Increased interleukin (IL)-7Rα expression in salivary glands of patients with primary Sjogren's syndrome is restricted to T cells and correlates with IL-7 expression, lymphocyte numbers and activity.

OBJECTIVE: To identify interleukin (IL)-7Rα expression in the labial salivary gland (LSG) of patients with primary Sjögren's syndrome (pSS) and non-Sjögren's syndrome sicca (nSS-sicca) and to study its correlation with glandular inflammation and IL-7 expression. METHODS: The presence of infiltrating immune cells and IL-7Rα cells in inflamed LSG of patients with pSS (n=12) and nSS-sicca controls (n=7) was studied by immunohistochemistry and fluorescence activated cell sorting analysis upon tissue digestion (n=15 and n=13, respectively). Additionally, the correlations of IL-7Rα cells with hallmark disease parameters of pSS, major infiltrating inflammatory cells and IL-7 were assessed. RESULTS: In the LSG of patients with pSS increased numbers of IL-7Rα cells were found as compared with nSS-sicca patients. IL7Rα cells strongly correlated with the lymphocytic focus score, IL-7 expression, the decrease in percentage of IgA plasma cells and numbers of CD3 T cells, CD20 B cells, and CD1a and CD208 myeloid dendritic cells. Analysis of isolated cells from the LSG demonstrated strongly increased percentages of IL-7Rα CD3 T cells in pSS as compared with nSS, showing abundant IL-7Rα expression on both CD4 and CD8 T cells. Other CD45 leucocytes and CD45- tissue cells scarcely expressed IL-7Rα. Percentages of IL-7Rα T cells also significantly correlated with glandular inflammation. CONCLUSIONS: This study shows the presence of increased IL-7Rα T cells in the LSG of patients with pSS and their association with the severity of sialadenitis, disease parameters and IL-7 expression. Considering the immunostimulatory ability of IL-7Rα T cells and IL-7, this suggests that IL-7(R)-dependent T cell-driven immune activation plays an important role in inflammation in pSS.

Increased frequency of CD16+ monocytes and the presence of activated dendritic cells in salivary glands in primary Sjögren syndrome.

OBJECTIVES: In the salivary glands of patients with primary Sjögren Syndrome (pSjS) an accumulation of dendritic cells (DCs) is seen, which is thought to play a role in stimulating local inflammation. Aberrancies in subsets of monocytes, generally considered the blood precursors for DCs, may play a role in this accumulation of DCs. This study is aimed at determining the level of mature CD14lowCD16+ monocytes in pSjS and their contribution to the accumulation of DCs in pSjS. METHODS: Levels of mature and immature monocytes in patients with pSjS (n = 19) and controls (n = 15) were analysed by flow cytometry. The reverse transmigration system was used for generation of DCs generated from monocyte subsets. The phenotype of DCs in pSjS salivary glands was analysed using immunohistochemistry. In vivo tracking of monocyte subsets was performed in a mouse model. RESULTS: Increased levels of mature CD14lowCD16+ monocytes were found in patients with pSjS (mean (SD) 14.5 (5.5)% vs 11.4 (3.4)%). These cells showed normal expression of chemokine receptor and adhesion molecules. Mature monocytes partly developed into DC-lysosome-associated membrane glycoprotein (LAMP)+ (19.6 (7.5)%) and CD83+ (16 (9)%) DCs, markers also expressed by DCs in pSjS salivary glands. Monocyte tracking in the non-obese diabetic (NOD) mouse showed that the homologue population of mature mouse monocytes migrated to the salivary glands, and preferentially developed into CD11c+ DCs in vivo. CONCLUSIONS: Mature monocytes are increased in pSjS and patient and mouse data support a model where this mature monocyte subset migrates to the salivary glands and develops into DCs.

An increased MRP8/14 expression and adhesion, but a decreased migration towards proinflammatory chemokines of type 1 diabetes monocytes.

