Cell proliferation in the bone marrow and blood of two patients with metastatic breast cancer who were treated with granulocyte colony-stimulating factor was studied by using [3H]thymidine labeling and autoradiography. Additionally, the fate of neutrophils labeled with "Tc-hexamethylpropyleneamineoxime was observed following granulocyte colony-stimulating factor infusion. Proliferation increased in all stages of granulopoiesis, but a significant amount of the increased production stemmed from a greater input to the myeloblast compartment. Changes in the myelogram combined with the increased labeling indicated a faster throughput of cells, which resulted in labeled cells appearing in the circulation within 1 day compared to the normal 4 or 5 days. The ""'Tc studies demonstrated no sequestration of circulating neutrophils by spleen, lungs, or liver. The halflife of the circulating neutrophils was not significantly changed, and calculations from the flow of labeled cells to the peripheral blood indicated an increase of 3.2 extra amplification divisions during neutrophil development. The dramatic neutrophil response to granulocyte colony-stimulating factor can therefore be accommodated by a relatively modest increase in granulopoietic activity.The recent availability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (1) has stimulated considerable interest in its potential applications for promoting hemopoietic regeneration following bone marrow damage by cytoreductive agents. Stimulation, in vivo, of granulopoiesis by rhG-CSF was demonstrated in mice (2) and in humans (3-7). In patients, 2 or 3 days of continuous infusion or repeated injections of rhG-CSF resulted in a 10-fold increase in peripheral neutrophil levels, which were maintained for the duration of infusion and, following chemotherapy, significantly shortened the duration of the subsequent neutropenia (3,(5)(6)(7). These neutrophils demonstrated normal function and competence (4). Only a small, nonsignificant increase in granulocyte/macrophage colony-forming cell (GM-CFC) cycling and in the GM-CFC/burst-forming unit-erythroid ratio was observed, suggesting that most of the expansion in neutrophil production probably arises after the GM-CFC stage (4). Our intention was to investigate the changes in kinetics induced by G-CSF and required to maintain this elevated neutrophilia. We therefore treated two patients with rhG-CSF and, by means of autoradiography, studied the kinetics of marrow cell proliferation and efflux into the peripheral blood following labeling in vivo with tritiumlabeled thymidine. Since rhG-CSF in vivo initially produces an early fall in peripheral neutrophils followed by rapid influx of mature neutrophils into the circulatory pool (4), we also studied the early effects of this growth factor on circulating neutrophils by labeling them with 99mTc-hexamethylpropyleneamineoxime (9mTc-HMPAO) (8) and observing their fate with a y camera. MATERIALS AND METHODSPatients and Therapy. Two patients with metastatic breast c...
Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1 2/2 mice significantly increased visceral pleural thickness (P , 0.001), elastance (P , 0.05), and total lung resistance (P , 0.05), while decreasing lung compliance (P , 0.01) and lung volumes (P , 0.05). Collagen, a-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1 2/2 mice. Colocalization of a-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1 2/2 mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1 2/2 mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.
Pleural organization follows acute injury and is characterized by pleural fibrosis, which may involve the visceral and parietal pleural surfaces. This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to pleural fibrosis and loculation. Pleural mesothelial cells (PMCs) undergo a process called mesothelial mesenchymal transition (MesoMT), by which PMCs acquire a profibrotic phenotype characterized by cellular enlargement and elongation, increased expression of α-smooth muscle actin (α-SMA), and matrix proteins including collagen-1. Although MesoMT contributes to pleural fibrosis and lung restriction in mice with carbon black/bleomycin-induced pleural injury and procoagulants and fibrinolytic proteases strongly induce MesoMT in vitro, the mechanism by which this transition occurs remains unclear. We found that thrombin and plasmin potently induce MesoMT in vitro as does TGF-β. Furthermore, these mediators of MesoMT activate phosphatidylinositol-3-kinase (PI3K)/Akt and NF-κB signaling pathways. Inhibition of PI3K/Akt signaling prevented TGF-β-, thrombin-, and plasmin-mediated induction of the MesoMT phenotype exhibited by primary human PMCs. Similar effects were demonstrated through blockade of the NF-κB signaling cascade using two distinctly different NF-κB inhibitors, SN50 and Bay-11 7085. Conversely, expression of constitutively active Akt-induced mesenchymal transition in human PMCs whereas the process was blocked by PX866 and AKT8. Furthermore, thrombin-mediated MesoMT is dependent on PAR-1 expression, which is linked to PI3K/Akt signaling downstream. These are the first studies to demonstrate that PI3K/Akt and/or NF-κB signaling is critical for induction of MesoMT.
