HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic

Background

Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia.

Methods

Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later.

Results

In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis.

Conclusions

HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.     

Modulation of pulmonary neuroendocrine cells in idiopathic interstitial pneumonia

In order to reveal modulation of the number of pulmonary neuroendocrine cells (PNEC) in interstitial lung diseases and to clarify significance of cell proliferation activity in occurrence of PNEC, we counted airway PNEC of the patients of idiopathic interstitial pneumonia, secondary interstitial pneumonia and control lungs, and compared the number of PNEC with airway Ki-67 labeling. The lung tissue samples were obtained by video-assisted thoracoscopic surgery from 22 patients with usual interstitial pneumonia (UIP), 7 with non-specific interstitial pneumonia (NSIP), 8 with chronic hypersensitivity pneumonia (CHP), 13 with collagen vascular disease (CVD), and were compared with age-matched control lungs. The tissues were immunostained for chromogranin A and for Ki-67. Average incidence of bronchiolar PNEC in normal, UIP, NSIP, CHP, CVD lungs was 0.169%, 0.348%, 0.326%, 0.175% and 0.201%, respectively, and average Ki-67 labeling index in them was 0.241%, 1.186%, 1.605%, 1.058%, and 2.353%, respectively. And, in UIP lungs, PNEC incidence or Ki-67 labeling index was different according to pathological lesions. Thus, PNEC increase in the bronchiole of UIP, and the incidence of PNEC varies according to degree of activity of epithelial cell proliferation probably related to epithelial cell injury. Moreover, enhanced expression of human homolog of achaete-scute complex (hASH1) mRNA in UIP lungs suggests that hASH1 could play roles in the regulation of PNEC.     

Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

Background

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP).

Methods

We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining).

Results

The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP.

Conclusion

Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen.     

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process.     

Active remodeling in idiopathic interstitial pneumonias: evaluation of collagen types XII and XIV.

Fibril-associated collagens with interrupted triple helices (FACITs) XII and XIV act as fibril organizers and assist in the maintenance of uniform fibril size. We investigated the spatial expression patterns of collagens XII and XIV in cryptogenic organizing pneumonia (COP)/organizing pneumonia (OP) and in idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) and compared them to normal human lung. Study subjects included 10 patients with COP/OP, 10 patients with IPF/UIP, and 8 control subjects. Immunostaining for collagens XII and XIV was carried out in paraffin-embedded human lung tissue sections. Picrosirius red histochemical staining for collagen I expression and electron microcopy to evaluate fibril diameter were also performed. In normal lung, collagens XII and XIV were expressed in perivascular and subpleural connective tissue. In COP/OP, both collagens showed intense staining in perivascular connective tissue, thickened alveolar septae, and subpleural areas. In IPF/UIP, XII and XIV were expressed in perivascular connective tissue, in areas of established fibrosis, and in areas of subpleural thickening. Only collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblastic foci in IPF/UIP. Collagen type I was overexpressed in fibrotic areas. Electron micrographs revealed obvious fibril diameter alteration and fusion in the same areas. FACITs XII and XIV are expressed in normal and fibrotic lung. Unlike collagen XIV, collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblast foci in IPF/UIP. This may suggest a possible distinct role for both collagens in the modulation of the extracellular matrix during the onset of fibrotic process.     

Expression of Fibroblast Growth Factor 9 in Normal Human Lung and Idiopathic Pulmonary Fibrosis

The fibroblast growth factor (FGF) family of signaling ligands contributes significantly to lung development and maintenance in the adult. FGF9 is involved in control of epithelial branching and mesenchymal proliferation and expansion in developing lungs. However, its activity and expression in the normal adult lung and by epithelial and interstitial cells in fibroproliferative diseases like idiopathic pulmonary fibrosis (IPF) are unknown. Tissue samples from normal organ donor human lungs and those of a cohort of patients with mild to severe IPF were sectioned and stained for the immunolocalization of FGF9. In normal lungs, FGF9 was confined to smooth muscle surrounding airways, alveolar ducts and sacs, and blood vessels. In addition to these same sites, lungs of IPF patients expressed FGF9 in a population of myofibroblasts within fibroblastic foci, hypertrophic and hyperplastic epithelium of airways and alveoli, and smooth muscle cells surrounding vessels embedded in thickened interstitium. The results demonstrate that FGF9 protein increased in regions of active cellular hyperplasia, metaplasia, and fibrotic expansion of IPF lungs, and in isolated human lung fibroblasts treated with TGF-β1 and/or overexpressing Wnt7B. The cellular distribution and established biologic activity of FGF9 make it a potentially strong candidate for contributing to the progression of IPF.     

The Role of Catalase in Pulmonary Fibrosis

Background

Catalase is preferentially expressed in bronchiolar and alveolar epithelial cells, and acts as an endogenous antioxidant enzyme in normal lungs. We thus postulated epithelial damage would be associated with a functional deficiency of catalase during the development of lung fibrosis.

Methods

The present study evaluates the expression of catalase mRNA and protein in human interstitial pneumonias and in mouse bleomycin-induced lung injury. We examined the degree of bleomycin-induced inflammation and fibrosis in the mice with lowered catalase activity.

Results

In humans, catalase was decreased at the levels of activity, protein content and mRNA expression in fibrotic lungs (n = 12) compared to control lungs (n = 10). Immunohistochemistry revealed a decrease in catalase in bronchiolar epithelium and abnormal re-epithelialization in fibrotic areas. In C57BL/6J mice, catalase activity was suppressed along with downregulation of catalase mRNA in whole lung homogenates after bleomycin administration. In acatalasemic mice, neutrophilic inflammation was prolonged until 14 days, and there was a higher degree of lung fibrosis in association with a higher level of transforming growth factor-β expression and total collagen content following bleomycin treatment compared to wild-type mice.

Conclusions

Taken together, these findings demonstrate diminished catalase expression and activity in human pulmonary fibrosis and suggest the protective role of catalase against bleomycin-induced inflammation and subsequent fibrosis.     

Hyper-responsiveness of IPF/UIP fibroblasts: interplay between TGFbeta1, IL-13 and CCL2

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.     

Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts

The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF.     

A plasminogen activator inhibitor-1 promoter polymorphism and idiopathic interstitial pneumonia

The normal fibrinolytic activity within the alveolar space is suppressed in fibrotic lung diseases in part because of increased levels of plasminogen activator inhibitor-1 (PAI-1). Studies with animals have shown that inhibition of the plasminogen system by PAI-1 increases the generation of pulmonary fibrosis. To determine if a similar relationship occurs in human fibrotic lung diseases, we took advantage of a polymorphism (4G/5G) that occurs in the promoter region of the human PAI-1 gene and influences the expression of PAI-1. We hypothesized that the 4G/4G genotype, because of its association with higher levels of PAI-1, would occur in patients with idiopathic interstitial pneumonia more frequently than in a control population. PAI-1 promoter genotype was determined in 88 well-characterized patients with idiopathic interstitial pneumonia consisting of 62 patients with usual interstitial pneumonia and 26 with nonspecific interstitial pneumonia. DNA was extracted from paraffin-embedded biopsy tissue and the genotype identified by polymerase chain reaction and restriction endonuclease digestion. We found that the distribution of PAI-1 genotypes in the idiopathic interstitial pneumonia population was similar to that of a large control population. However, subgroup analysis showed that patients with nonspecific interstitial pneumonia were more likely than the control population to have the promoter genotype (4G/4G) that is associated with higher levels of PAI-1. A similar pattern in PAI-1 polymorphism was not seen in the usual interstitial pneumonia subgroup. The results of this study support the conclusion that PAl-1 expression influences the development of nonspecific interstitial pneumonia in a similar manner to what occurs in animal models of pulmonary fibrosis. Patients with usual interstitial pneumonia did not show the same relationship with PAl-1 genotype.     

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.     

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

Dual-immunohistochemistry provides little evidence for epithelial–mesenchymal transition in pulmonary fibrosis

epithelial–mesenchymal transition (EMT) has been considered to be involved in organ fibrogenesis. However, there is few direct evidence of this process in the pathophysiology of pulmonary fibrosis in vivo. Therefore, we tried to verify the involvement of this process in the development of pulmonary fibrosis. Since the co-expressions of epithelial and mesenchymal markers are thought to be a marker of EMT, we performed dual-immuunohistochemistry to assess the co-expressions of these proteins in lung tissues from bleomycin-induced pulmonary fibrosis in mice, and from patients with idiopathic pulmonary fibrosis, and nonspecific interstitial pneumonia. Double positive cells for epithelial markers including E-cadherin, T1α, or aquaporin 5, and a mesenchymal markers α-smooth muscle actin or vimentin were not found in bleomycin-induced pulmonary fibrosis in mice. Double positive cells for E-cadherin, ICAM-1, LEA, CD44v9, or SP-A and α-smooth muscle actin or vimentin were not found in lung tissues from normal lung parenchyma, idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia. These results offer at least two possibilities. One is that EMT does not occur in IPF or bleomycin-induced pulmonary fibrosis in mice. Another is that EMT may occur in pulmonary fibrosis but the time during this transition in which cells express detectable levels of epithelial and mesenchymal markers is too small to be detected by double immunohistochemistry.     

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs)

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively). In IPF, significantly increased percentage of AM FasL(+) and CD8(+)FasL(+) cells as well as sFasL levels in BAL were found. Increased sFasL levels were also observed in HP. NSIP and asbestosis were characterized by higher AM FasL(+) relative number; CD8(+)FasL(+) population was expanded in asbestosis only. There was a significant decline in AL FasL(+) percentage in PS and HP. Vital capacity was negatively correlated with sFasL levels, AM FasL(+) and CD8(+)FasL(+) cell relative count. CD4(+)FasL(+) and CD8(+)FasL(+) percentage strongly correlated with BAL neutrophilia, an unfavorable prognostic factor in lung fibrosis. The concurrent comparative BAL analysis of FasL expression indicates that FasL(+) AM and AL (mainly Tc cells) comprise an important element of the fibrotic process, mostly in IPF. FasL might play a crucial role in other fibrosis-complicated ILDs, like NSIP and asbestosis.     

Mechanical stretch induces epithelial-mesenchymal transition in alveolar epithelia via hyaluronan activation of innate immunity

Epithelial injury is a central event in the pathogenesis of many inflammatory and fibrotic lung diseases like acute respiratory distress syndrome, pulmonary fibrosis, and iatrogenic lung injury. Mechanical stress is an often underappreciated contributor to lung epithelial injury. Following injury, differentiated epithelia can assume a myofibroblast phenotype in a process termed epithelial to mesenchymal transition (EMT), which contributes to aberrant wound healing and fibrosis. We demonstrate that cyclic mechanical stretch induces EMT in alveolar type II epithelial cells, associated with increased expression of low molecular mass hyaluronan (sHA). We show that sHA is sufficient for induction of EMT in statically cultured alveolar type II epithelial cells and necessary for EMT during cell stretch. Furthermore, stretch-induced EMT requires the innate immune adaptor molecule MyD88. We examined the Wnt/β-catenin pathway, which is known to mediate EMT. The Wnt target gene Wnt-inducible signaling protein 1 (wisp-1) is significantly up-regulated in stretched cells in hyaluronan- and MyD88-dependent fashion, and blockade of WISP-1 prevents EMT in stretched cells. In conclusion, we show for the first time that innate immunity transduces mechanical stress responses through the matrix component hyaluronan, and activation of the Wnt/β-catenin pathway.     

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.     

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.