The contribution of PARs to inflammation and immunity to fungi.

During inflammation, host- and microbial-derived proteases trigger the activation of protease-activated receptors (PARs), a family of G-protein-coupled receptors. We report here that activation of Toll-like receptors (TLRs) by fungi unmasks an essential and divergent role for PAR(1) and PAR(2) in downstream signaling and inflammation. TLRs activated PARs and triggered distinct signal transduction pathways involved in inflammation and immunity to Candida albicans and Aspergillus fumigatus. Inflammation was promoted by PAR(1) and PAR(2) activation in response to Candida and by PAR(2) inhibition in response to Aspergillus. This occurred by TLR regulation of PAR signaling, with TLR2 promoting PAR(1) activity, and TLR4 suppressing PAR(2) activity. Thus, tissue injury and pathogens induce signals that are integrated at the level of distinct TLR/PAR-dependent pathways, the exploitation or subversion of which contributes to divergence in microbial promotion of inflammatory response.

Immunity to Aspergillus fumigatus: the basis for immunotherapy and vaccination.

Efficient responses to fungi require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) immune responses. Murine and human DCs phagocytose conidia and hyphae of Aspergillus fumigatus through distinct recognition receptors. The engagement of distinct receptors translates into disparate downstream signaling events, ultimately affecting cytokine production and co-stimulation. Adoptive transfer of different types of DCs activates protective and non-protective Th cells as well as regulatory T cells, ultimately affecting the outcome of the infection in mice with invasive aspergillosis. The infusion of fungus-pulsed or RNA-transfected DCs also accelerates recovery of functional antifungal Th 1 responses in mice with allogeneic hematopoietic stem cell transplantation. Patients receiving T cell-depleted allogeneic hematopoietic stem cell transplantation are unable to develop antigen-specific T cell responses soon after transplant due to defective DC functions. Our results suggest that the adoptive transfer of DCs may restore immunocompetence in hematopoietic stem cell transplantation by contributing to the educational program of T cells. Thus, the remarkable furictional plasticity of DCs can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to the fungus.

Synthesis and secretion of transforming growth factor-beta1 by human desmoid fibroblast cell line and its modulation by toremifene.

The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

Bronchial epithelial cell matrix production in response to silica and basic fibroblast growth factor.

BACKGROUND: Previous studies show that macrophages, lung fibroblasts, and their soluble mediators are responsible for the onset and development of pulmonary fibrosis. This study was conducted to determine whether airway epithelial cells are also directly involved in response to fibrogenic agents and consequently in the pathogenesis of lung fibrosis. To verify the hypothesis, we determined whether silica acts directly on human bronchial epithelial cells by stimulating cytokine and growth factor release and by modifying matrix production. MATERIALS AND METHODS: An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o (16HBE), was used. The expression profile of some proinflammatory interleukins (ILs), such as IL-1alpha, IL-1beta and IL-6 and their modulation by silica, were evaluated by polymerase chain reaction (PCR) analysis. Transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) mRNA levels were tested by Northern blotting in the presence and in the absence of silica. The silica- and/or bFGF-induced effects on matrix components (total proteins, collagen, and fibronectin) were also evaluated using radio-labeled precursors. RESULTS: The results demonstrated 16HBE internalized silica particles. Silica induced a little IL-6 secretion, without affecting IL-1 and TGFbeta isoform production and strongly stimulated bFGF mRNA level and bFGF protein secretion. Silica also induced changes in 16HBE production of total proteins, collagen, and fibronectin production. When added in combination with the growth factor, it strengthened bFGF stimulation of matrix component secretion. CONCLUSIONS: These results support the hypothesis that the changes in matrix components are due to a direct effect of silica on bronchial epithelial cells. Silica-induced over-secretion of bFGF suggests that autocrine and paracrine differentiation loops for bFGF may also be operative and that these mechanisms may be involved in the pathogenesis of pulmonary fibrosis. In the future, cytokine-directed therapeutic strategies might find a place in clinical practice.

Silica and its antagonistic effects on transforming growth factor-beta in lung fibroblast extracellular matrix production.

