Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis

Purpose

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation.

Methods

Platelet–monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n = 19), interstitial lung disease (ILD) other than IPF (n = 9), and control subjects without pulmonary fibrosis (n = 14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA.

Results

Significantly elevated levels of platelet–monocyte binding were found in patients with IPF (35.6 ± 4.34 % [mean ± SEM]) compared with patients with non-IPF ILD (23.5 ± 3.68 %) and non-ILD control subjects (16.5 ± 2.26 %; P < 0.01). There was a trend towards increased divalent cation-independent platelet–monocyte binding in IPF (6.0 ± 0.77 % [mean ± SEM]) compared with non-IPF ILD (4.3 ± 1.38 %) and control subjects without ILD (3.1 ± 1.75 %; P = 0.058). There was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet–monocyte binding had no significant correlation with percent predicted TLco or FVC.

Conclusions

Platelet–monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF.     

Chronic Low-Grade Systemic Inflammation Causes DNA Damage in the Lungs of Mice

Background

Whether systemic inflammation compromises the pulmonary system is largely unknown. We tested the hypothesis that chronic low-grade systemic inflammation damages alveolar wall cells.

Methods

A chronic low-grade systemic inflammatory state was induced in 8-week-old male C57/BL6J mice by administration of lipopolysaccharide (LPS, 44.4???g/day) for a 90-day period by subcutaneous implantation of a delayed-release pellet system. Acute systemic inflammation was induced in another group of mice by a single intraperitoneal injection of LPS (125???g/body). The lungs of mice were examined for histologic changes and genetic damage to alveolar wall cells.

Results

Chronic LPS exposure for a 30-day period or a 90-day period did not cause any obvious architectural changes in the lungs except for a mild level of alveolar macrophage infiltration. Despite the lack of architectural changes in the lung, immunofluorescence staining for ??H2AX and phosphorylated 53BP1 showed that chronic LPS exposure resulted in an almost doubling of the number of DNA double-strand breaks (DSBs) in type 1 and type 2 alveolar epithelial cells and in alveolar endothelial cells. Acute LPS exposure also resulted in a doubling of the number of DSBs in type 1 and type 2 alveolar epithelial cells and in alveolar endothelial cells at 24?h, but the increased number of DSBs returned to the baseline level by 48?h.

Conclusions

These results suggest that chronic systemic low-grade inflammation induces persistent DNA damage in alveolar epithelial and endothelial cells before architectural changes in the lung become evident.     

Obstructive sleep apnea is common in patients with interstitial lung disease

Purpose

The incidence of obstructive sleep apnea (OSA) in interstitial lung disease (ILD) has been reported at different frequencies in several studies. The aims of our study were to evaluate the frequency of OSA in ILD and to analyze the relationship between polysomnography (PSG) findings and pulmonary function, disease severity, parenchymal involvement, and Epworth Sleepiness Scale (ESS) scores.

Methods

ILD patients with parenchymal involvement were evaluated. The disease severity was assessed using an index consisting of body mass index (BMI), carbon monoxide diffusion capacity, the Modified Medical Research Council dyspnea scale, and the 6-min walking distance. All of the patients had lung function, chest X-ray, PSG, ESS scoring, and an upper airway examination. Patients with a BMI?≥?30 or significant upper airway pathologies were excluded.

Results

Of 62 patients, 50 patients comprised the study group (14 male, 36 female; mean age 54?±?12.35 years, mean BMI 25.9?±?3.44 kg/m²) with diagnoses of idiopathic pulmonary fibrosis (IPF; n?=?17), stage II–III sarcoidosis (n?=?15), or scleroderma (n?=?18). The frequency of OSA was 68 %. The mean apnea–hypopnea index (AHI) was 11.4?±?12.5. OSA was more common in IPF patients (p?=?0.009). The frequency of rapid eye movement-related sleep apnea was 52.9 %. The frequency of OSA was higher in patients with a disease severity index ≥3 (p?=?0.04). The oxygen desaturation index and the AHI were higher in patients with diffuse radiological involvement (p?=?0.007 and p?=?0.043, respectively).

Conclusions

OSA is common in ILD. PSG or at minimum nocturnal oximetry should be performed, particularly in patients with functionally and radiologically severe disease.     

L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury

Background

Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of l-arginine to l-citrulline in endothelial cells. We investigated whether administering l-citrulline by raising the serum levels of l-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.

Methods

Newborn rats were exposed to FiO₂?=?0.6 or room air for 14?days to induce lung derangement and then were administered l-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.

