Mononuclear Phagocytes and Airway Epithelial Cells: Novel Sources of Matrix Metalloproteinase-8 (MMP-8) in Patients with Idiopathic Pulmonary Fibrosis

Objectives

Matrix metalloproteinase-8 (MMP-8) promotes lung fibrotic responses to bleomycin in mice. Although prior studies reported that MMP-8 levels are increased in plasma and bronchoalveolar lavage fluid (BALF) samples from IPF patients, neither the bioactive forms nor the cellular sources of MMP-8 in idiopathic pulmonary fibrosis (IPF) patients have been identified. It is not known whether MMP-8 expression is dys-regulated in IPF leukocytes or whether MMP-8 plasma levels correlate with IPF outcomes. Our goal was to address these knowledge gaps.

Methods

We measured MMP-8 levels and forms in blood and lung samples from IPF patients versus controls using ELISAs, western blotting, and qPCR, and assessed whether MMP-8 plasma levels in 73 IPF patients correlate with rate of lung function decline and mortality. We used immunostaining to localize MMP-8 expression in IPF lungs. We quantified MMP-8 levels and forms in blood leukocytes from IPF patients versus controls.

Results

IPF patients have increased BALF, whole lung, and plasma levels of soluble MMP-8 protein. Active MMP-8 is the main form elevated in IPF lungs. MMP-8 mRNA levels are increased in monocytes from IPF patients, but IPF patients and controls have similar levels of MMP-8 in PMNs. Surprisingly, macrophages and airway epithelial cells are the main cells expressing MMP-8 in IPF lungs. Plasma and BALF MMP-8 levels do not correlate with decline in lung function and/or mortality in IPF patients.

Conclusion

Blood and lung MMP-8 levels are increased in IPF patients. Active MMP-8 is the main form elevated in IPF lungs. Surprisingly, blood monocytes, lung macrophages, and airway epithelial cells are the main cells in which MMP-8 is upregulated in IPF patients. Plasma and BALF MMP-8 levels are unlikely to serve as a prognostic biomarker for IPF patients. These results provide new information about the expression patterns of MMP-8 in IPF patients.     

Macrophage derived chemokine (CCL22), thymus and activation-regulated chemokine (CCL17), and CCR4 in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients.

Methods

We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization.

Results

BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients.

Conclusion

We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.     

Identification of annexin 1 as a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vbeta genes were identified in sequential materials obtained at 1-3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vbeta genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.     

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

ABSTRACT: BACKGROUND: Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases. METHODS: Elastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11). RESULTS: The sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p < 0.001) and 124% higher in IPF patients (p < 0.0001) compared with controls. ELN-441 had better diagnostic value in COPD patients (AUC 97%, p = 0.001) than in IPF patients (AUC 90%, p = 0.0001). The odds ratios for differentiating controls from COPD or IPF were 24 [2.06-280] for COPD and 50 [2.64-934] for IPF. CONCLUSIONS: MMP-9 and -12 time-dependently released the ELN-441 epitope from elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings.     

Lung infection with gamma-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease of unknown etiology. A viral pathogenesis in IPF has been suggested since >95% of IPF patients have evidence of chronic pulmonary infection with one or more herpesviruses. To determine whether pulmonary infection with herpesvirus can cause lung fibrosis, we infected mice with the murine gamma-herpesvirus 68 (MHV68). Because IPF patients have a T helper type 2 (Th2) pulmonary phenotype, we used IFN-gammaR-/-, a strain of mice biased to develop Th2 responses. Chronic MHV68 infection of IFN-gammaR-/- mice resulted in progressive deposition of interstitial collagen as shown by light and electron microscopy. A significant decrease in tidal volume paralleled the collagen deposition. Five features typically seen in IPF, increased transforming growth factor-beta expression, myofibroblast transformation, production of Th2 cytokines, hyperplasia of type II cells, and increased expression of matrix metalloproteinase-7, were also present in chronically infected IFN-gammaR-/- mice. There also was altered synthesis of surfactant proteins, which is seen in some patients with familial IPF. MHV68 viral protein was found in type II alveolar epithelial cells, especially in lung areas with extensive alveolar remodeling. In summary, chronic herpesvirus pulmonary infection in IFN-gammaR-/- mice causes progressive pulmonary fibrosis and many of the pathological features seen in IPF.     

