the rheumatoid synovium.

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-beta1) and tumour necrosis factor-alpha (TNF-alpha) 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-alpha), IFN-beta and IFN-gamma 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.