Results

Serum l-arginine rose in the L-citr?+?hyperoxia group (p?=?0.05), as well as the Von Willebrand factor stained vessels count (p?=?0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the l-citr?+?hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with l-citrulline under exposure to hyperoxia (p?=?0.0001). Lung VEGF and eNOS increased in the l-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p?=?0.003).

Conclusions

We conclude that administering l-citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.     

Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis.

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.     

Anti-pituitary antibodies against corticotrophs in IgG4-related hypophysitis

Purpose

IgG4-related disease is a systemic inflammatory disease characterized by infiltration of IgG4-positive plasma cells into multiple organs, including the pituitary gland. Autoimmunity is thought to be involved in the pathogenesis of IgG4-related disease. The diagnosis of IgG4-related hypophysitis (IgG4-RH) is difficult because its clinical features, such as pituitary swelling and hypopituitarism, are similar to those of other pituitary diseases, including lymphocytic hypophysitis and sellar/suprasellar tumors. The presence and significance of anti-pituitary antibodies (APA) in IgG4-RH is unclear.

Methods

In this case-control study, we used single indirect immunofluorescence on human pituitary substrates to assess the prevalence of serum APA in 17 patients with IgG4-RH, 8 control patients with other pituitary diseases (lymphocytic infundibulo-neurohypophysitis, 3; craniopharyngioma, 2; germinoma, 3), and 9 healthy subjects. We further analyzed the endocrine cells targeted by the antibodies using double indirect immunofluorescence.

Results

APA were found in 5 of 17 patients with IgG4-RH (29%), and in none of the pituitary controls or healthy subjects. The endocrine cells targeted by the antibodies in the 5 IgG4-RH cases were exclusively corticotrophs. Antibodies were of the IgG1 subclass, rather than IgG4, in all 5 cases, suggesting that IgG4 is not directly involved in the pathogenesis. Finally, antibodies recognized pro-opiomelanocortin in 2 of the cases.

Conclusions

Our study suggests that autoimmunity is involved in the pathogenesis of IgG4-RH and that corticotrophs are the main antigenic target, highlighting a possible new diagnostic marker for this condition.

     

Surfactant Protein A Influences Reepithelialization in an Alveolocapillary Model System

Purpose

Restoring the barrier integrity of the alveolar epithelium after injury is pivotal. In the current study, we evaluated the effects of surfactant, surfactant protein A (SP-A), transforming growth factor ?? (TGF-??), and analogues of SP-A on alveolar epithelial repair. Additionally, we assessed the influence of microvascular endothelial cells on reepithelialization.

Methods

Repair was studied in an in vitro model system consisting of a bilayer coculture of A549 and human pulmonary microvascular endothelial cells (HPMECs), which stably expressing fluorescent proteins. The epithelial repair was assessed in a scratch assay using vital fluorescence microscopy and compared with a monolayer of A549 cells.

Results

HMPEC cells differentially modulated the response of the A549 cells. Surfactant and SP-A augmented the reepithelialization in the presence of HPMECs, whereas in the absence of HPMECs, surfactant inhibited wound healing and SP-A failed to alter the response. Like SP-A, a structural analogue of its collagenous tail domain augmented the reepithelialization in the model system, whereas an analogue of its head domain did not alter the response. Additionally, we demonstrated that TGF-?? associated with SP-A was able to initiate the Smad-dependent TGF-?? pathway and that both TGF-?? and TGF-?? free SP-A were able to stimulate wound healing in the bilayer model.

Conclusions

These data show that surfactant, SP-A and TGF-??, influence epithelial repair in vitro and that the microvascular endothelial cells can modulate the response. This indicates that surfactant and SP-A could play a role in alveolar epithelial repair and that the microvascular endothelium may be involved in these processes.     

ADAM33 Gene Polymorphisms are Associated with the Risk of Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive dyspnea and worsening lung function due to remodeling of the lung, including epithelial mesenchymal transition. ADAM33 is a disintegrin and metalloprotease domain-containing protein, which may be related to lung fibrosis by exerting angiogenesis and remodeling of the lung. Thus, we evaluated the association of single-nucleotide polymorphisms (SNPs) of ADAM33 with the risk of IPF.

Methods

A total of 237 patients with IPF and 183 healthy subjects participated in the present study. Nine polymorphisms were selected. Genotyping was performed by single-base extension. Polymorphisms and haplotypes were analyzed for associations with the risk of IPF using multiple logistic regression models controlling for age, gender, and smoking status as covariates.

Results

All SNPs were in Hardy-Weinberg equilibrium. The minor allele frequency (MAF) of rs628977G>A in intron 21 was significantly lower in subjects with surgical IPF than in normal controls in the recessive model [33.2 vs. 38.0 %, p = 0.02, OR = 0.40 (0.19–0.84)]. When the subjects with clinical IPF were included, the difference in MAF persisted with a p value of 0.03 [OR = 0.50 (0.27–0.94)].