The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of “immune coagulation” and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as well as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.     

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.     

Expression of bcl-2 protein in bronchoalveolar lavage cell populations from patients with idiopathic pulmonary fibrosis.

OBJECTIVE: To investigate changes in the expression of the antiapoptotic protein bcl-2 in bronchoalveolar lavage fluid (BALF) cell populations in patients with idiopathic pulmonary fibrosis (IPF). STUDY DESIGN: Ten patients with IPF underwent fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) in the area of maximal radiographic shadowing (based on high-resolution computed tomography findings). Results were compared with those of 10 normal people in the control group. Cellular bcl-2 expression was identified using an immunoperoxidase staining method. RESULTS: A statistically significant (P < .001) increase in the expression of bcl-2 in BALF neutrophils and eosinophils was observed in patients with IPF as compared with controls. BAL macrophages exhibited only a slight (statistically insignificant) increase in bcl-2 expression in IPF patients. No bcl-2 expression was observed in BAL lymphocytes from IPF patients in contrast to the control group. CONCLUSION: The overexpression of bcl-2 on BALF neutrophils and eosinophils, cells that characterize the special cellular profile of alveolitis in IPF, could be one of the pathophysiologic mechanisms of this disease.     

TLR4 protein contributes to cigarette smoke-induced matrix metalloproteinase-1 (MMP-1) expression in chronic obstructive pulmonary disease

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease and alters expression of proteolytic enzymes that contribute to disease pathology. Previously, we reported that smoke exposure leads to the induction of matrix metalloproteinase-1 (MMP-1) through the activation of ERK1/2, which is critical to the development of emphysema. To date, the upstream signaling pathway by which cigarette smoke induces MMP-1 expression has been undefined. This study demonstrates that cigarette smoke mediates MMP-1 expression via activation of the TLR4 signaling cascade. In vitro cell culture studies demonstrated that cigarette smoke-induced MMP-1 was regulated by TLR4 via MyD88/IRAK1. Blockade of TLR4 or inhibition of IRAK1 prevented cigarette smoke induction of MMP-1. Mice exposed to acute levels of cigarette smoke exhibited increased TLR4 expression. To further confirm the in vivo relevance of this signaling pathway, rabbits exposed to acute cigarette smoke were found to have elevated TLR4 signaling and subsequent MMP-1 expression. Additionally, lungs from smokers exhibited elevated TLR4 and MMP-1 levels. Therefore, our data indicate that TLR4 signaling, through MyD88 and IRAK1, plays a predominant role in MMP-1 induction by cigarette smoke. The identification of the TLR4 pathway as a regulator of smoke-induced protease production presents a series of novel targets for future therapy in chronic obstructive pulmonary disease.     

Alterations in Adenosine Metabolism and Signaling in Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Background

Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.

Methodology/Principal Findings

The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A_2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A_2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A_2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A_2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.

Conclusions/Significance

These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A_2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.     

The role of ChemR23 in the induction and resolution of cigarette smoke-induced inflammation

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11b(hi)CD103(-) and CD11b(lo)CD103(+) dendritic cells (DCs), and CD4(+) and CD8(+) T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11b(hi)CD103(-) DCs, and activated CD4(+) T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8(+) T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.     

Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.     

Tumstatin,a Matrikine Derived from Collagen Type IVα3, is Elevated in Serum from Patients with Non–Small Cell Lung Cancer