Pleural loculation affects about 30,000 patients annually in the United States and in severe cases can resolve with restrictive lung disease and pleural fibrosis. Pleural mesothelial cells contribute to pleural rind formation by undergoing mesothelial mesenchymal transition (MesoMT), whereby they acquire a profibrotic phenotype characterized by increased expression of α-smooth muscle actin and collagen 1. Components of the fibrinolytic pathway (urokinase plasminogen activator and plasmin) are elaborated in pleural injury and strongly induce MesoMT in vitro. These same stimuli enhance glycogen synthase kinase (GSK)-3β activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3β mobilization from the cytoplasm to the nucleus. GSK-3β down-regulation blocked induction of MesoMT. Likewise, GSK-3β inhibitor 9ING41 blocked induction of MesoMT and reversed established MesoMT. Similar results were demonstrated in a mouse model of Streptococcus pneumoniae-induced empyema. Intraperitoneal administration of 9ING41, after the induction of pleural injury, attenuated injury progression and improved lung function (lung volume and compliance; P < 0.05 compared with untreated and vehicle controls). MesoMT marker α-smooth muscle actin was reduced in 9ING41-treated mice. Pleural thickening was also notably reduced in 9ING41-treated mice (P < 0.05). Collectively, these studies identify GSK-3β as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3β signaling in the control of MesoMT and pleural injury.
Background Pleural infection affects about 65,000 patients annually in the US and UK. In this and other forms of pleural injury, mesothelial cells (PMCs) undergo a process called mesothelial (Meso) mesenchymal transition (MT), by which PMCs acquire a profibrogenic phenotype with increased expression of α-smooth muscle actin (α-SMA) and matrix proteins. MesoMT thereby contributes to pleural organization with fibrosis and lung restriction. Current murine empyema models are characterized by early mortality, limiting analysis of the pathogenesis of pleural organization and mechanisms that promote MesoMT after infection. Methods A new murine empyema model was generated in C57BL/6 J mice by intrapleural delivery of Streptococcus pneumoniae (D39, 3 × 10 7 –5 × 10 9 cfu) to enable use of genetically manipulated animals. CT-scanning and pulmonary function tests were used to characterize the physiologic consequences of organizing empyema. Histology, immunohistochemistry, and immunofluorescence were used to assess pleural injury. ELISA, cytokine array and western analyses were used to assess pleural fluid mediators and markers of MesoMT in primary PMCs. Results Induction of empyema was done through intranasal or intrapleural delivery of S. pneumoniae . Intranasal delivery impaired lung compliance (p < 0.05) and reduced lung volume (p < 0.05) by 7 days, but failed to reliably induce empyema and was characterized by unacceptable mortality. Intrapleural delivery of S. pneumoniae induced empyema by 24 h with lung restriction and development of pleural fibrosis which persisted for up to 14 days. Markers of MesoMT were increased in the visceral pleura of S. pneumoniae infected mice. KC, IL-17A, MIP-1β, MCP-1, PGE 2 and plasmin activity were increased in pleural lavage of infected mice at 7 days. PAI-1 −/− mice died within 4 days, had increased pleural inflammation and higher PGE 2 levels than WT mice. PGE 2 was induced in primary PMCs by uPA and plasmin and induced markers of MesoMT. Conclusion To our knowledge, this is the first murine model of subacute, organizing empyema. The model can be used to identify factors that, like PAI-1 deficiency, alter outcomes and dissect their contribution to pleural organization, rind formation and lung restriction.
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