Human Idiopathic Pulmonary Fibrosis Research Articles

Chronic Pulmonary Interstitial Fibrosis in a Blue-Fronted Amazon Parrot (Amazona aestiva aestiva)

A 30-yr-old blue-fronted Amazon parrot (Amazon aestiva aestiva) was presented to the clinic with a history of sneezing more often during the last 2 mo. Physical examination revealed only a mild nasal discharge. Complete hematologic and plasma biochemical examination showed no abnormalities. Computerized tomography (CT) of the complete bird showed generalized lung alterations consistent with lung fibrosis. Two lung biopsies were taken. The results of the histologic examination of the biopsies confirmed the tentative CT diagnosis of pulmonary interstitial fibrosis. To our knowledge this is the first reported case of chronic pulmonary interstitial fibrosis diagnosed by means of a lung biopsy in an avian species. The histologic characteristics are discussed and compared with those of human idiopathic pulmonary fibrosis.

Role of Galectin-3 in Human Pulmonary Fibrosis

Galectin-3 is a beta-galactoside-binding protein which is implicated in diverse physiological and pathological processes including human liver cirrhosis and a mouse lung fibrosis model. The aim of this study is to determine whether galectin-3 is involved in human lung fibrosis. We measured galectin-3 concentration in bronchoalveolar lavage fluid (BALF) and examined its expression in alveolar macrophages from patients with interstitial lung disorders using ELISA and immunohistochemical staining, respectively. Using monocyte/macrophage cell lines in vitro, we examined the effect of cytokines on galectin-3 expression, and the opposite similarly by RT-PCR and Western blotting. Finally, we performed Micro Boyden chamber assay and Sircoll assay to determine whether galectin-3 induces migration and collagen synthesis, respectively, in fibroblasts. Galectin-3 was specifically increased in BALF from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia associated with collagen vascular disease (CVD-IP). Galectin-3 levels in BALF seemed to be lower in IPF and CVD-IP patients receiving corticosteroid therapy. Alveolar macrophages from IPF patients expressed more galectin-3 compared with those from control. Galectin-3 expression was induced by tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-gamma in a monocytic cell line U937. Galectin-3 also induced mRNA expression and protein production of TNF-alpha and interleukin (IL)-8 in a macrophage cell line THP-1. This lectin stimulated NIH-3T3 fibroblast to induce migration and collagen synthesis in vitro. These results suggest that galectin-3 is involved in the pathogenesis of human IPF and CVD-IP by activating macrophages and fibroblasts.

Three‐dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.

Evolving Concepts of Apoptosis in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. Significant progress has been made in recent years in elucidating key aspects of the pathobiology of IPF. Insights into disease pathogenesis have come from studies of cell biology, growth factor/cytokine signaling, animal models of pulmonary fibrosis, and human IPF cells and tissue. A consistent finding in the ultrastructural pathology of IPF is alveolar epithelial cell injury and apoptosis. Another consistent finding in the histopathology of human IPF, described as usual interstitial pneumonia, is the accumulation of aggregates of myofibroblasts in fibroblastic foci. The extent or profusion of fibroblastic foci in lung biopsies is strongly correlated with increased mortality in patients with IPF. There is emerging evidence that myofibroblasts in IPF/usual interstitial pneumonia, both in the in vivo microenvironment and during the process of differentiation in vitro, acquire resistance to apoptosis. Here, we review the current evidence and mechanisms for this apparent "apoptosis paradox" in the pathogenesis of IPF.

Cutting Edge: Nonproliferating Mature Immune Cells Form a Novel Type of Organized Lymphoid Structure in Idiopathic Pulmonary Fibrosis

Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation.

Idiopathic Pulmonary Fibrosis Related to Endothelial Injury and Antiendothelial Cell Antibodies

