Tadashi Mio

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix.

SummaryFibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E2 (PGE2), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE2 inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.

Human bronchial epithelial cells modulate collagen gel contraction by fibroblasts

Connective tissue contraction is an important aspect of both normal wound healing and fibrosis. This process may contribute to small airway narrowing associated with certain airway diseases. Fibroblast-mediated contraction of a three-dimensional collagen gel has been considered a model of tissue contraction. In this study, the ability of primary cultured human bronchial epithelial cells (HBEC) obtained by bronchial brushings to modulate fibroblast gel contraction was evaluated. Human lung fibroblasts (HFL1) were cast into type I collagen gels. The gels were floated both in dishes containing a monolayer of HBEC or in dishes without HBEC. Contraction assessed by measuring the area of gels was increased at all time points from 24 h up to 96 h of coculture. At 48 h, coculture of HBEC with fibroblasts resulted in significantly more contraction than fibroblasts alone (36.6 +/- 1.2 vs. 20.4 +/- 1.7%, P < 0.05). Lipopolysaccharide (LPS, 10 micrograms/ml) stimulation of the HBEC augmented the contraction (44.9 +/- 1.0%, P < 0.05 vs. HBEC). In the presence of indomethacin, the augmentation by LPS was increased further (52.2 +/- 4.3%, P < 0.05 vs. HBEC with LPS), suggesting that prostaglandins (PGs) are present and may inhibit contraction. Consistent with this, PGE was present in HBEC-conditioned medium. Bronchial epithelial cell conditioned medium had an effect similar to coculturing. SG-150 column chromatography revealed augmentive activity between 20 and 30 kDa and inhibitory activity between 10 and 20 kDa. Measurement by enzyme-linked immunosorbent assay confirmed the presence of the active form of transforming growth factor (TGF)-beta 2. The stimulatory activity of conditioned medium was blocked by adding anti-TGF-beta antibody. These data demonstrate that, through the release of factors including TGF-beta 2 which can augment and PGE which can inhibit, HBEC can modulate fibroblast-mediated collagen gel contraction. In this manner, HBEC may modulate fibroblast activities that determine the architecture of bronchial tissue.Connective tissue contraction is an important aspect of both normal wound healing and fibrosis. This process may contribute to small airway narrowing associated with certain airway diseases. Fibroblast-mediated contraction of a three-dimensional collagen gel has been considered a model of tissue contraction. In this study, the ability of primary cultured human bronchial epithelial cells (HBEC) obtained by bronchial brushings to modulate fibroblast gel contraction was evaluated. Human lung fibroblasts (HFL1) were cast into type I collagen gels. The gels were floated both in dishes containing a monolayer of HBEC or in dishes without HBEC. Contraction assessed by measuring the area of gels was increased at all time points from 24 h up to 96 h of coculture. At 48 h, coculture of HBEC with fibroblasts resulted in significantly more contraction than fibroblasts alone (36.6 ± 1.2 vs. 20.4 ± 1.7%, P < 0.05). Lipopolysaccharide (LPS, 10 μg/ml) stimulation of the HBEC augmented the contraction (44.9 ± 1.0%, P < 0.05 vs. HBEC). In the presence of indomethacin, the augmentation by LPS was increased further (52.2 ± 4.3%, P< 0.05 vs. HBEC with LPS), suggesting that prostaglandins (PGs) are present and may inhibit contraction. Consistent with this, PGE was present in HBEC-conditioned medium. Bronchial epithelial cell conditioned medium had an effect similar to coculturing. SG-150 column chromatography revealed augmentive activity between 20 and 30 kDa and inhibitory activity between 10 and 20 kDa. Measurement by enzyme-linked immunosorbent assay confirmed the presence of the active form of transforming growth factor (TGF)-β2. The stimulatory activity of conditioned medium was blocked by adding anti-TGF-β antibody. These data demonstrate that, through the release of factors including TGF-β2 which can augment and PGE which can inhibit, HBEC can modulate fibroblast-mediated collagen gel contraction. In this manner, HBEC may modulate fibroblast activities that determine the architecture of bronchial tissue.

IL-4 induces ICAM-1 expression in human bronchial epithelial cells and potentiates TNF-α

