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Byline: I. StAiz (1), A. Slavcev (1), J. Kalanin (1), S. I. Rennard (2) Keywords: macrophages; epithelial cells; phenotype Abstract: Interactions of macrophages with epithelium represent one of the pathways involved in regulating local immune mechanisms. We studied the effect of cell--cell contact with an epithelial monolayer on the phenotype of macrophages. Human monocytes and THP-1 macrophages were co-cultured with monolayers of human bronchial epithelial cells (HBECs), the alveolar type II-like cell line A549, renal adenocarcinoma epithelial cells (RA), and the lung fibroblast strain HFL-1. The expression of CD11b, CD14, CD54, and HLA-DR was measured by immunocytochemistry and flow cytometry and showed epithelial cell induction of CD54 and HLA-DR in monocytes and of all antigens in THP-1 cells. Co-culture with fibroblasts did not change the phenotype of macrophages. Separation by a filter insert inhibited most of the effects. Culture supernatants did not induce prominent phenotypic changes. Cell--cell contacts with epithelium appear to be of importance in regulating the phenotype of macrophages. Author Affiliation: (1) Department of Immunology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic (2) Pulmonary and Critical Care Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska Article History: Registration Date: 17/10/2004

Byline: I. StAiz (1), T. Mio (2), Y. Adachi (2), R. A. Robbins (3), D. J. Romberger (3), S. I. Rennard (2) Abstract: Cytokine networks are important in regulating the traffic of inflammatory cells in the airways. Interleukin-8 (IL-8) released by human bronchial epithelial cells (HBECs) is thought to be of particular importance in attracting neutrophils and monocytes to sites of inflammation. Increased release of IL-8 by HBECs in response to Th-1 cytokines such as TNF alpha and IL-1 beta may be an important pathophysiologic pathway. The present study was designed to explore the role of the Th2 cytokine IL-4 and the functionally related interleukins IL-10, and IL-13 on the regulation of IL-8 release by HBECs. HBECs (passage 4--6) were cultured in LHC9/RPMI and when confluent cells were stimulated in unsupplemented medium LHCD/RPMI by IL-4, IL-10, and IL-13 at 10 ng/ml concentration for all cytokines. TNF alpha stimulation was used as a positive control. After 24 hours supernatants were collected and tested for IL-8 by a sandwich ELISA. Unstimulated HBECs spontaneously released limited amounts of IL-8 (11 +- 1 pM) and significantly increased cytokine production in response to IL-4 (42 +- 1 pM), IL-13 (30 +- 1 pM) and TNF (128 +- 11 pM). Stimulation with IL-10 (11 +- 1 pM) did not change basal production of IL-8. When HBECs were co-stimulated with IL-4 plus TNF, the production of IL-8 was further increased (204 +- 5 pM). In contrast, IL-10 attenuated the effect of TNF during co-stimulation (82 +- 5 pM). IL-13 did not affect the release of IL-8 induced by TNF (111 +- 9 pM). Northern blot analysis of IL-8 mRNA levels showed the highest induction of IL-8 mRNA in HBECs co-stimulated with TNF and IL-4. We conclude from our study that IL-4 directly induces IL-8 release from HBECs and amplifies the release of IL-8 in response to TNF alpha. IL-13 is less active and IL-10 has an inhibitory effect. Airway epithelial cells are able to interact, therefore, with products of both Th1 and Th2 cells with respect to modulating release of IL-8. Author Affiliation: (1) Department of Immunology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic (2) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (3) Veterans Administration Hospital, Omaha, Nebraska Article History: Registration Date: 30/09/2004

TGF-beta plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-beta and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-beta in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-beta for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E2 (PGE2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE2 release by HFL-1 fibroblasts. TGF-beta significantly augmented gel contraction but also induced a 30% increase in PGE2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-beta1 induced-PGE2 release, and enhanced TGF-beta augmented gel contraction without significantly affecting TGF-beta augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-beta augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-beta in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE2 production. Such interactions between glucocorticoids and TGF-beta may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.