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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

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-[Alpha], interleukin-1 [Beta], and interferon-[Gamma]), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% (P [is less than] 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% (P [is less than] 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% (P [is less than] 0.01). [[Alpha].sub.1]-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema. matrix metalloproteinases; collagen; fibroblasts; interleukin-1; tumor necrosis factor

Skold, C. M., X. D. Liu, T. Umino, Y. K. Zhu, R. F. Ertl, D. J. Romberger, and S. I. Rennard. Blood monocytes attenuate lung fibroblast contraction of three-dimensional collagen gels in coculture. Am J Physiol Lung Cell Mol Physiol 279: L667-L674, 2000.--Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors ([is greater than] 95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of [PGE.sub.2], a well-known inhibitor of gel contraction, was higher (P [is less than] 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1[Beta] and tumor necrosis factor-[Alpha], whereas fibroblasts increased synthesis of [PGE.sub.2]. Antibodies to interleukin-1[Beta] and tumor necrosis factor-[Alpha] blocked the monocyte inhibitory effect and reduced the amount of [PGE.sub.2] produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling. interleukin-1[Beta]; matrix; prostaglandin [E.sub.2]; remodeling; tumor necrosis factor-[Alpha]

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway. Am J Physiol Lung Cell Mol Physiol 278: L1032-L1038, 2000.--Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 [micro]M) significantly augmented collagen gel contraction by HFL1 cells (78.5 [+ or -] 0.8 vs. 58.3 [+ or -] 2.1, P [is less than] 0.01), whereas S-nitroso-N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-l,2,3-oxadiazolium chloride, NONOate, and [N.sup.G] monomethyl-L-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1H-[1,2,4]-oxadiazolo-[4,3-[Alpha]]-quinoxalin-l-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and [PGE.sub.2] production were measured by immunoassay after 2 days of gel contraction. SNP inhibited [PGE.sub.2] production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and [PGE.sub.2] production. Addition of exogenous [PGE.sub.2] blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased [PGE.sub.2] production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway. nitric oxide; guanosine 3',5'-cyclic monophosphate