Mechanisms underlying idiopathic pulmonary fibrosis are not well understood. This paper presents data supporting the hypothesis that microvascular endothelial cell injury and antiendothelial cell antibodies play roles in human idiopathic pulmonary fibrosis. Serologic and pathologic features of 40 patients diagnosed with idiopathic pulmonary fibrosis were evaluated. All patients had open lung biopsies indicating either usual or nonspecific interstitial pneumonitis. All biopsies had morphologic evidence of microvascular injury to the endothelium, and direct immunofluorescence testing revealed variable deposition of IgG, IgM, or IgA within septal microvasculature suggestive of humorally mediated microvascular injury. Ultrastructural studies revealed changes of endothelial cell injury and necrosis and evidence of repetitive episodes of microvascular injury characterized by basement membrane zone collagen deposition and lamellation. Serum samples demonstrated reactivity to multiple endothelial cell antigenic epitopes, and indirect immunofluorescent testing demonstrated a prominent pattern of fluorescence in pulmonary endothelial cell preparations. Serum samples were positive in 37/40 patients for antiphospholipid antibodies with one fourth having positive lupus anticoagulant tests accompanied by thrombotic episodes. In patients with idiopathic pulmonary fibrosis, Factor VIII levels and C-reactive protein levels were also elevated, supporting the presence of endothelial cell injury and inflammation. These data underscore a potential role for immune-based microvascular injury in the evolution of usual or nonspecific interstitial pneumonitis and indicate that those patients have evidence of microvascular injury and endothelial cell necrosis. The high prevalence of antiphospholipid antibodies in these patients may lead to an inherent thrombophilic tendency.

RAGE: A beneficial role in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease with a poor prognosis. There are currently no effective therapies for IPF, thus the identification of new therapeutic targets is needed. The receptor for advanced glycation end products (RAGE) is a cell surface receptor whose activation has been linked to many diseases. Although expressed at the highest levels in the lungs of adult animals, the role of RAGE has not been examined in pulmonary disease. This study examined the potential role of RAGE in IPF pathogenesis. Methods: RAGE expression was examined in two mouse models of pulmonary fibrosis and in human IPF lungs. Pulmonary histology and hydroxyproline levels of RAGE null mice was also compared with that of wild-type mice. In addition, cultured human pulmonary fibroblasts were treated with RAGE ligands and α-smooth muscle actin expression was examined. Results: Membrane RAGE and its soluble (decoy) isoform, sRAGE, are significantly depleted in both murine and human fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, histologic and hydroxyproline studies on aged RAGE null mice indicate that they spontaneously develop pulmonary fibrosis-like alterations. In vitro studies suggest that RAGE signaling may inhibit myofibroblastic phenotypes in pulmonary fibroblasts. Conclusions: Combined with data from mouse models of pulmonary fibrosis and human IPF tissues, these data indicate that loss of RAGE/sRAGE plays an important role in the pathogenesis of IPF.

Different susceptibilities of PECAM-deficient mouse strains to spontaneous idiopathic pneumonitis

Platelet Endothelial Cell Adhesion Molecule (PECAM) is an adhesion and signaling molecule used for leukocyte extravasation. We have generated two strains of PECAM-deficient mouse, one in the original C57BL/6 and a second by backcrossing nice generations into the FVB/n strain. The FVB/n strain has reduced responses in models of acute inflammation. We show here that this strain is also susceptible to a chronic pneumonia which leads to pulmonary fibrosis. In contrast, PECAM-deficient C57BL/6 mice do not develop this lung disease and have normal responses in acute models of inflammation. This demonstrates that PECAM-dependent and -independent mechanisms are found in both acute and chronic inflammation. Further, the PECAM-deficient FVB/n strain has many pathologic similarities to the human disease Idiopathic Pulmonary Fibrosis, suggesting that similar molecular mechanisms may play a role in human disease.

Role of the Chemokine Receptors CXCR3 and CCR4 in Human Pulmonary Fibrosis

The chemokine receptors CXCR3 and CCR4 have recently been described as playing a pivotal role in the mouse model of bleomycin-induced fibrosis. To evaluate the role of these receptors in human idiopathic pulmonary fibrosis (IPF). We studied 57 patients: 18 with IPF, 17 with non-IPF (nIPF), 12 with sarcoidosis, and 10 healthy control subjects. We evaluated the expression of CXCR3 and CCR4 in blood and bronchoalveolar lavage (BAL) T lymphocytes by flow cytometry and the chemokine CXCL10, CXCL11 and CCL17 BAL concentration by singular immunoassay. Patients with IPF had a significantly lower CXCR3 and a higher CCR4 expression on BAL CD4 T cells compared with the other groups. Among patients with IPF, those treated with corticosteroids exhibited higher CXCR3 and lower CCR4 expression compared with untreated patients. CXCR3 expression correlated with BAL lymphocytes and CCR4 with BAL neutrophils and eosinophils. CXCL10 levels correlated with the expression of CXCR3 on BAL CD4 cells. CXCL11 was undetectable in almost all patients, whereas CCL17 was primarily detectable in patients with IPF. The percentage of BAL CCR4CD4 cells negatively correlated with DL(CO). The changes in the total lung capacity, VC, and of the alveolar-arterial PO2 gradient in patients with IPF and those with nIPF 6 to 12 mo after the first evaluation were associated with CD4CXCR3 percentage on BAL cells. We found an imbalance in CXCR3/CCR4 expression on BAL CD4 lymphocytes and reduced CXCL10 BAL levels in patients with IPF, suggesting a pivotal role of these molecules in IPF.

Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung.Methods and FindingsUsing oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7.ConclusionsOur results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease.

The -2518 Promotor Polymorphism in the MCP-1 Gene Is Associated with Systemic Sclerosis

Factors influencing the initiation or progression of sclerosis in patients with systemic sclerosis (SSc) are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, which is upregulated in fibroblasts during development of sclerosis. In this study, we investigated the frequency of the functional -2518G MCP-1 promoter polymorphism in 18 patients with SSc and 139 healthy controls. In the lesional skin of the same SSc patients, expression of MCP-1 protein was examined by immunohistochemistry. To investigate a genotype/phenotype correlation, basal as well as tumor necrosis factor (TNF)-induced MCP-1 expression was analyzed in fibroblasts isolated from the skin of SSc patients with different MCP-1 genotypes by quantitative RT-PCR and ELISA. Genotyping for the -2518 (A/G) MCP-1 promotor polymorphism showed that GG homozygotes were significantly more frequent in patients with SSc than in controls (28%vs 6%). Results of immunohistochemistry revealed that MCP-1 was expressed in keratinocytes, infiltrating inflammatory cells, fibroblasts, and endothelial cells in scleroderma skin, whereas normal control skin showed no MCP-1 expression. MCP-1 expression in fibroblasts from GG-homozygote individuals tended to be stronger as compared to AG or AA genotypes. Furthermore, basal as well as TNF-induced MCP-1 expression of fibroblasts isolated from a GG-homozygote SSc patient was significantly higher than MCP-1 expression of fibroblasts isolated from heterozygote or AA-homozygote donors. The A -2518G polymorphism of the MCP-1 gene appears to affect MCP-1 expression of skin fibroblasts of patients with SSc. In accordance, the G/G genotype may predispose patients to SSc.

Identification and Characterization of an Idiopathic Pulmonary Fibrosis‐Like Condition in Cats

Interstitial lung diseases are a heterogeneous group of disorders with a variety of causes. In veterinary medicine, such lung diseases with a prominent fibrotic component of unknown etiology are often called idiopathic pulmonary fibrosis (IPF). In human medicine, this term is reserved for a distinct disease entity with specific histologic findings labeled as usual interstitial pneumonia (UIP). We identified 23 cats displaying histologic criteria of UIP. The purpose of this retrospective study is to describe the presentation and response to therapy of these cats to better define this disease entity. All but 2 cats were middle aged to older (median 8.7 years), with no apparent sex or breed predisposition. Complaints included respiratory distress (n = 18) and cough (13). Duration of signs was less than 6 months in 17 cats. Physical‐examination abnormalities included tachypnea, inspiratory or mixed inspiratory and expiratory effort, and adventitial lung sounds. No consistent hematologic or biochemical abnormalities, parasites, or positive serologic results for feline retroviruses, heartworms, or toxoplasmosis were present. Radiographic changes included dense patchy or diffuse interstitial, bronchiolar, and alveolar infiltrates. Analysis of bronchial lavage fluid revealed mild neutrophilic inflammation (n = 6) with no consistent pathogen growth. Clinical condition of 5 cats worsened after lavage. Coincident pulmonary neoplasia was identified in 6 cats. Response to therapy (corticosteroids, antibiotics, bronchodilators, and diuretics) was poor, and most cats died within days to months. Cats with histologic changes compatible with UIP had signs that mimicked many of the clinical findings of human IPF, and treatment response was similarly unrewarding.

Identification of Spontaneous Feline Idiopathic Pulmonary Fibrosis: Morphology and Ultrastructural Evidence for a Type II Pneumocyte Defect