In the early development of type 1 diabetes macrophages and dendritic cells accumulate around the islets of Langerhans at sites of fibronectin expression. It is thought that these macrophages and dendritic cells are derived from blood monocytes. Previously, we showed an increased serum level of MRP8/14 in type 1 diabetes patients that induced healthy monocytes to adhere more strongly to fibronectin (FN). Here we show that MRP8/14 is expressed and produced at a higher level by type 1 diabetes monocytes, particularly after adhesion to FN, creating a positive feedback mechanism for a high fibronectin-adhesive capacity. Also adhesion to endothelial cells was increased in type 1 diabetes monocytes. Despite this increased adhesion the transendothelial migration of monocytes of type 1 diabetes patients was decreased towards the proinflammatory chemokines CCL2 and CCL3. Because non-obese diabetic (NOD) mouse monocytes show a similar defective proinflammatory migration, we argue that an impaired monocyte migration towards proinflammatory chemokines might be a hallmark of autoimmune diabetes. This hampered monocyte response to proinflammatory chemokines questions whether the early macrophage and dendritic cell accumulation in the diabetic pancreas originates from an inflammatory-driven influx of monocytes. We also show that the migration of type 1 diabetes monocytes towards the lymphoid tissue-related CCL19 was increased and correlated with an increased CCR7 surface expression on the monocytes. Because NOD mice show a high expression of these lymphoid tissue-related chemokines in the early pancreas it is more likely that the early macrophage and dendritic cell accumulation in the diabetic pancreas is related to an aberrant high expression of lymphoid tissue-related chemokines in the pancreas.

Short course dexamethasone treatment following injury inhibits bleomycin induced fibrosis in rats.

BACKGROUND: Corticosteroids are routinely used in patients with pulmonary fibrosis. The timing for initiation of treatment is likely to be crucial for corticosteroids to exert an antifibrotic effect. Experimental studies in animals have examined the effect of corticosteroid treatment starting before or at the time of lung injury. However, this is not representative of the human condition as treatment only begins after disease has been established. We examined the effect of a short course corticosteroid treatment starting 3 days after bleomycin induced lung injury on the development of pulmonary fibrosis. METHODS: Bleomycin (1.5 mg/kg) was instilled intratracheally into rats to induce pulmonary fibrosis. The effect of a 3-day course of dexamethasone (0.5 mg/kg) initiated 3 days after bleomycin induced lung injury on cell proliferation and collagen deposition was examined by analysing bronchoalveolar lavage (BAL) fluid and lung tissue. RESULTS: Treating bleomycin exposed animals after injury with dexamethasone for 3 days inhibited lung collagen deposition compared with animals exposed to bleomycin without dexamethasone treatment (15.2 (2.2) mg collagen/lung v 22.5 (2.1) mg/lung; p<0.05). Dexamethasone treatment reduced pulmonary parenchymal cell proliferation in bleomycin exposed rats but did not influence BAL fluid mitogenic activity for lung fibroblasts or alter the BAL fluid levels of the fibrogenic mediators transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin. CONCLUSIONS: A 3 day course of dexamethasone treatment initiated 3 days after bleomycin induced lung injury reduces lung cell proliferation and collagen deposition by mechanisms other than through reduction of transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin levels in BAL fluid. We propose that an early short course treatment with dexamethasone may be useful in inhibiting pulmonary fibrosis.

Dexamethasone treatment does not inhibit fibroproliferation in chronic lung disease of prematurity.

Pulmonary fibrosis results from excessive fibroblast proliferation and increased collagen deposition and occurs in chronic lung disease of prematurity (CLD). Platelet-derived growth factor (PDGF)-BB is mitogenic for fibroblasts and levels are increased in fibrotic lung disorders. Systemic dexamethasone (DEX) treatment improves pulmonary function and reduces inflammation in infants with or at risk of CLD. However, the effect of DEX treatment on fibroblast activity, PDGF-BB and collagen synthesis in the lungs of CLD patients is uncertain. Bronchoalveolar lavage (BAL) fluids, obtained from 15 infants at risk of CLD before and after DEX treatment, were analysed for fibroblast mitogenicity, PDGF-BB, N-terminal propeptide of collagen type III (PIIINP) and interleukin (IL)-1beta levels and inflammatory cell numbers. After DEX treatment, the mitogenic activity of BAL fluid for fibroblasts was not reduced but increased. The change in mitogenicity correlated with a change in BAL fluid PDGF-BB levels. Furthermore, BAL fluid-induced fibroblast proliferation was blocked using an inhibitor of the PDGF receptor. DEX treatment did not influence PIIINP levels, but reduced IL-1beta levels and inflammatory cell numbers in BAL fluid. This study suggests that dexamethasone treatment does not reduce fibroblast proliferation despite apparent downregulation of inflammation. The present findings do not support the use of dexamethasone for prevention of the fibrotic response in infants at risk of chronic lung disease of prematurity.