Interleukin (IL)-4 is thought to contribute to the Th2 type of immune response and hence the development of allergic reactions such as asthma. In asthmatic patients, the airway epithelium expresses increased amounts of the cell surface adhesion molecule intercellular adhesion molecule (ICAM)-1 (CD54). One cytokine capable of inducing ICAM-1 in airway epithelial cells, tumor necrosis factor-α (TNF-α), is present in asthma. This study evaluated if IL-4 either alone or together with TNF-α costimulation might modulate CD54 expression by human bronchial epithelial cells (HBECs). CD54 positivity increased in response to IL-4 (16 ± 2% positive vs. 3 ± 1%, P < 0.01); greater induction of CD54 resulted from TNF-α (45 ± 2%, P < 0.001). Costimulation with TNF-α plus IL-4 further augmented expression (56 ± 1%, P < 0.05). Immunoperoxidase results were confirmed by flow cytometry. RT-PCR revealed no increase in ICAM-1 mRNA expression under control conditions or after stimulation with IL-4 alone. TNF-α increased IL-4 mRNA, and IL-4 potentiated this. Functionally, IL-4 augmented the adhesion of THP-1 monocyte/macrophage cells to monolayers of HBECs both alone and in the presence of TNF-α. We conclude that 1) IL-4 augments epithelial cell ICAM-1 expression, 2) IL-4 potentiates the adhesion of THP-1 monocyte/macrophage cells to epithelial cells, and 3) modulation of epithelial cell ICAM-1 expression by IL-4 may play a role in the immunopathology of bronchial asthma.

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture.

SummaryIn vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.

Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells.

Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.

IL-4 and IL-13 Stimulate Human Bronchial Epithelial Cells to Release IL-8

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.

The CD14 molecule participates in regulation of IL-8 and IL-6 release by bronchial epithelial cells

The soluble form of the leukocyte membrane antigen CD14 is known to increase the sensitivity of endothelial and epithelial cell lines to bacterial lipopolysaccharide (LPS). This molecule also directly induces cytokine production in monocytes. Here, the effect of sCD14 and LPS on the release of IL-6 and IL-8 by human bronchial epithelial cells (HBECs) was studied. Soluble CD14 induced cytokine production both in the presence and absence of LPS. In addition, neither sCD14 nor anti-CD14 monoclonal antibody which blocks the interaction of LPS with CD14 had any effect on the binding of LPS to HBECs. These data suggest that sCD14 may induce the release of IL-6 and IL-8 from HBECs. However, the binding of LPS to bronchial epithelium appears to be mediated by CD14-independent mechanisms.

Th2-type cytokines modulate IL-6 release by human bronchial epithelial cells.

The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial cells (HBECs), has been found to play a role in IL-4 dependent IgE synthesis. Since the allergic reaction in bronchial asthma is associated with the upregulation of IL-4 and Th2 type of immune response, the purpose of our study was to assess whether IL-4 and related cytokines IL-10 and IL-13 regulate IL-6 release by HBEC s. HBECs were obtained by bronchial brushing, cultured in LHC-9/RPMI 1640. At the third passage the cells were stimulated with cytokines (0.1-20 ng/ml) diluted in unsupplemented media for 24 h. The supernatants were tested for IL-6 content by sandwich ELISA. Unstimulated HBECs produced detectable amounts of IL-6 (368+/-25 pg/ml). Exposure to IL-10 (368+/-22 pg/ml) and IL-13 (395+/-6 pg/ml) resulted in little changes. IL-4 caused a slight but significant increase in IL-6 release (530+/-45 pg/ml), P<0.05, TNFalpha (1657+/-85 pg/ml) and IFNgamma (1953+/-37 pg/ml) showed strong induction of IL-6 release in HBECs (P<0.005 and P<0.001, respectively). Both IL-4 and IL-13 significantly inhibited TNF induced IL-6 release (P<0.01 for both) while augmenting the effect of IFNgamma (P<0.005 and P<0.01, respectively.). IL-10 was without a significant effect. We conclude that Th2-type cytokines IL-4 and IL-13 affect the release of IL-6 by HBECs in response to TNFalpha (inhibition) and IFgamma (augmentation). IL-10 had no effect on the regulation of IL-6 release. Modulation of IL-6 levels by Th2-type cytokines may play a role in allergic reactions through the IL-6 promoting effect on IL-4 mediated IgE production.

Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity

Fibronectin is a major product of fibroblasts and can mediate diverse functions including wound healing. Chronic bacterial infections are generally associated with a marked decreased in the ability to repair. We therefore hypothesized that bacterial endotoxin, lipopolysaccharide (LPS), might alter fibroblast fibronectin production. LPS augmented fibronectin production by fibroblasts and also stimulated the release of fibronectin from cell layers. An increase in new protein synthesis appeared to account for part of the increased fibronectin, because the inhibitor of protein synthesis, cycloheximide, inhibited the increase in total production of fibronectin. Cycloheximide did not attenuate the increased release of fibronectin into the culture medium. This increased release appeared to be caused, at least in part, by fragmentation of fibronectin by proteases contained in LPS preparations. In this regard all preparations of LPS tested were found to cleave fibronectin. Finally, zymograms indicated that LPS could also cleave gelatin with at least two bands of proteolytic activity but that it did not cleave bovine serum albumin or ovalbumin. These results indicate that the ability of bacterial products to alter fibronectin production and to degrade this macromolecule may account for altered wound repair that occurs with chronic bacterial infection.