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Endogenous inhibitors tightly control the activity of proteinases in the extracellular space. Proteinase/antiproteinase imbalance may be caused by predominance of proteinases, resulting in severe tissue damage or abundance of proteinase inhibitors, leading to a shift in the balance of synthesis and degradation of extracellular matrix proteins and accumulation of these matrix components. Lung fibrosis is characterised by accumulation of fibrous matrix proteins in the alveolar interstitium. The activity of cathepsin D and amounts of cathepsins D and B in bleomycin-injured rat lung tissue and alveolar macrophages were examined. In addition, the activities of cathepsins and cysteine proteinase inhibitors (CPIs) in bronchoalveolar lavage fluid (BALF) were determined. No cathepsin but high CPI activity and large amounts of procathepsin B were detected in the BALF. In the alveolar lumen, the disturbed proteinase/antiproteinase balance for cysteine proteinases was clearly dominated by CPIs. In alveolar macrophages, the main source of increased cathepsin levels, large changes in cathepsin B and D content were observed during the inflammatory phase, corresponding to the occurrence of procathepsin B in BALF. With the end of the phase of tissue remodelling, imbalances in cathepsin and CPI activities were largely eliminated. Immunoblot data, revealing an increase in cathepsin D levels in myofibroblast-like cells compared to fibroblasts and in resting fibroblasts compared to proliferating cells, implicate this proteinase in the differentiation and conversion processes occurring at the beginning of the fibrotic phase of lung injury. The results show that cathepsin amounts and activities are increased transiently in lung tissue during regeneration processes in bleomycin-induced lung injury. Imbalances of cathepsin and cysteine proteinase inhibitors activities are also a phenomenon of the phase of tissue remodelling initiated by lung injury.

Lung fibrosis is the end-point of numerous lung disorders induced by a pneumonia or by a variety of different noxes, one of which is the cytostatic drug bleomycin (BLM). Fibrosis is characterized by excessive extracellular matrix accumulation. Macrophage-fibroblast interactions are suggested to play an important role in the development of this disease. The present study was addressed to investigate one possible pathway of this interaction, the influence of soluble mediators produced by BLM-stimulated macrophages on lung fibroblast collagen synthesis and modification. Conditioned media (CM) of BLM-exposed macrophages of the cell line NR8383 submitted to rat lung fibroblast cultures increased the activity of prolyl 4-hydroxylase (P4H) in fibroblasts in a dose dependent manner. CM of stimulated macrophages increased the collagen concentration in fibroblast culture supernatant. The level of mRNAs specific for the alpha-subunit of P4H and that for alpha1(I) collagen were found to be increased by about two-fold, that for lysyloxidase (LO) by about 2.5-fold in fibroblasts cultured in CM of stimulated macrophages. Pre-incubation of CM of BLM-exposed macrophages with neutralizing antibodies against TGF-beta or against PDGF resulted in a partial reversal of the increasing effect of the CM on P4H- and LO-activities in fibroblasts. Both growth factors, TGF-beta and PDGF, added to fibroblast cultures led to significant increases of P4H activity in the treated cells. We conclude that TGF-beta and PDGF produced by stimulated macrophages are involved in the regulation of the expression of alpha1(I) collagen, of P4H-alpha-subunit and LO in lung fibroblasts. The results indicate that this is not a direct effect but involves the action of a so far unidentified mediator responsible for autocrine stimulation of collagen production.

In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1.

Natterjack toads (Bufo calamita) form metapopulations consisting of interacting local and temporal breeding assemblages. The gene flow between simultaneously reproductive local populations is limited at the adult stage to about 2% roaming males and around 20% of females. The gene flow between temporal populations breeding successively at the same locality is based on about 0-15% of males and 0-29% of females reproducing in different temporal breeding assemblages. The annual mortality rate of males ranges between 50 and 60%, corresponding to a maximum life expectancy of about 7 years. The main cause of local extinction is always deterioration of the habitat, namely destruction of the breeding habitat or a dry climate.

The anti-cancer drug bleomycin (BLM) induces lung injury and triggers apoptosis of alveolar epithelial cells. In epithelia, among other functions, the adhesion protein CD44 promotes the contact to components of the extracellular matrix like hyaluronate. A functional link between apoptosis and the loss of CD44 has been observed in colon carcinoma cells and involvement of CD44 in apoptosis of lung cells has been reported in several studies. The present in vitro study examined the expression of CD44s (CD44 standard) in two human epithelial lung cell lines, L132 and A549, during BLM-induced apoptosis. A loss of CD44s by lung epithelial cells and an increase of the soluble form of this adhesion protein in culture supernatants upon exposure to BLM were observed. Apoptosis was characterized by an activation of caspase-3 as well as by release of cytochrome C into the cytosol as shown for L132 cells. Inhibition of apoptosis by the broad-range caspase inhibitor Z-VAD-fmk reduced CD44 release by both cell lines demonstrating that CD44 release is a result of apoptotic processes. Kinetic experiments failed to discriminate between the initiation of apoptosis and CD44 release. Blocking experiments using antagonistic anti-CD95 receptor antibodies revealed that BLM may cause apoptosis and CD44 release in a CD95-independent manner.

Summary The cytostatic drug bleomycin (BLM) induces pulmonary fibrosis as its main side effect. Fibroblasts in fibrotic foci are the main cellular source for extracellular matrix accumulation that typifies fibrosis. In vitro studies demonstrated the ability of cytotoxic drugs to induce terminal differentiation of fibroblasts. These postmitotic cells are very active in regard to production of collagens. The present study was addressed to investigate the potential of BLM to induce terminal differentiation of rat lung fibroblasts in vitro and the consequences for collagen production and for the expression and activity of the collagen modifying enzyme prolyl 4-hydroxylase (P4H). The BLM effects were compared with those of mitomycin C (MMC), another cytotoxic agent with known potential for initiation of postmitotic differentiation of fibrobasts. BLM induced postmitotic differentiation of rat lung fibroblasts. The capacity of the cells to form clones was diminished by BLM or MMC in a concentration dependent manner. Both drugs initiated the formation of an increasing number of postmitotic cell clones. The postmitotic differentiation was accompanied by an increase in total collagen production by the cells. Administration of BLM to cultures of lung fibroblasts at concentrations of 1 or 10 mU/ml resulted in an increase of the collagen amount to about the 1.5-fold and 1.6-fold of controls, respectively. Treatment of fibroblasts with MMC elevated the collagen level to about the 2-fold. P4H activity and P4Hα mRNA levels in cells exposed to BLM or MMC were found to be increased. We conclude that terminally differentiated fibroblasts might be part of the heterogeneous population of fibroblast-like cells in fibrotic foci responsible for the increased production of collagen during the fibrotic phase of the development of pulmonary fibrosis.

Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

The transdifferentiation of hepatic stellate cells into myofibroblast-like cells and the proliferation of the transdifferentiated cells are controlled by TGF-β1. Little is known about the intracellular signal transducers of TGF-β1. In this paper we show that in cultured hepatic stellate cells TGF-β1 induces activation of Ras, Raf-1, MEK and MAPK p42 and p44. The activation of MAPK depends on the activation of MEK. Our data exclude that the observed effects are mediated by a bFGF or PDGF autocrine loop.© 1997 Federation of European Biochemical Societies.