BACKGROUND: Silicosis, a pneumoconiosis marked by interstitial pulmonary fibrosis, is caused by inhalation of free crystalline silica particles. When silica particles are injected into the lower lung, they are translocated across the epithelium into the interstitial space, where macrophage-derived growth factors affect lung fibroblast proliferation and collagen deposition. We hypothesized that silica may act directly on pulmonary fibroblasts modifying extracellular matrix (ECM) synthesis and that the effects of silica may be mediated by transforming growth factor-beta (TGFbeta) overproduction. METHODS: To test this hypothesis, we studied a human lung fibroblast cell line (WI-1003) exposed to silica in vitro. We investigated cell morphology by electron microscopic procedure, cell growth, collagen production, and glycosaminoglycans (GAG) composition by radiolabeled precursors. Cytokine and growth factor synthesis were evaluated by specific enzyme-linked immunoadsorbent assay kits and Northern blotting analysis. RESULTS: Pulmonary fibroblasts internalized silica particles without detectable cell damage. Silica directly stimulated collagen synthesis and decreased the amount of 3H-glucosamine-labeled GAG. Silica-treated fibroblasts secreted less TGFbeta than untreated controls, antagonized the stimulatory effect of TGFbeta on ECM synthesis, and reversed TGFbeta-induced inhibition of cell proliferation. Northern blotting analysis showed increased interleukin-1alpha (IL-1alpha) mRNA after silica treatment. IL-1alpha had no influence on collagen synthesis but increased the number of WI-1003 fibroblasts. CONCLUSIONS: These results support our hypothesis that lung fibroblasts are direct silica targets. However, contradicting our hypothesis, silica antagonized TGFbeta activities through a TGFbeta downregulation and an IL-1alpha upregulation. The complex pattern of TGFbeta and IL-1alpha regulation in pulmonary fibroblasts is imbalanced by silica exposure and might play a key role in silica-mediated pulmonary fibrosis.

In vitro cytotoxic effects of orthodontic appliances.

The objective of this study was to evaluate the effects of an orthodontic appliance and of its components (brackets, bands, and arch wires) on some cell functions. Fibroblasts were cultured either in the presence of one unwashed orthodontic appliance, or one orthodontic appliance immersed in MEM for 28 days before use (washed appliance), or in the presence of MEM in which the appliances had been immersed. At the end of in vitro maintenance, morphological studies were carried out with SEM and TEM. Cell proliferation and GAG synthesis and secretion by radio-labeled precursors were assessed. The data indicated that unwashed appliances were more cytotoxic than washed ones. Moreover, the arch wire was the most biocompatible component of the orthodontic appliance, and the bracket was the least biocompatible. A comparative study into the effects on cell proliferation of the most common metal ions released by the appliances was also carried out. At the concentration released by one orthodontic appliance immersed for 28 days, the highest reduction in DNA synthesis was observed in the presence of Cu(++).

Interleukin secretion, proteoglycan and procollagen alpha(1)(I) gene expression in Crouzon fibroblasts treated with basic fibroblast growth factor.

The present study provides the first evidence that fibroblasts obtained from patients affected by Crouzon syndrome, a rare craniosynostosis, despite mutations in the high-affinity bFGF receptor retain their capacity to respond to bFGF. The growth factor reduces IL-1 secretion, downregulates biglycan and procollagen alpha(1)(I), and increases betaglycan expression. Since betaglycan is a co-receptor for bFGF signalling, an alternative signal transduction pathway is suggested in Crouzon fibroblasts, to explain the documented changes in ECM macromolecule production.

Biocompatibility of alloys used in orthodontics evaluated by cell culture tests.

The cytotoxicity of the most common alloys used in orthodontic appliances was determined by cell culture testing. Human gingival fibroblasts were cultured on 304 and 316 stainless steel, on brazing alloy composed of palladium (Pd), copper (Cu), and silver (Ag), and on plastic substrate (control). Studies were carried out with SEM and radiolabeled precursor incorporation. Cells were cultured in MEM without serum but with the addition of (3)H-thymidine to evaluate cell proliferation and (3)H-glucosamine to evaluate glycosaminoglycan (GAG) synthesis and secretion in the culture medium. Moreover, gingival fibroblasts were cultured in the presence of some metal ions generally released by orthodontic appliances to evaluate the cytotoxic effects of single ions. Morphologic observations with SEM and radiolabeled incorporation studies showed that 304 and 316 stainless steel were more biocompatible than the brazing alloy. Among the metal ions tested, Ag and Pd, constituents of the brazing alloy, showed the highest cytotoxicity.

TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins.

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity.