SPARC oppositely regulates inflammation and fibrosis in bleomycin-induced lung damage

Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc(-/-) and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc(-/-) >WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc(-/-) macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc(-/-) donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis.     

Intercellular adhesion molecule-1 and L-selectin regulate bleomycin-induced lung fibrosis

The development of bleomycin-induced lung injury, a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. At present, the identity and role of the adhesion molecules involved in the fibrotic process are unknown. Therefore, bleomycin-induced pulmonary fibrosis was examined in mice lacking L-selectin (L-selectin(-/-)) expression, intercellular adhesion molecule-1 (ICAM-1) expression, or both. After 16 days of intratracheal bleomycin challenge, collagen deposition was inhibited in both L-selectin(-/-) and ICAM-1(-/-) mice when compared with wild-type littermates. Interestingly, collagen deposition was virtually eliminated in L-selectin/ICAM-1(-/-) mice relative to either the L-selectin(-/-) or ICAM-1(-/-) mice. Decreased pulmonary fibrosis was associated with reduced accumulation of leukocytes, including neutrophils and lymphocytes. Decreased mRNA expression of proinflammatory cytokines and transforming growth factor (TGF)-beta1 paralleled the inhibition of collagen deposition. The present study indicates that L-selectin and ICAM-1 play a critical role in pulmonary fibrosis by mediating the accumulation of leukocytes, which regulate the production of proinflammatory cytokines and TGF-beta1. This suggests that these adhesion molecules are potential therapeutic targets for inhibiting human pulmonary fibrosis.     

Time course of bleomycin-induced lung fibrosis

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time-course of bleomycin-induced lung fibrosis in mice using computer-assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post-IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin-treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer-assisted morphometry demonstrated a 3-fold increase in fibrosis fraction and a 1.3-fold increase in wall area fraction in bleomycin-treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer-assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin-induced lung injury.     

Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2

Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohn's disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.     

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.     

Progress of bleomycin-induced lung fibrosis in rabbits

Progression of lung fibrosis induced in rabbits by intratracheal bleomycin (BLM, 10 mg/kg) was monitored by biochemical and morphological measurements. These indicated that the evolution of fibrosis in the rabbit was slower than in other experimental animals. Histology revealed cellular and fibrocellular lesions 4 weeks after BLM. At 8 weeks these lesions still predominated, indicating continuing active disease accompanied by a progression to fibrosis. The gradual progression of the response was reflected in alterations in lung composition. Changes in lung content of collagen, protein and DNA were evident at 8 weeks after BLM, at which time concentration (micrograms/mg dry weight) of collagen and protein were also elevated (P less than 0.05). Plasma angiotensin converting enzyme (ACE), a marker of endothelial injury, decreased 1 week after BLM (P less than 0.01) and then returned to normal, indicating that endothelial injury does not parallel the fibrotic response. In summary, the continuing active inflammation and slow progress of fibrosis induced by i.t. BLM in the rabbit suggests that this model has advantages over others for the serial study of the cellular and biochemical evolution of lung fibrosis.     

A TSP-1 synthetic peptide inhibits bleomycin-induced lung fibrosis in mice

Bleomycin showed toxicity to lung and was recognized to induce a well model of lung fibrosis. Activated alveolar macrophages released increased amounts of transforming growth factor-β1(TGF-β1) in response to bleomycin-induced lung injury. Thrombospondin-1(TSP-1) was involved in the activation of latent TGF-β1(L-TGF-β1) through the association of the TSP-1/L-TGF-β1 complex with the cell receptor of TSP-1, CD36. The antagonistic effects of the synthetic peptides were studied by the administration of TSP-1 (447–452) synthetic peptides to the mouse model. The hydroxyproline contents of the TSP-1-treated groups were significantly lower than those of other experimental groups. Inflammation, fibrotic degree and distribution of collagen fibers in the interstitial and alveolar in the TSP-1-treated groups were less than those of the other experimental groups. The expressions of collagen I and III in TSP-1-treated groups were significantly lower than in the other experimental groups. TSP-1 synthetic peptide reduced the tissue fibrotic pathologies and collagen accumulation in the model, resulting in the decreased severity of bleomycin-induced lung injury.     

