Interleukin-1beta and interferon-gamma differentially regulate release of monocyte chemotactic protein-1 and interleukin-8 by human bronchial epithelial cells.

Airway inflammation is characterized by an accumulation of activated leukocytes. Bronchial epithelial cells may contribute to this process by releasing chemokines and by expressing surface membrane molecules involved in the adhesion and activation of the recruited leukocytes. In this study, we analyzed the effects of cytokines and glucocorticoids on the release of monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for predominantly monocytes and lymphocytes, by human bronchial epithelial cells and compared this with the release of interleukin-8 (IL-8), which potently attracts neutrophils. In addition, we analyzed the effects of cytokines and glucocorticoids on the epithelial expression of intercellular adhesion molecule (ICAM)-1, CD40, and human leukocyte antigen (HLA) class II molecules. Primary cultures of human bronchial epithelial cells constitutively released MCP-1 and IL-8. IFN-gamma greatly increased MCP-1 release, which was accompanied by increased expression of MCP-1 mRNA and an increased monocyte chemotactic potential. In contrast, IFN-gamma had no effect on the release of IL-8, but it did increase the epithelial expression of ICAM-1, CD40, and HLA class II molecules. IL-1beta increased both MCP-1 and IL-8 release, and increased the expression of ICAM-1 and CD40, but not HLA class II molecules. Dexamethasone partially inhibited the cytokine-induced release of MCP-1 and IL-8 and the expression of ICAM-1, CD40, and HLA class II molecules by human bronchial epithelial cells. Our results indicate that IFN-gamma and IL-1beta differentially regulate the MCP-1 and IL-8 release by human bronchial epithelial cells. In addition, IL-1beta and particularly IFN-gamma increase the expression of ICAM-1, HLA class II and/or CD40 molecules, which are involved in the adhesion and possibly activation of the recruited leukocytes. Finally, the beneficial effect of glucocorticoid therapy in airway inflammatory diseases may be mediated in part by inhibition of chemokine release and ICAM-1, CD40, and HLA class II expression by bronchial epithelial cells.

Identification of regulatory sequences in the promoter of the PDGF B-chain gene in malignant mesothelioma cell lines.

Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor. Coexpression of the PDGF beta-receptor and its ligand may lead to an autocrine growth stimulating loop in the malignant cell type. In nuclear run off experiments, PDGF B-chain mRNA was detectable in MM cells only, indicating an increased level of transcription in this cell type. The proximal promoter of the PDGF B-chain gene contains DNaseI hypersensitive (DH) sites and mediates reporter gene activation in both normal and malignant cells. Nuclear proteins, extracted from both cell types, interact with DNA sequences within the proximal promoter around bp-64 to -61 relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) indicate that these factors are more abundantly present in the malignant than in the normal cell type. A DH site around -9.9 kb was found in both cell types. When tested in CAT assays, this region exerted a stimulatory effect on transcription in malignant cells. The elevated level of transcription of the PDGF B-chain gene in malignant cells may well be the result of interaction of regulatory sites in the proximal promoter and an enhancing element located at -9.9 kb from the transcription start site.

Expression of platelet-derived growth factor (PDGF) and PDGF receptors in human malignant mesothelioma in vitro and in vivo.

The expression of platelet-derived growth factor (PDGF) and PDGF receptors was studied in human normal and malignant mesothelial cells in vitro and in vivo. Staining with anti-cytokeratin and ME1 antibodies and ultrastructural analysis confirmed the mesothelial nature of the cell lines used to study PDGF and PDGF receptor expression in vitro. Using antibodies, mesothelioma cell lines were found to express PDGF and both the PDGF alpha- and the PDGF beta-receptor, whereas cultured normal mesothelial cells expressed PDGF and PDGF alpha-receptor. This PDGF and PDGF receptor staining pattern largely reflects the earlier described mRNA expression in these cell lines. The only exception was the immunocytochemical detection of PDGF alpha-receptors in the mesothelioma cell lines, which is different from the inability to detect alpha-receptor transcripts on Northern blots. Expression was also investigated in mesothelial cells in vivo. Expression of PDGF was observed in malignant mesothelioma cells on frozen tissue sections. In pleural effusions, a double immunofluorescence staining procedure for PDGF and epithelial membrane antigen (EMA) revealed PDGF expression by EMA-positive malignant mesothelioma cells. PDGF beta-receptors and occasionally PDGF alpha-receptors were detected in frozen tissue sections of malignant mesotheliomas, whereas mesothelioma cells in effusions showed faint expression of only the PDGF beta-receptor. In contrast, in effusions containing non-malignant mesothelial cells, only a very low level of PDGF alpha-receptor could be detected. Taken together, these results indicate that the pattern of PDGF and PDGF receptor expression in mesothelial cells in vivo largely corresponds to expression of PDGF and its receptors in vitro. Malignant mesothelioma cell lines thus constitute a good model system for studies on the role of PDGF in this malignancy. Furthermore, the data reported in this paper are consistent with the idea that an autocrine growth stimulatory effect of PDGF via PDGF receptors may play a role in the pathogenesis of malignant mesothelioma.

Regulation of differential expression of platelet-derived growth factor alpha- and beta-receptor mRNA in normal and malignant human mesothelial cell lines.

In earlier studies we showed that the expression of patterns of platelet-derived growth factor (PDGF) alpha- and beta-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF alpha-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF beta-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF beta-receptor mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF beta-receptor transcription but of alpha-receptor transcription as well, as evidenced from run off analysis and RT-PCR using alpha-receptor specific primers. However, the fact that PDGF alpha-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF alpha-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF alpha-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF beta-receptor mRNA expression was seen. The half-life of the PDGF beta-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF beta-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in beta-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF beta-receptor transcripts in these cells. Transforming growth factor beta-1 (TGF-beta 1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-beta 1 revealed decreased PDGF alpha-receptor mRNA expression in normal mesothelial cells. The effect on PDGF beta-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-beta 1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-beta 1 may nevertheless play a role in downregulation of an (already) low PDGF alpha-receptor mRNA level in malignant mesothelioma cell lines.