OBJECTIVES: Fibrosis and cancer are characterized by extracellular matrix (ECM) remodeling. The basement membrane is mainly composed by collagen type IV and laminin. Tumstatin is a matrix metalloproteinase-9 (MMP-9) generated matrikine of collagen type IV α3 chain. We evaluated the potential of tumstatin as a diagnostic biomarker of lung disorders. METHODS: A monoclonal antibody was raised against the neo-epitope tumstatin. A novel competitive enzyme-linked immunosorbent assay for detection of tumstatin (TUM), was developed and technically characterized. Levels of TUM were measured in serum of patients with idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and non–small cell lung cancer (NSCLC) belonging to two cohorts. RESULTS: The developed TUM enzyme-linked immunosorbent assay (ELISA) was technically robust. In cohort 1, levels of TUM were significantly higher in NSCLC compared to healthy controls, IPF, and COPD (P = 0.007, P = 0.03 and P = 0.001, respectively). The area under the receiver operating characteristics (AUROC) for separation of patients with NSCLC from healthy controls was 0.97, for separation of NSCLC and IPF patients was 0.98, and for separation of NSCLC and COPD patients was 1.0. In cohort 2, levels of TUM were also significantly higher in patients with NSCLC compared to healthy controls (P = 0.002), and the AUROC for separation of NSCLC and healthy controls was 0.73. CONCLUSIONS: We developed a technically robust competitive ELISA targeting the fragment tumstatin. The level of TUM in circulation was significantly higher in patients with NSCLC compared to patients with IPF, COPD and healthy controls. The assay provided high diagnostic accuracy in separating NSCLC patients from other lung disorders and from healthy controls.     

Increase in T-Cells Bearing CD25 in Bronchoalveolar Lavage Fluid from HAM/TSP Patients and HTLV-I Carriers

To determine the immunologic characteristics of T-cells in local pulmonary lesions of human T-cell lymphotropic virus type I (HTLV-I) carriers, we investigated lymphocyte surface markers in peripheral blood and bronchoalveolar lavage fluid (BALF) of 38 HTLV-I carriers, 8 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 44 HTLV-I seronegative patients with pulmonary diseases and 7 healthy volunteers using two-color flow cytometric analysis. In peripheral blood, activated T-cells, CD4+HLA-DR +, CD8 + HLA-DR + and CD3 + CD25 +, and CD4+CD29+ cells increased significantly in carriers and HAM/TSP patients compared with healthy volunteers and seronegative patients. In BALF, T-cells, especially CD25+ cells, increased significantly in carriers and HAM/TSP patients, compared with healthy volunteers and seronegative patients. These findings indicated that T-cells in the lungs, as well as in peripheral blood, are activated in carriers and HAM/TSP patients. Interestingly, there was dissociation between expression of CD3 + CD25+ cells in BALF and peripheral blood from these patients. These results suggest that T-cells activated probably by HTLV-I accumulate in the lungs in some carriers and HAM/TSP patients, and HTLV-I may be involved in the immunologic dysfunction in the lungs of these patients. However, we did not find any correlation between the degree of clinical features and the elevation of CD3 + CD25+ cells in BALF, or its characteristic features on chest roentgenograms.     

Diesel Exhaust Particle Exposure In Vitro Alters Monocyte Differentiation and Function

Air pollution by diesel exhaust particles is associated with elevated mortality and increased hospital admissions in individuals with respiratory diseases such as asthma and chronic obstructive pulmonary disease. During active inflammation monocytes are recruited to the airways and can replace resident alveolar macrophages. We therefore investigated whether chronic fourteen day exposure to low concentrations of diesel exhaust particles can alter the phenotype and function of monocytes from healthy individuals and those with chronic obstructive pulmonary disease. Monocytes were purified from the blood of healthy individuals and people with a diagnosis of chronic obstructive pulmonary disease. Monocyte-derived macrophages were generated in the presence or absence of diesel exhaust particles and their phenotypes studied through investigation of their lifespan, cytokine generation in response to Toll like receptor agonists and heat killed bacteria, and expression of surface markers. Chronic fourteen day exposure of monocyte-derived macrophages to concentrations of diesel exhaust particles >10 µg/ml caused mitochondrial and lysosomal dysfunction, and a gradual loss of cells over time both in healthy and chronic obstructive pulmonary disease individuals. Chronic exposure to lower concentrations of diesel exhaust particles impaired CXCL8 cytokine responses to lipopolysaccharide and heat killed E. coli, and this phenotype was associated with a reduction in CD14 and CD11b expression. Chronic diesel exhaust particle exposure may therefore alter both numbers and function of lung macrophages differentiating from locally recruited monocytes in the lungs of healthy people and patients with chronic obstructive pulmonary disease.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice

Increased numbers of T lymphocytes are observed in the lungs of patients with chronic obstructive pulmonary disease, but their role in the disease process is not known. We investigated the role of CD8+ T cells in inflammatory cell recruitment and lung destruction in a cigarette smoke-induced murine model of emphysema. In contrast to wild-type C57BL/6J mice that displayed macrophage, lymphocyte, and neutrophil recruitment to the lung followed by emphysema in response to cigarette smoke, CD8+ T cell-deficient (CD8-/-) mice had a blunted inflammatory response and did not develop emphysema when exposed to long-term cigarette smoke. Further studies supported a pathogenetic pathway whereby the CD8+ T cell product, IFN-gamma-inducible protein-10, induces production of macrophage elastase (matrix metalloproteinase 12) that degrades elastin, both causing lung destruction directly and generating elastin fragments that serve as monocyte chemokines augmenting macrophage-mediated lung destruction. These studies demonstrate a requirement for CD8+ T cells for the development of cigarette smoke-induced emphysema and they provide a unifying pathway whereby CD8+ T cells are a central regulator of the inflammatory network in chronic obstructive pulmonary disease.     

沙美特罗联合噻托溴铵对慢性阻塞性肺疾病患者血清MMP-2, MMP-9及IL-8水平的影响

目的:探讨沙美特罗联合噻托溴铵对慢性阻塞性肺疾病患者血清炎症因子水平及肺功能的影响。方法:选择2014年5月-2016年5月我院收治的慢性阻塞性肺病患者83例作为研究对象,根据治疗方法不同,将所选患者分为研究组(45例)和对照组(38例)。研究组患者采用沙美特罗联合噻托溴铵吸入治疗,对照组患者采用沙美特罗治疗。观察并比较两组患者治疗前后血清MMP-2,MMP-9及IL-8水平及肺功能指标的变化情况。结果:治疗前两组患者血清MMP-2,MMP-9及IL-8水平比较,差异无统计学意义(P0.05);治疗后两组患者血清MMP-2,MMP-9及IL-8水平均低于治疗前,且研究组低于对照组,差异均具有统计学意义(P0.05)。与治疗前比较,两组患者治疗后FEV1/FVC,FEV1及MVV均升高,差异具有统计学意义(P0.05);与对照组比较,研究组患者治疗后FEV1/FVC,FEV1及MVV较高,差异具有统计学意义(P0.05)。结论:沙美特罗联合噻托溴铵治疗慢性阻塞性肺疾病的临床效果显著,不仅能够降低患者血清炎症因子水平,还可改善患者肺功能,值得临床推广应用。     

Lung Collagens Perpetuate Pulmonary Fibrosis via CD204 and M2 Macrophage Activation

Idiopathic pulmonary fibrosis is characterized by abundant collagen production and accumulation of alternatively activated macrophages (M2) in the lower respiratory tract. Mechanisms as to how alveolar macrophages are activated by collagen breakdown products are unknown. Alveolar macrophages were obtained by bronchoalveolar lavage from 30 patients with idiopathic pulmonary fibrosis (IPF) and 37 healthy donors (HD). Alveolar macrophages were cultured in the presence of collagen type I, III, IV and V monomers w/wo a neutralizing antibody against scavenger receptor I class A (CD204). Culture supernatants were assayed for the M2 markers CCL18, CCL2, and interleukin-1 receptor antagonist (IL-1ra) by ELISA. Furthermore, expression of phospho-Akt was measured using ELISA and expression of CD204 by RT-PCR and flow cytometry. Stimulation with collagen type I and III monomers significantly up-regulated CCL18, IL-1ra production of alveolar macrophages. Furthermore, expression of CCL2 and CD204 were up-regulated by collagen type I exposure. In addition, collagen type I stimulation increased pospho-Akt expression. Collagen type I effects were abrogated by neutralizing antiCD204 and a non-selective Phosphatidylinositide 3-kinase inhibitor (LY294002). Spontaneous CD204 expression of alveolar macrophages was significantly increased in patients with IPF. In conclusion, our findings demonstrate that monomeric collagen type I via CD204 induces phospho-Akt expression shifting alveolar macrophages to the profibrotic M2 type. Innate immune responses induced by collagen monomers might perpetuate pulmonary fibrosis.