Localization and potential role of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 and -2 in different phases of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) can evolve in prematurely born infants who require mechanical ventilation because of hyaline membrane disease (HMD). The development of BPD can be divided in an acute, a regenerative, a transitional, and a chronic phase. During these different phases, extensive remodeling of the lung parenchyma with re-epithelialization of the alveoli and formation of fibrosis occurs. Matrix metalloproteinase-1 (MMP-1) is an enzyme that is involved in re-epithelialization processes, and dysregulation of MMP-1 activity contributes to fibrosis. Localization of MMP-1 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were investigated in lung tissue obtained from infants who died during different phases of BPD development. In all studied cases (n = 50) type-II pneumocytes were found to be immunoreactive for MMP-1, TIMP-1, and TIMP-2. During the acute and regenerative phase of BPD, type-II pneumocytes re-epithelialize the injured alveoli. This may suggest that MMP-1 and its inhibitors, expressed by type-II pneumocytes, play a role in the re-epithelialization process after acute lung injury. Although MMP-1 staining intensity remained constant in type-II pneumocytes during BPD development, TIMP-1 increased during the chronic fibrotic phase. This relative elevation of TIMP-1 compared with MMP-1 is indicative for reduced collagenolytic activity by type-II pneumocytes in chronic BPD and may contribute to fibrosis. Fibrotic foci in chronic BPD contained fibroblasts immunoreactive for MMP-1 and TIMP-1 and -2. This may indicate that decreased collagen turnover by fibroblasts contributes to fibrosis in BPD development.

Abnormal organogenesis in salivary gland development may initiate adult onset of autoimmune exocrinopathy.

OBJECTIVES: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues. METHODS: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice. RESULTS: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein. DISCUSSION: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity.

HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways.

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.

Two different types of sialoadenitis in the NOD- and MRL/lpr mouse models for Sjögren's syndrome: a differential role for dendritic cells in the initiation of sialoadenitis?

Sjögren's syndrome is an autoimmune disease that primarily affects the salivary and lacrimal glands. In these glands, focal lymphocytic infiltrates develop. Little is known about the initiation of this autoimmune disease. Antigen-presenting cells (APC) such as dendritic cells (DC) can play a role in the initiation of autoimmunity. To date, no data on the presence of DC in Sjögren's syndrome are available. Several mouse strains, the nonobese diabetic (NOD) and the MRL/Ipr mouse, can be used as models for Sjögren's syndrome. We compared the development of sialoadenitis in the submandibular glands (SMG) of NOD and MRL/Ipr mice with particular focus on the presence of APC. DC, macrophages, T cells, and B cells in the SMG were studied by means of immunohistochemistry, after which positively stained cells were quantified. NOD-severe combined immunodeficiency (SCID) mice were used to study the presence of APC in the SMG in the absence of lymphocytes. Before lymphocytic infiltration, increased numbers of DC were detected in the SMG of NOD mice compared with those numbers in control mice and MRL/Ipr mice, which suggests that DC play a role in the initiation of sialoadenitis in NOD mice. In the SMG of NOD mice, lymphocytic infiltrates organized in time. In MRL/Ipr mice, however, lymphocytic infiltrates were already organized at the time of appearance. This organization was lost over time. In conclusion, two types of sialoadenitis are described in two mouse models for Sjögren's syndrome. Differences exist with regard to early events that may lead to the development of sialoadenitis and to the composition and organization of inflammatory infiltrates. It is possible that different types of sialoadenitis also exist in humans and that the pathogenetic process in both the early and late phases of the autoimmune reaction differs among patients.

Professional antigen presenting cells in minor salivary glands in Sjögren's syndrome: potential contribution to the histopathological diagnosis?