Human placental mesenchymal stem cells of fetal origins-alleviated inflammation and fibrosis by attenuating MyD88 signaling in bleomycin-induced pulmonary fibrosis mice

Pulmonary fibrosis is a progressive lung disease that its pathogenic mechanism currently is incompletely understood. Toll-like receptor (TLR) signaling has recently been identified as a regulator of inflammation and pulmonary fibrosis. In addition, mesenchymal stem cells (MSCs) of different origins offer a great promise in treatment of idiopathic pulmonary fibrosis (IPF). However mechanisms of pathogenic roles of TLR signaling and therapeutic effects of MSCs in the IPF remain elusive. In present study, the involvement of TLR signaling and the therapeutic role of MSCs were interrogated in MyD88-deficient mice using human placental MSCs of fetal origins (hfPMSCs). The results showed an alleviated pulmonary inflammation and fibrosis in myeloid differentiation primary response gene 88 (MyD88)-deficient mice treated with bleomycin (BLM), accompanied with a reduced TGF-β signaling and production of pro-fibrotic cytokines, including TNF-α, IL-1β. An exposure of HLF1 lung fibroblasts, A549 epithelial cells and RAW264.7 macrophages to BLM led an increased expression of key components of MyD88 and TGF-β signaling cascades. Of interest, enforced expression and inhibition of MyD88 protein resulted in an enhanced and a reduced TGF-β signaling in above cells in the presence of BLM, respectively. However, the addition of TGF-β1 showed a marginally inhibitory effect on MyD88 signaling in these cells in the absence of BLM. Importantly, the administration of hfPMSCs could significantly attenuate BLM-induced pulmonary fibrosis in mice, along with a reduced hydroxyproline (HYP) deposition, MyD88 and TGF-β signaling activation, and production of pro-fibrotic cytokines. These results may suggest an importance of MyD88/TGF-β signaling axis in the tissue homeostasis and functional integrity of lung in response to injury, which may offer a novel target for treatment of pulmonary fibrosis.     

Progress of bleomycin-induced lung fibrosis in rabbits.

Progression of lung fibrosis induced in rabbits by intratracheal bleomycin (BLM, 10 mg/kg) was monitored by biochemical and morphological measurements. These indicated that the evolution of fibrosis in the rabbit was slower than in other experimental animals. Histology revealed cellular and fibrocellular lesions 4 weeks after BLM. At 8 weeks these lesions still predominated, indicating continuing active disease accompanied by a progression to fibrosis. The gradual progression of the response was reflected in alterations in lung composition. Changes in lung content of collagen, protein and DNA were evident at 8 weeks after BLM, at which time concentration (micrograms/mg dry weight) of collagen and protein were also elevated (P less than 0.05). Plasma angiotensin converting enzyme (ACE), a marker of endothelial injury, decreased 1 week after BLM (P less than 0.01) and then returned to normal, indicating that endothelial injury does not parallel the fibrotic response. In summary, the continuing active inflammation and slow progress of fibrosis induced by i.t. BLM in the rabbit suggests that this model has advantages over others for the serial study of the cellular and biochemical evolution of lung fibrosis.     

Isolation and characterization of lung mast cells from rats with bleomycin-induced pulmonary fibrosis

To study the nature and extent of mast cell heterogeneity within a single species, we have developed methodologies to isolate rat lung mast cells (LMC) and have compared these to peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC). In normal and athymic nude (rnu/rnu) rats, a single intratracheal administration of bleomycin (5 U/kg) leads to pulmonary fibrosis accompanied by parenchymal hyperplasia of mast cells that are histochemically like PMC rather than IMMC. Using collagenase digestion of fibrotic rat lungs (30-80 days after bleomycin treatment), we recovered an average of 58.1 x 10(6) viable cells per rat, containing 2.5% mast cells. Control experiments in which PMC were subjected to the isolation procedure used for LMC showed that there was no qualitative effect on PMC, but that a reduction of 26-60% in responsiveness to secretagogues occurred. Isolated LMC secreted histamine in response to 48/80, A23187, substance P, VIP and somatostatin and bradykinin, but at lower levels than PMC. The anti-allergic compound theophylline, which does not inhibit antigen-induced histamine secretion by IMMC, was effective against both LMC and PMC. Taken together, the thymus independence of pulmonary mast cell hyperplasia, the histochemical characteristics and the responsiveness to secretagogues and anti-allergic compounds indicate that the majority of dispersed LMC are similar to PMC rather than to IMMC. Whether LMC should be considered analogous to PMC or, because of their size, histamine content and responsiveness to many secretagogues, intermediate between PMC and IMMC, remains to be determined through additional studies.     

