Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture.

Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5-6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14-21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.

Cytokines induce urokinase-dependent adhesion of human myeloid cells. A regulatory role for plasminogen activator inhibitors.

Differentiation of monocytic precursors often results in adhesive properties thought to be important in migration. In this study, the influence of cytokines, known to induce macrophage differentiation, on the adhesiveness of the monocytic cell line U937 was examined in vitro. Despite development of a macrophage morphology, < 5% of cytokine-stimulated U937 cells were adherent at 24 h. Addition of 1-10 nM urokinase-type plasminogen activator (uPA) induced adherence in the presence of transforming growth factor type beta-1, 1,25-(OH)2 vitamin D3, granulocyte macrophage colony-stimulating factor, or tumor necrosis factor alpha. uPA-dependent adhesiveness was reversible after 24 h of stimulation with cytokines and uPA as adherence was prevented by the subsequent addition of anti-uPA antibodies. Adherence induced by diisopropylfluorophosphate-inactivated uPA was severalfold greater than that seen with active uPA. This difference was largely due to cell-surface turnover of active uPA complexed with plasminogen activator inhibitor (PAI). These data indicate that cytokines prime monocyte progenitors for uPA receptor-mediated signals leading to adherence, continued uPA receptor occupancy is required for adherence, and PAI decreases adherence by promoting clearance of uPA/PAI complexes. Thus the interaction of uPA and PAI at the cell surface, known to affect extracellular matrix proteolysis and hence myeloid cell migration, also regulates adhesion. The coordinated regulation of these two uPA functions by PAI may enhance the migratory potential of monocytic cells.

Cigarette smoking induces an elastolytic cysteine proteinase in macrophages distinct from cathepsin L.

Degradation of the interstitium of the lung by elastolytic enzymes is thought to be a critical component of the pathogenesis of emphysema. Alveolar macrophages are increased in numbers in cigarette smokers and contain the elastolytic cysteine proteinase cathepsin L. We sought to determine if cigarette smoking induces a change in cathepsin L levels in alveolar macrophages which would, in turn, alter the expression of elastolytic activity. Lysates of smokers' macrophages, assayed at pH 5.50, degraded more than seven times as much [3H]elastin as did lysates from nonsmokers' macrophages (44 +/- 20.8 vs. 6 +/- 1.6 micrograms.10(6) cells-1.24 h-1). Little or no activity was demonstrable at neutral pH. Immunoblots of macrophage lysates demonstrated that smokers' cells contain 3.7 +/- 1.1 times as much 25-kDa cathepsin L antigen as nonsmokers' cells. However, as judged by active site labeling, levels of active cathepsin L in smokers and nonsmokers are indistinguishable, suggesting that most of the 25-kDa antigen found in smokers' macrophages is inactive. Inhibitors of cathepsin L had little effect on lysate elastolytic activity, confirming that an enzyme other than cathepsin L is responsible for the increased elastolytic activity seen in smokers' macrophages. Further experiments demonstrated that this second enzyme(s) has a profile of inhibition indicating that it is a cysteine proteinase with optimal activity at pH 5.50. It is this second elastolytic cysteine proteinase(s) that is induced by exposure to cigarette smoke and is responsible for the sevenfold increase in elastolytic activity found in smokers' macrophage lysates.

Alveolar macrophage urokinase receptors localize enzyme activity to the cell surface.

Human alveolar macrophages are known to synthesize urokinase (uPA) and a specific plasminogen activator inhibitor, PAI-2. In this study we have identified a uPA receptor expressed by these cells and defined the influence of PAI-2 on the interaction of uPA with its receptor. Alveolar macrophages from four normal volunteers were incubated with 55 kDa 125I-labeled uPA (0.24-8 nM) in the presence or absence of excess unlabeled uPA. Specific and saturable binding was demonstrable in all cases. Scatchard plots were linear; regression analysis revealed a mean Kd of 5.25 nM (range 3.2-6.7) and mean Bmax of 30.7 femtomoles/10(5) cells (range 21.5-34.5). The structure of the uPA receptor was defined by electroblotting membrane fractions of macrophages and sequentially exposing filters to uPA and uPA antibodies. Membranes from macrophages demonstrated binding of either uPA or a 15-kDa amino-terminal fragment of uPA to a 55- to 60-kDa glycosylated membrane protein. Binding of uPA to filters was blocked by a synthetic oligopeptide containing the known receptor binding region of native uPA. Preincubation of 125I-uPA with PAI-2 dramatically reduced the rate of association of uPA with macrophage uPA receptor. Conversely, receptor-bound uPA activity was less susceptible to inhibition by PAI-2 than soluble uPA activity. These data indicate that normal alveolar macrophages express uPA receptors. The receptor preferentially binds and protects free uPA over complexed enzyme, indicating that one function of the receptor is to allow the cells to express active uPA in an inhibitor-rich environment.