Sjögren's syndrome is an autoimmune disease in which lymphocytic infiltrates develop in the salivary and lacrimal glands. We have shown that dendritic cells (DC) infiltrate the submandibular gland of the nonobese diabetic (NOD) mouse, a mouse model for Sjögren's syndrome, before lymphocytic infiltration, suggesting that these antigen-presenting cells (APC) may play a role in the initiation of Sjögren's syndrome. In later stages, DC and macrophages also form an important part of the infiltrate of the NOD sialoadenitis. To find out if DC and macrophages form part of the infiltrate in Sjögren's syndrome as well, and to determine whether they may be useful in the histopathological diagnosis of Sjögren's syndrome, we studied their presence in minor salivary glands (MSG) of patients with Sjögren's syndrome and patients with focal lymphocytic sialoadenitis (FLS), but without clinical or serological criteria of Sjögren's syndrome. Immunohistochemistry was applied, followed by semiquantitative analysis. DC and macrophages were present in all MSG; however, there were clear differences in marker expression between Sjögren's syndrome and FLS, on the one hand, and control tissue, on the other hand. CD1a+ DC and RFD9+ macrophages were mainly observed in MSG in which a focal lymphocytic infiltrate was present. In fact, the diffuse presence of single CD1a+ DC and RFD9+ macrophages correlated closely with the presence of a focal lymphocytic infiltrate in the MSG. This indicates that these cells could be of help during the evaluation of a MSG. Because the detection of APC is technically less cumbersome than a focal score, this parameter may perhaps replace the focal score in the histopathological diagnosis of Sjögren's syndrome. This study therefore prompts further investigation focusing on the presence of CD1a+ and RFD9+ cells in the MSG of a large cohort of patients.

Peptidase activities in serum and bronchoalveolar lavage fluid from allergic asthmatics--comparison with healthy non-smokers and smokers and effects of inhaled glucocorticoids.

BACKGROUND: Neuropeptides may be involved in the pathogenesis of asthma by evoking neurogenic inflammation. Since the effects of neuropeptides are limited by peptidases, reduced activity of peptidases may contribute to the inflammatory process. OBJECTIVE: We hypothesized that soluble peptidase activities are decreased in asthmatics and that inhaled glucocorticoids exert part of their anti-inflammatory action by increasing soluble peptidase activities. METHODS: Serum and bronchoalveolar lavage (BAL) fluid was obtained from non-smoking and smoking volunteers and from allergic asthmatics both before and after treatment for 12 weeks with placebo or inhaled fluticasone propionate. Activities of neutral endopeptidase (NEP), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV) were determined using colourometric assays. RESULTS: Reduced DPP IV activity in serum and reduced NEP activity in BAL fluid were found in healthy smokers compared with non-smokers. In contrast, no differences in peptidase activities in serum or BAL fluid were observed between allergic asthmatics and healthy non-smokers. Fluticasone propionate treatment did not affect peptidase activities in the asthmatic patients. CONCLUSIONS: We conclude that reduced peptidase activities in serum or BAL fluid can be found in healthy smokers, but not in allergic asthmatics, and that inhaled glucocorticoids do not affect peptidase activities in BAL fluid or serum of asthmatics. Our results do not support the hypothesized dysfunction of peptidases in the asthmatic airways.

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.

Interleukin 4 receptors on human bronchial epithelial cells. An in vivo and in vitro analysis of expression and function.

Asthma is considered a Th2-like disease, characterized by locally increased levels of interleukin (IL) 4. The bronchial epithelium plays an important role in the initiation and perpetuation of inflammatory reactions within the airways. However, little is known about the presence of IL-4 receptors on human bronchial epithelial cells, or the effects of IL-4 on these cells. In this report, definitive evidence of IL-4 receptor expression on human bronchial epithelial cells using several methods is presented. IL-4 receptor expression on human bronchial epithelial cells in vivo was demonstrated using in situ hybridization and immunohistochemistry. No difference in IL-4 receptor protein expression was observed between bronchial biopsies of healthy subjects compared to allergic asthmatics. Cultured human bronchial epithelial cells also expressed IL-4 receptor mRNA and protein (as determined by RT-PCR analysis and flow cytometry, respectively). IL-4 receptor protein expression by bronchial epithelial cells could be increased by stimulation with PMA+calcium ionophore, whereas IL-1beta and IL-6 decreased IL-4 receptor expression. A cyclic AMP analogue and IL-4 had no effect. Finally, it is shown that the IL-4 receptor is functionally active as IL-4 stimulates the release of IL-8, monocyte chemoattractant protein 1, and particularly IL-1 receptor antagonist by human bronchial epithelial cells. It is concluded that human bronchial epithelial cells express IL-4 receptors both in vivo and in vitro. Stimulation of human bronchial epithelial cells by IL-4 may result in the release of both pro- and anti-inflammatory mediators known to be upregulated in asthmatic airways.

