Transforming growth factor-beta-induced collagen synthesis by human liver myofibroblasts is inhibited by alpha2-macroglobulin.

BACKGROUND: Transforming growth factor-beta (TGFbeta) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFbeta in different systems has been shown to be influenced by alpha2-macroglobulin (alpha2M), a serum protein with strong protease-scavenging and cytokine-binding properties. AIMS: In the present study, alpha2M derived from normal human plasma has been tested for its ability to modulate the TGFbeta-induced collagen production by human liver fat-storing cells (FSC), which had transformed into alpha-smooth muscle actin-expressing myofibroblasts in culture. METHODS: Alpha2M has been tested after activation with methylamine (alpha2M-Me), an in vitro equivalent of protease activated alpha2M. The binding of 125I-TGFbeta1 to activated forms of alpha2M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of 3H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of alpha2M was studied by means of immunofluorescence. RESULTS: TGFbeta (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFbeta. Alpha2M-Me reduced this TGFbeta-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 microg/ml alpha2M-Me onward. Upon addition of 100 microg/ml alpha2M-Me the effect of TGFbeta was reduced by 60% to 128+/-31% (mean+/-SD) of control values in the absence of TGFbeta. Human liver myofibroblasts endocytosed alpha2M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFbeta-activity by alpha2M-Me may be explained by receptor-mediated clearance of alpha2M-TGFbeta complexes by the cells. CONCLUSIONS: TGFbeta-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated alpha2M. From these results, we hypothesize that alpha2M may have an antifibrogenic effect in vivo by interference with TGFbeta-induced matrix synthesis during liver fibrosis.

Localization of alpha 2-macroglobulin protein and messenger RNA in rat liver fibrosis: evidence for the synthesis of alpha 2-macroglobulin within Schistosoma mansoni egg granulomas.

alpha 2-Macroglobulin (alpha 2M) in the rat is a strong-reacting acute-phase protein with potent protease-inhibiting and cytokine-binding properties. Production of alpha 2M is ascribed mainly to liver parenchymal cells. In the present study, we investigated, by means of immunohistochemistry and in situ hybridization, whether fibrosis in the rat liver induced by Schistosoma mansoni eggs leads to local production of alpha 2M. alpha 2M protein and messenger RNA (mRNA) in the unaffected liver tissue, as well as serum values of alpha 2M, were comparable in control rats and egg-injected rats, at 1, 3, and 8 weeks after injection of the eggs. alpha 2M was homogeneously distributed across the liver lobule. In contrast, at the sites of the granulomas, a strong increase in alpha 2M was observed. alpha 2M mRNA was expressed by granuloma cells, but not by the surrounding liver parenchymal cells. Within the granulomas, alpha 2M protein was present in numerous spindle-shaped cells and was diffusely distributed in the extra-cellular matrix. Using double-staining techniques, a subpopulation of the alpha 2M-positive cells in the granulomas appeared to be desmin-positive, suggesting a myofibroblast origin. In addition, parenchymal cells directly surrounding the granulomas contained alpha 2M protein in approximately 50% of the granulomas 1 week after injection of the eggs. In situ hybridization on consecutive sections revealed that these parenchymal cells showed only background activity of alpha 2M mRNA, suggesting uptake of alpha 2M-protein by these parenchymal cells and previous activation of alpha 2M by proteases within the granuloma. The significance of the present study is that alpha 2M is produced locally at sites of inflammation and liver fibrosis, without measurable increase of serum levels of alpha 2M. Unexpectedly, alpha 2M present at the sites of the granulomas is not produced by the liver parenchymal cells, but rather by granuloma cells.

Interleukin-6 production by human liver (myo)fibroblasts in culture. Evidence for a regulatory role of LPS, IL-1 beta and TNF alpha.