Developmental expression of plasminogen activator inhibitor type 1 by human alveolar macrophages. Possible role in lung injury.

Urokinase activity is regulated by the specific endogenous plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2). One of these inhibitors, PAI-1, has been directly implicated in connective tissue metabolism by virtue of its ability to bind extracellular matrix proteins. Because the normal lung is relatively rich in urokinase and abnormalities in urokinase activity have been associated with fibrotic lung diseases, we have explored the possibility of local production of PAI-1 and PAI-2 in human lung. Reverse transcription and subsequent amplification by the polymerase chain reaction of total lung RNA revealed PAI-1 mRNA in each of three normal samples and in two specimens from patients with the adult respiratory distress syndrome (ARDS). In situ hybridizations of lung biopsy specimens from a patient with ARDS with cRNA probes to PAI-1 and PAI-2 indicated that alveolar macrophages express PAI-1 mRNA during the acute injury phase. Subsequent reverse transcription and PCR amplification of normal human monocyte and alveolar macrophage mRNA revealed that neither cell type expressed mRNA for urokinase inhibitors. However, after 24 h stimulation with endotoxin in vitro, monocytes were strongly positive for PAI-2 but negative for PAI-1 mRNA whereas, under the same conditions, alveolar macrophages exhibited mRNA for both PAI-1 and PAI-2. Metabolic labeling of endotoxin-stimulated alveolar macrophages with 35S-methionine followed by immunoprecipitation with PAI-1 and PAI-2 antibodies revealed that macrophages synthesized both PAI-1 and PAI-2. As judged by immunoprecipitation and functional studies, PAI-2 was found to be the major intracellular PA inhibitor whereas PAI-1 was found to predominate outside the cell. Thus, mononuclear phagocytes exhibit a developmental potential for PAI-1 expression. The release of PAI-1 by stimulated macrophages, as observed in the setting of ARDS, may be one mechanism by which these cells promote connective tissue accumulation.

Uptake of extracellular enzyme by a novel pathway is a major determinant of cathepsin L levels in human macrophages.

The phorbol myristate acetate (PMA)-differentiated myelomonocytic cell line, THP-1, and human alveolar macrophages contain the cysteine proteinase cathepsin L. This enzyme is synthesized as a 43-kD proenzyme and processed to the active 25-kD form. Differentiation of THP-1 cells in the presence of human serum resulted in an increase in the size of the vacuolar compartment and the accumulation of more 25-kD cathepsin L antigen, as compared with THP-1 cells differentiated in the presence of fetal calf serum. Cells cultured in both types of sera have equivalent levels of cathepsin L mRNA. Metabolic labeling experiments demonstrated equivalent rates of synthesis, processing to the active form, and persistence in both culture conditions. An extracellular source of enzyme was documented by immunoblotting human serum which demonstrated 25-kD cathepsin L antigen; furthermore, we demonstrated that both THP-1 cells, differentiated in human serum, and human alveolar macrophages take up the 43-kD proenzyme and process it to the 25-kD form. Thus, human serum contains a factor(s) that induces both a marked increase in the size of the vacuolar compartment in differentiated THP-1 cells and a novel pathway that is responsible for the uptake and processing of extracellular cathepsin L. The activity of this inducible pathway is a major determinant of levels of intracellular cathepsin L. Cathepsin L is a potent elastase and the regulation of its uptake and processing may play a role in the pathogenesis of disease processes characterized by the destruction of elastin, such as pulmonary emphysema.