Glucocorticoid receptor expression in human bronchial epithelial cells: effects of smoking and COPD.

Previously, we found that inflammatory mediators modulated the number and binding affinity of glucocorticoid receptors (GR) in human bronchial epithelial cell lines. In this study we investigated whether smoking and chronic obstructive pulmonary disease (COPD), both characterized by airway inflammation with increased levels of inflammatory mediators, affect GR characteristics in cultured human bronchial epithelial cells (HBEC). A statistically significant difference was found between the dissociation constant (Kd) values in HBEC from smoking (Kd = 0.98+/-0.08 nM; n = 6) and nonsmoking controls (Kd = 0.76+/-0.10 nM, P = 0.03; n = 5), but no significant difference was found between the mean number of binding sites. Our results are the first indication that cultured HBEC from smokers possess GR with a lower binding affinity. This may result from the inflammation found in the airways from smokers. Furthermore, these results provide further evidence that the bronchial epithelium may be an actual target for inhaled glucocorticoid therapy.

Expression of aminopeptidase N and dipeptidyl peptidase IV in the healthy and asthmatic bronchus.

BACKGROUND: Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and chronic inflammation of the airways. Since peptides are able to produce many of the pathophysiological features which are characteristic of asthma, peptide-mediated inflammation is thought to play a role in this disease. The effects of peptides are modulated by peptidases, which are able to degrade peptides, mostly resulting in their inactivation. OBJECTIVES: In this study, we investigated the distribution of two peptidases, aminopeptidase N and dipeptidyl peptidase IV, in the human bronchus and determined whether their expression was altered in allergic asthmatics. METHODS: We first determined the distribution of aminopeptidase N and dipeptidyl peptidase IV in the human bronchus using immuno- and enzymehistochemistry and compared this with the distribution of neutral endopeptidase. Secondly, the expression of aminopeptidase N and dipeptidyl peptidase IV was determined in bronchial biopsies of healthy subjects (n = 8) and allergic asthmatics (n = 12). RESULTS: Aminopeptidase N was localized in connective tissue, blood vessels, gland ducts, perichondrium, nerves and leucocytes (mainly mononuclear phagocytes, dendritic cells, and eosinophils). Dipeptidyl peptidase IV was localized in serosal glands, blood vessels, and T cells. Immunohistochemistry and enzymehistochemistry gave similar results. Comparison of the expression of aminopeptidase N and dipeptidyl peptidase IV in bronchial biopsies of healthy controls and atopic asthmatics revealed no significant differences in the lamina propria. In contrast, in the bronchial epithelium of atopic asthmatics an increased number of aminopeptidase N-positive cells could be found. Double-staining identified these cells as L25+ dendritic cells and eosinophils. CONCLUSIONS: We conclude that expression of aminopeptidase N and dipeptidyl peptidase IV is restricted to specific sites within the human bronchus. Furthermore, in the bronchial epithelium of allergic asthmatics an increased number of aminopeptidase N-expressing dendritic cells and eosinophils can be found.

Cytokines and glucocorticoids modulate human bronchial epithelial cell peptidases.