BACKGROUND/AIMS: Interleukin-6 is a major trigger for the synthesis of acute phase proteins by liver parenchymal cells. Acute phase proteins may contribute to the regulation of liver fibrosis by inhibition of proteases (e.g. collagenase) and by binding of cytokines. Since liver (myo)fibroblasts play an important role in the production of extracellular matrix in fibrotic livers, a study was undertaken into whether these cells are able to synthesize interleukin-6, which would give them the opportunity to contribute to regulation of synthesis of acute phase proteins by neighbouring parenchymal cells. METHODS: In the present study we investigated interleukin-6 production by two cell types obtained from human liver tissue: human fat-storing cells obtained from 5-15% Percoll fractions, which transformed in culture into myofibroblasts co-expressing alpha-smooth muscle actin and vimentin (VA cells) and fibroblasts obtained from 30-40% Percoll fractions which express vimentin only (V vells). Interleukin-6 production was measured in culture media of these cells using an enzyme-linked immunosorbent assay after incubation with lipopolysaccharide, and mediators like interleukin-1 beta, tumor necrosis factor-alpha, transforming growth factor-beta and interferon-gamma, known to be present in elevated concentrations in fibrotic livers. RESULTS: Unstimulated human liver (myo)fibroblasts produced considerable amounts of interleukin-6 (287 ng/mg cellular protein (VA cells), and 54 ng/mg cellular protein (V cells), within 48 h). Biological activity of these high concentrations of interleukin-6 measured in the enzyme-linked immuno-sorbent assay was confirmed in the B9-bioassay for interleukin-6 and by stimulation of alpha 2-macroglobulin production in rat liver parenchymal cell cultures. Lipopolysaccharide, interleukin-1 beta and tumor necrosis factor-alpha were potent stimulators of basal interleukin-6 production by VA and V cells, 1 microgram/ml lipopolysaccharide enhanced basal interleukin-6 production 3-fold within 48 h. 100 U/ml interleukin-1 beta and 1000 U/ml tumor necrosis factor-alpha each stimulated basal interleukin-6 production by VA cells 2-5 fold, whereas V cells were stimulated 10-25 fold. These effects were specific since the stimulation by lipopolysaccharide was completely inhibited by polymyxin B and the enhancing effects of interleukin-1 beta and tumor necrosis factor-alpha were neutralized by specific antibodies. Transforming growth factor-beta and interferon gamma did not influence interleukin-6 synthesis by either cell type in culture. CONCLUSIONS: These results indicate that transformed fat-storing cells (VA cells) and fibroblasts (V cells) may function as a local source of interleukin-6 in the human liver. Since interleukin-6 plays a key role in the regulation of the production of acute phase proteins by liver parenchymal cells, we hypothesize that human liver (myo)fibroblasts may stimulate local production of acute phase proteins in the fibrotic liver, thus contributing to local regulation of inflammatory and fibrogenic reactions.

Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1 beta, IL-4, TGF beta and IFN gamma.