CD19 regulates skin and lung fibrosis via Toll-like receptor signaling in a model of bleomycin-induced scleroderma

Mice subcutaneously injected with bleomycin, in an experimental model of human systemic sclerosis, develop cutaneous and lung fibrosis with autoantibody production. CD19 is a general "rheostat" that defines signaling thresholds critical for humoral immune responses, autoimmunity, and cytokine production. To determine the role of CD19 in the bleomycin-induced systemic sclerosis model, we investigated the development of fibrosis and autoimmunity in CD19-deficient mice. Bleomycin-treated wild-type mice exhibited dermal and lung fibrosis, hyper-gamma-globulinemia, autoantibody production, and enhanced serum and skin expression of various cytokines, including fibrogenic interleukin-4, interleukin-6, and transforming growth factor-beta1, all of which were inhibited by CD19 deficiency. Bleomycin treatment enhanced hyaluronan production in the skin, lung, and sera. Addition of hyaluronan, an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, stimulated B cells to produce various cytokines, primarily through TLR4; CD19 deficiency suppressed this stimulation. These results suggest that bleomycin induces fibrosis by enhancing hyaluronan production, which activates B cells to produce fibrogenic cytokines mainly via TLR4 and induce autoantibody production, and that CD19 deficiency suppresses fibrosis and autoantibody production by inhibiting TLR4 signals.     

Protease-activated receptor-2 induces myofibroblast differentiation and tissue factor up-regulation during bleomycin-induced lung injury: potential role in pulmonary fibrosis

Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2-/- mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2-/- mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.     

Aggravation of bleomycin-induced pulmonary inflammation and fibrosis in mice lacking peroxiredoxin I

Oxidative stress plays an important role in the pathogenesis of acute lung injury and pulmonary fibrosis. Peroxiredoxin (Prx) I is a cellular antioxidant enzyme induced under stress conditions. In the present study, the protective effects of Prx I on the development of bleomycin-induced acute pulmonary inflammation and pulmonary fibrosis were investigated using Prx I-deficient mice. Survival of Prx I-deficient mice after bleomycin administration was significantly lower than that of wild-type mice, corresponding with enhanced acute pulmonary inflammation and fibrosis. The level of inflammatory cytokines and chemokines, such as TNF-α, macrophage inflammatory protein-2, and monocyte chemotactic protein-1, was significantly elevated in the bronchoalveolar lavage fluid of Prx I-deficient mice after bleomycin administration. Furthermore, the level of 8-isoprostane, an oxidative stress marker, and the concentration and alveolar macrophage expression of macrophage migration inhibitory factor were elevated in the lungs of Prx I-deficient mice after bleomycin administration. The exacerbation of bleomycin-induced pulmonary inflammation and fibrosis in Prx I-deficient mice was inhibited by treatment with N-acetyl-L-cysteine, a radical scavenger, or with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, a tautomerase inhibitor of macrophage migration inhibitory factor. These findings suggest that mice lacking Prx I are highly susceptible to bleomycin-induced pulmonary inflammation and fibrosis because of increases in pulmonary oxidant levels and macrophage migration inhibitory factor activity in response to bleomycin.     