Peptidases play an important role in the regulation of peptide-mediated effects. Modulation of peptidase activity may therefore be a major mechanism to control peptide actions. Our aim was to analyse the effects of cytokines and glucocorticoids on peptidases expressed by human bronchial epithelial cells, which have been shown to be an important site for peptidase activity. The effects of cytokines [interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), IL-4, interferon gamma (INF-gamma), and epidermal growth factor (EGF)] and/or dexamethasone (DEX) on both expression and activity of neutral endopeptidase (NEP) and aminopeptidase N (APN) by BEAS 2B cells were determined using flow cytometry and activity assays, respectively. IL-1 beta, and to a lesser extent, TNF-alpha and IL-4 increased NEP activity and expression, whereas IFN-gamma decreased NEP. The effect of IL-1 beta was mediated, at least in part, via a cAMP-dependent pathway which did not involve prostaglandin E2 synthesis. APN was increased after 24-h stimulation with IFN-gamma, whereas other stimuli had no effect. DEX strongly increased NEP and APN expression and activity, both in the absence and in the presence of cytokines. We conclude that cytokines and glucocorticoids are able to modulate the activity of NEP and APN on BEAS 2B cells. Our results suggest a role for the human bronchial epithelium in the control of inflammation and indicate that one beneficial effect of glucocorticoids on asthma may be upregulation of peptidases expressed by bronchial epithelial cells.

The gene for the cyclin-dependent-kinase-4 inhibitor, CDKN2A, is preferentially deleted in malignant mesothelioma.

Cytogenetic deletions of the short arm of chromosome 9, 9p, have been detected in cell lines of malignant mesothelioma as well as in tumor material. Many tumor types carry deletions of chromosome 9 or more specifically of 9p21. The tumor-suppressor genes, CDKN2A and CDKN2B, each of which encodes a structurally and functionally similar cyclin-dependent kinase inhibitor, were mapped to the commonly deleted region. The tumor-suppressive effect of these genes, or of CDKN2A alone, requires functional retinoblastoma protein, pRb. Malignant mesothelioma expresses pRb, which, together with the cytogenetic data, suggests the involvement of CDKN2A and/or CDKN2B in its tumorigenesis. We present data on the deletion status of chromosome 9 in malignant mesothelioma cell lines and tumor tissue. A deletion map of the 9p21.3-p23 region was constructed for 12 cell lines. Homozygous deletions of chromosomal regions containing CDKN2A were detected in all cell lines. The smallest region of overlap for deletion is approximately 24 kb, and does not include CDKN2B. The frequency of deletion of the centromeric region of chromosome 9 was compared with that of chromosomes 1, 6, and 10 by genomic in situ hybridization. Deletion of the centromere of chromosome 9 is the predominant event at a frequency of 73 +/- 3%. Our data show that deletions of a critical region of chromosome 9, including the CDKN2A but not the CDKN2B locus, are common among malignant mesothelioma. Such deletions may be involved in tumorigenesis of mesothelium.

Cytokine regulation of mesothelial cell proliferation in vitro and in vivo.

Previous studies have demonstrated mitogenic effects of several mediators on mesothelial cells in vitro, but their effects in vivo have not been investigated. The aim of this study was to examine the effects of various cytokines on normal mesothelial cell proliferation in vitro and in vivo and correlate the findings in both assay systems. In vitro proliferation was assessed using a technique based on the uptake and subsequent release of methylene blue. Autoradiographic methods were applied in a murine model to assess mitogenic activity of these factors on mesothelium in vivo. In vitro data demonstrated a dose-dependent increase in human mesothelial cell proliferation by all mediators examined: at optimal concentrations, proliferation was enhanced between 26.53 +/- 3.77% standard deviation (SD), p < 0.001 for fibroblast growth factor-2 (FGF-2) and 114.58 +/- 6.97%, p < 0.001 for platelet-derived growth factor-AB (PDGF-AB) above control medium. In vivo, DNA synthesis in mesothelial cells was stimulated by FGF-2 (29.52 +/- 5.85% labeled cells, compared with 7.04 +/- 4.36% for control medium; p < 0.001), tumor necrosis factor-alpha (TNF-alpha; 13.14 +/- 4.55% compared with 7.23 +/- 2.85; p < 0.005) and PDGF-BB (11.53 +/- 4.74% compared with 4.67 +/- 3.48%; p < 0.005). Transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) had no effect on DNA synthesis in mesothelial cells in vivo. It is concluded that FGF2, TNF-alpha, and PDGF stimulate mesothelial cell proliferation in vitro and in vivo, whereas TGF-beta1 and EGF only had a mitogenic effect in vitro at the concentrations examined. The mitogenic potency of the different PDGF isoforms in vitro was consistent with PDGF-alpha and beta receptor expression.