BACKGROUND/AIMS: Different cytokines have been described in fibrotic livers, including interleukin-1, interleukin-4 and interferon gamma, which are capable of regulating collagen production in human skin and lung fibroblasts. METHODS: To investigate possible involvement of interleukin-1, interleukin-4 and interferon gamma in the regulation of collagen production in human liver fibrosis, we studied the effects of these cytokines on collagen synthesis by nonparenchymal human liver cells in vitro. The effects of interleukin-1, interleukin-4 and interferon gamma were compared with the effect of transforming growth factor-beta, a well-known stimulator of collagen synthesis in liver fibrosis. Using a Percoll gradient we isolated two types of fibroblast-like cells from human liver tissue: fat-storing cells, which transformed in culture into myofibroblasts co-expressing vimentin and alpha-smooth muscle actin (VA-cells), and fibroblasts expressing vimentin only (V-cells). Production of collagen was measured in confluent cell cultures by incorporation of 3H-proline into collagenase degradable proteins. RESULTS: The cytokines studied had comparable effects on collagen synthesis in confluent cultures of VA-cells obtained from three different human livers and in confluent cultures of V-cells. Interleukin-1 beta and interleukin-4 enhanced collagen synthesis dose-dependently. 100 U/ml interleukin-1 beta stimulated collagen synthesis up to 174 +/- 25% (mean +/- sd, VA-cells) and 140 +/- 7% (V-cells) of control values. 1000 U/ml interleukin-4 enhanced collagen formation up to 195 +/- 58% (mean +/- sd, VA-cells) and 153 +/- 4% (V-cells) of control values after 48 h. These values were comparable to the stimulatory effects induced by transforming growth factor-beta (235 +/- 33% (mean +/- sd, VA-cells) and 150 +/- 18% of control values (V-cells) after incubation with 10 ng/ml transforming growth factor-beta for 48 h). Interferon gamma reduced both basal (36 +/- 29% (mean +/- sd) of control values in VA-cells, and 59 +/- 9% in V-cells) and transforming growth factor-beta induced collagen synthesis. CONCLUSIONS: These results indicate that in addition to the well-known role of transformed fat-storing cells (VA-cells) in collagen synthesis, fibroblasts (V-cells) may contribute to collagen production in human liver tissue. Moreover, these data demonstrate that in addition to the extensively documented collagen-inducing mediator transforming growth factor-beta, other cytokines present in fibrotic liver tissue like interleukin-1 beta and interleukin-4 may contribute to the enhanced synthesis of collagen, whereas interferon gamma may reduce collagen formation during liver fibrosis in man.

Th1 lymphokine production profiles of nickel-specific CD4+T-lymphocyte clones from nickel contact allergic and non-allergic individuals.

Panels of nickel-specific T-lymphocyte clones (TLC) were prepared from nickel-allergic and non-allergic donors. TLC from both panels showed similar levels of expression of TCR alpha/beta, CD4, CD2, CD25, and CD29 and recognized nickel in association with class II HLA molecules with restriction determinants in HLA-DR, HLA-DP, and HLA-DQ. The lymphokine secretion was analyzed in TLC from both panels upon antigen-specific or non-specific stimulation and was compared with the secretion profiles of representants of pre-established human atopen-specific Th1 and Th2 cells. Nickel-specific TLC from both panels showed a lymphokine secretion pattern similar to the atopen-specific Th1 cells, although there was some variation from clone to clone. Most TLC secreted substantial amounts of IFN-gamma, IL-2, TNF-alpha, and GM-CSF, but little or no IL-4 and IL-5. The variation observed mainly concerned IL-2 secretion that could be low or absent in some of the TLC. The general secretion pattern did not change upon different modes of stimulation, including activation via CD3, CD2, or CD28. Because nickel-specific TLC from allergic and non-allergic individuals show a similar Th1 secretion pattern, the present results give no evidence that aberrant lymphokine secretion by CD4+T cells determines the contact allergic state, as was found for atopic allergy in a previous study.

Mast cells in early stages of antigen-induced arthritis in rat knee joints.

The occurrence of mast cells has been investigated in inflamed and control knee joints of rats suffering from antigen-induced arthritis, an animal model of rheumatoid arthritis in man. Rats were immunized with methylated bovine serum albumin (mBSA) followed by an intra-articular injection of mBSA (arthritis, right joints) or saline (control, left joints). Rats developed severe acute synovitis associated with cartilage erosion in the arthritic joints, whereas control joints did not show any noticeable changes. Mast cells were counted in synovial and adjacent tissues in cryostat sections of whole knee joints stained with Toluidine Blue O. The area of the synovium of each knee joint was determined using survey photomicrographs and a morphometer. Both total numbers of mast cells and frequency of mast cells in inflamed synovia were decreased after 1 day after induction of arthritis. The frequency of mast cells remained decreased up to 14 days after induction of arthritis. Morphological indications for degranulation of mast cells were never found in inflamed joints. It is concluded, therefore, that mast cells do not play a significant role in the inflammatory process during the early phase of arthritis.