Upregulation of matrix metalloproteinase-1 and proteinase-activated receptor-1 promotes the progression of human gliomas

Matrix metalloproteinases (MMPs) have been proposed to be involved in remodeling the tumor-stromal microenvironment. The protease-activated receptors (PARs) are the latest MMP targets. Recent studies have revealed that stromal-derived MMP-1 acts as a signaling molecule by cleaving PAR1 to cause tumor migration and invasion of various cancers. However, the involvement of MMP-1/PAR1 signaling pathway in the progression and prognosis of human gliomas remains to be identified.Immunohistochemical staining was performed to detect the expression patterns of MMP-1 and PAR1 in biopsies from 108 patients with primary gliomas. Kaplan-Meier survival and Cox regression analyzes were performed to evaluate the prognosis of patients.Immunostaining revealed MMP-1 to be expressed in 83.3% (90/108) and PAR1 in 76.9% (83/108) of the biopsies. PAR1 expression was significantly correlated with that of MMP-1 (r = 0.786, p < 0.0001). The total IHC scores for MMP-1 and PAR1 were significantly higher in high-grade tumors than in low-grade tumors (both p = 0.001). In addition, patients with high MMP-1 and high PAR1 expression have lower Karnofsky performance scale (KPS) scores than patients with low MMP-1 and low PAR1 expression (both p = 0.008). Moreover, MMP-1 and PAR1 expression was shown to be a strong prognostic marker for decreased overall survival (p = 0.002 and 0.003, respectively). Furthermore, Cox multi-factor analysis showed that KPS (p = 0.008), WHO grade (p = 0.006), MMP-1 (p = 0.006), and PAR1 (p = 0.008) were independent prognostic factors for human gliomas.Our results suggest that in gliomas, the upregulation of MMP-1 and PAR1 correlates with histological malignancy grade and clinical outcome. Also, MMP-1 and PAR1 immunostaining supplements the current histological grading by offering additional prognostic and predictive information.     

Role of the chemokine receptor CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.     

Neutrophil and pathogen proteinases versus proteinase-activated receptor-2 lung epithelial cells: more terminators than activators

The proteinase-activated receptor-2 (PAR-2) is expressed by different lung cells, including bronchial and alveolar epithelial cells. Since its discovery in 1995, numerous in vivo and in vitro studies have demonstrated its involvement in lung inflammation, whether from infectious or allergic causes. However, its role is controversial because there is evidence of both pro- and anti-inflammatory activities. PARs, including PAR-2, display a unique activation process. Specific proteinases cleave the N-terminal extracellular domain at a particular site. The new N-terminal sequence functions as a tethered ligand and binds intramolecularly to activate the receptor. Recently, other specific proteinases have been shown to cleave the N-terminal exodomain at other sites, resulting in a disarming of the receptor. Some of these activating and disabling proteinases are produced by host cells and others by pathogens, and may be present in the airspaces under diverse pathophysiologic settings.     

Essential roles for angiotensin receptor AT1a in bleomycin-induced apoptosis and lung fibrosis in mice

Apoptosis of alveolar epithelial cells (AECs) has been implicated as a key event in the pathogenesis of lung fibrosis. Recent studies demonstrated a role for the synthesis and binding of angiotensin II to receptor AT1 in the induction of AEC apoptosis by bleomycin (BLEO) and other proapoptotic stimuli. On this basis we hypothesized that BLEO-induced apoptosis and lung fibrosis in mice would be inhibited by the AT1 antagonist losartan (LOS) or by targeted deletion of the AT1 gene. Lung fibrosis was induced by intratracheal administration of BLEO (1 U/kg) to wild-type C57BL/6J mice. Co-administration of LOS abrogated BLEO-induced increases in total lung caspase 3 activity detected 6 hours after in vivo administration and reduced by 57% BLEO-induced caspase 3 activity in blood-depleted lung explants exposed to BLEO ex vivo (both P < 0.05). Co-administration of LOS in vivo reduced DNA fragmentation and immunoreactive caspase 3 (active form) in AECs, measured at 14 days after intratracheal BLEO, by 66% and 74%, respectively (both P < 0.05). LOS also inhibited the accumulation of lung hydroxyproline by 45%. The same three measures of apoptosis and lung fibrosis were reduced by 89%, 85%, and 75%, respectively (all P < 0.01), in mice with a targeted disruption of the AT1a receptor gene (C57BL/6J-Agtr1a(tm1Unc)). These data indicate an essential role for angiotensin receptor AT1a in the pathogenesis of BLEO-induced lung fibrosis in mice and suggest that AT1 receptor signaling is required for BLEO-induced apoptosis of AECs in mice as it is in rat and human AECs.