Idiopathic pulmonary fibrosis (IPF) is a poorly understood chronic respiratory disease of humans, which has no correlate in other animals. Understanding the role that inflammation, alveolar epithelial cells, and myofibroblasts play in the progression of the disease is controversial, and hampered by the lack of an animal model. We have identified spontaneous IPF in domestic cats and hypothesized that this newly identified disease shares the pathology of human IPF; further, this work provides data suggesting that the disease is related to a defect in type II pneumocyte biology. Chronic respiratory disease with pathology consistent with usual interstitial pneumonia (UIP) spontaneously developed in 16 domestic cats. The histopathology of feline IPF consisted of the following: (1) interstitial fibrosis with fibroblast/myofibroblast foci, (2) honeycombing with alveolar epithelial metaplasia and type II pneumocyte hyperplasia, and (3) alveolar interstitial smooth-muscle metaplasia. Interstitial inflammation was not a prominent feature of the disease. alpha-Smooth muscle actin-positive myofibroblasts were prominent in myofibroblast foci, beneath honeycomb and hyperplastic epithelium, and in alveolar septa away from the remodeling. Feline IPF type II pneumocyte ultrastructure is similar to a heritable form of human IPF, with abnormal cytoplasmic lamellar body-like inclusions. We conclude the following: (1) chronic respiratory disease with clinical and pathology features of UIP/IPF occurs in the domestic cat; (2) as in human IPF, the type II pneumocyte and myofibroblasts are important cellular constituents of feline IPF; and (3) type II cell ultrastructure suggests feline IPF is a defect in the type II pneumocyte.

T Cell–specific Ablation of Fas Leads to Fas Ligand–mediated Lymphocyte Depletion and Inflammatory Pulmonary Fibrosis

To study the role of Fas–Fas ligand (FasL) interaction-mediated apoptosis in lymphocyte homeostasis, we generated a mutant fas allele allowing conditional inactivation of the fas gene through Cre-mediated recombination. Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues. Selective inactivation of Fas in T cells led to a severe lymphopenia over time, accompanied by up-regulation of FasL on activated T cells and apoptosis of peripheral lymphocytes. In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis. Inhibition of Fas–FasL interaction in vivo completely prevented the loss of lymphocytes and initial lymphocyte infiltration in the lungs. Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model.

Identification and Characterization of an Idiopathic Pulmonary Fibrosis–Like Condition in Cats

Interstitial lung diseases are a heterogeneous group of disorders with a variety of causes. In veterinary medicine, such lung diseases with a prominent fibrotic component of unknown etiology are often called idiopathic pulmonary fibrosis (IPF). In human medicine, this term is reserved for a distinct disease entity with specific histologic findings labeled as usual interstitial pneumonia (UIP). We identified 23 cats displaying histologic criteria of UIP The purpose of this retrospective study is to describe the presentation and response to therapy of these cats to better define this disease entity. All but 2 cats were middle aged to older (median 8.7 years), with no apparent sex or breed predisposition. Complaints included respiratory distress (n = 18) and cough (13). Duration of signs was less than 6 months in 17 cats. Physical-examination abnormalities included tachypnea, inspiratory or mixed inspiratory and expiratory effort, and adventitial lung sounds. No consistent hematologic or biochemical abnormalities, parasites, or positive serologic results for feline retroviruses, heartworms, or toxoplasmosis were present. Radiographic changes included dense patchy or diffuse interstitial, bronchiolar, and alveolar infiltrates. Analysis of bronchial lavage fluid revealed mild neutrophilic inflammation (n = 6) with no consistent pathogen growth. Clinical condition of 5 cats worsened after lavage. Coincident pulmonary neoplasia was identified in 6 cats. Response to therapy (corticosteroids, antibiotics, bronchodilators, and diuretics) was poor, and most cats died within days to months. Cats with histologic changes compatible with UIP had signs that mimicked many of the clinical findings of human IPF, and treatment response was similarly unrewarding.

Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis.

Administration of bleomycin into the lungs of experimental animals has been utilized as a model to understand human pulmonary fibrosis. Most of the studies, however, have focused on early stages of the lung reaction. We hypothesized that chronic stages of the model may not mimic idiopathic pulmonary fibrosis, since in preliminary studies, lung volume and compliance were not decreased. Eight male Sprague-Dawley rats receiving intratracheal bleomycin (0.5 U/100 g body weight) underwent measurement of FRC, inspiratory capacity, and lung compliance 120 d later. Lung histologic changes were evaluated using light microscopy. Eight rats without intervention served as controls. Results show that our model, in early stages, has histologic changes no different from those previously described elsewhere. In chronic stages, however, the model does not behave as a restrictive syndrome: FRC is normal or increased, whereas lung compliance is normal. Focal peribronchiolar inflammation and fibrosis associated with paracicatricial emphysematous changes are the main histologic features of long-term lung remodeling after bleomycin. We conclude that while the chronic stages of the model may be informative in understanding mechanisms of fibrosis, care should be taken not to extrapolate to human idiopathic pulmonary fibrosis. We speculate that the model might resemble a particular subgroup of human interstitial lung disease, namely, those involving peribronchiolar structures.

A new function of green tea: prevention of lifestyle-related diseases.

In the normal human life span, there occur lifestyle-related diseases that may be preventable with nontoxic agents. This paper deals with the preventive activity of green tea in some lifestyle-related diseases. Green tea is one of the most practical cancer preventives, as we have shown in various in vitro and in vivo experiments, along with epidemiological studies. Among various biological effects of green tea, we have focused on its inhibitory effect on TNF-alpha gene expression mediated through inhibition of NF-kappaB and AP-1 activation. Based on our recent results with TNF-alpha-deficient mice, TNF-alpha is an endogenous tumor promoter. TNF-alpha is also known to be a central mediator in chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. We therefore hypothesized that green tea might be a preventive agent for chronic inflammatory diseases. To test this hypothesis, TNF-alpha transgenic mice, which overexpress TNF-alpha only in the lungs, were examined. The TNF-alpha transgenic mouse is an animal model of human idiopathic pulmonary fibrosis which also frequently develops lung cancer. Expressions of TNF-alpha and IL-6 were inhibited in the lungs of these mice after treatment with green tea in drinking water for 4 months. In addition, judging from the results of a prospective cohort study in Saitama Prefecture, Japan, green tea helps to prevent cardiovascular disease. In this study, a decreased relative risk of death from cardiovascular disease was found for people consuming over 10 cups of green tea a day, and green tea also had life-prolonging effects on cumulative survival. These data suggest that green tea has preventive effects on both chronic inflammatory diseases and lifestyle-related diseases (including cardiovascular disease and cancer), resulting in prolongation of life span.

TNF and IL-6 mediate MIP-1α expression in bleomycin-induced lung injury

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

MOLECULAR PATHOGENESIS OF INTERSTITIAL PNEUMONITIS WITH TNF-α TRANSGENIC MICE

Tumour necrosis factor α (TNF-α) transgenic mice, which overexpress TNF-α only in the lungs, develop interstitial pneumonitis resembling idiopathic pulmonary fibrosis (IPF) in humans. Transgenic mice were used to study molecular pathogenesis of interstitial pneumonitis with regard to sequential histological changes and cytokine network induced by TNF-α. The authors divided the histological process of interstitial pneumonitis into three stages: early stage with lymphocytic infiltration in alveolar septa, middle stage with recruitment of macrophages, and late stage with hyperplasia of epithelial cells and mild fibrosis. As for cytokine network, prolonged overexpression of TNF-α along with increasing interleukin 6 (IL-6) were associated with the progression of interstitial pneumonitis. Increasing IL-1 was found only in the early stage, the beginning of lymphocyte proliferation. The mRNA level of an anti-inflammatory cytokine, IL-10, was constantly enhanced in the lungs of transgenic mice. However, transforming growth factor β1 (TGF-β1) protein decreased, which is closely associated with prolonged TNF-α synthesis, resulting in development of chronic inflammation and less severe fibrosis in the lungs of this animal model, analogous to inflammatory stage of human IPF. TNF-α transgenic mice enabled the analysis of the sequential process of interstitial pneumonitis as a model of IPF pathogenesis in humans, the results of which will give rise to new therapeutic measures for human IPF.

Effects of neutrophil elastase inhibitor on bleomycin-induced pulmonary fibrosis in mice.

Neutrophils play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To elucidate the possible involvement of neutrophil elastase (NE) in pulmonary fibrosis, we investigated the efficacy of a new specific NE inhibitor (ONO-5046 Na) in a murine model of human IPF, bleomycin-induced pulmonary fibrosis. Bronchoalveolar lavage (BAL) and histopathological analysis were performed on bleomycin-treated mice (group A), bleomycin and ONO-5046 Na-treated mice (group B), and saline control groups at 1, 15, and 29 d after the end of bleomycin treatment. At 29 d, multifocal fibrosis was observed in group A, whereas no fibrotic regions were observed in group B. Interleukin-1 beta and macrophage inflammatory protein-2 mRNA levels in BAL cells on day 1, and platelet-derived growth factor-A and insulin-like growth factor-1 mRNA levels on days 1 and 15, were significantly lower in group B than in group A. Thus, we demonstrated an inhibitory effect of ONO-5046. Na on pulmonary fibrosis in mice, indicating the involvement of NE in the pathogenesis of pulmonary fibrosis. We propose that this effect might be related to suppressed expression of particular cytokines in alveolar macrophages and that this specific NE inhibitor could be a novel therapeutic agent for IPF.