Conclusions

ADAM33 rs628977G>A was marginally associated with a decreased risk of IPF in a recessive model.     

Gremlin-1 for the Differential Diagnosis of Idiopathic Pulmonary Fibrosis Versus Other Interstitial Lung Diseases: A Clinical and Pathophysiological Analysis

Purpose

The differential diagnosis of interstitial lung diseases (ILDs), particularly idiopathic pulmonary fibrosis (IPF) versus other non-IPF ILDs, is important for selecting the appropriate treatment. This retrospective study aimed to explore the utility of gremlin-1 for the differential diagnosis.

Methods

Serum gremlin-1 concentrations were measured using an ELISA in 50 patients with IPF, 42 patients with non-IPF ILD, and 30 healthy controls. The baseline clinical data, including pulmonary functions, prognosis, and three serum biomarkers (Krebs von den Lungen-6 [KL6], surfactant protein-D [SP-D], and lactate dehydrogenase [LDH]), were obtained through a medical record review for analyzing their associations with serum gremlin-1 concentrations. To evaluate the origin of gremlin-1, we performed immunostaining on lung sections.

Results

Serum gremlin-1 concentrations were significantly higher in patients with IPF (mean concentration, 14.4 ng/mL), followed by those with non-IPF ILD (8.8 ng/mL) and healthy controls (1.6 ng/mL). The area under the curve for IPF versus non-IPF ILDs was 0.759 (95% confidence interval, 0.661–0.857), which was superior to that of KL6/SP-D/LDH. The sensitivity and specificity for gremlin-1 (cutoff, 10.4 ng/mL) was 72 and 69%, respectively. By contrast, serum gremlin-1 concentrations were not associated with the pulmonary functions nor the prognosis in all patients with ILDs. In immunostaining, the gremlin-1 was broadly upregulated in IPF lungs, particularly at myofibroblasts, bronchiolar/alveolar epithelium, and CD163-positive M2-like macrophages.

Conclusions

Gremlin-1 may be a useful biomarker to improve the diagnostic accuracy for IPF compared to non-IPF ILDs, suggesting a role of this molecule in the pathogenesis of IPF.

     

Imbalance of circulating endothelial cells and progenitors in idiopathic pulmonary fibrosis

Background

Fibrogenesis during idiopathic pulmonary fibrosis (IPF) is strongly associated with abnormal vascular remodeling. Respective abundance of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair and potentially serve as biomarkers of the disease.

Objectives and Methods

We postulated that CEC and EPC subtypes might be differently modulated in IPF. Sixty-four consecutive patients with newly diagnosed IPF were prospectively enrolled and compared to thirteen healthy volunteers. CEC were counted with immunomagnetic CD146-coated beads; progenitors CD34+45^dim/CD34+133+/CD34+KDR+were assessed through flow cytometry and EPC (colony-forming-units-Endothelial Cells, CFU-EC, and endothelial colonies forming cells, ECFC) were quantified by cell culture assays.

Results

IPF patients were characterized by a marked increase in CEC associated to an EPC defect: both CD34⁺KDR⁺ cells and CFU-EC were decreased versus controls. Moreover, in IPF subjects with a low diffusing capacity of the lung for carbon monoxide (DL_CO) < 40 %, CFU-EC and ECFC were higher compared to those with DL_CO > 40 %. Finally, ECFC were negatively correlated with DL_CO. During an 18 month follow up, CEC levels increased in patients with exacerbation, including those who died during follow up. Finally, ECFC from patients with exacerbation proliferative potential was strongly increased.

Conclusion

IPF is basically associated with both a vascular injury and a repair defect. This study highlights an adaptative process of EPC mobilization in the most severe forms of IPF, that could reflect enhanced homing to the pulmonary vasculature, which clinical consequences remain to be determined.     

Small bile duct involvement in IgG4-related sclerosing cholangitis: liver biopsy and cholangiography correlation

Background

IgG4-related sclerosing cholangitis (IgG4-SC) needs to be differentiated from primary sclerosing cholangitis (PSC). In this study, we performed a retrospective study to reveal cases in which liver needle biopsy was useful for differential diagnosis.

Methods

Nineteen patients with IgG4-SC and 22 patients with PSC were studied. All patients underwent endoscopic retrograde cholangiography and liver needle biopsy. We defined small bile duct involvement of IgG4-SC histologically as damage to the small bile duct associated with infiltration of ??10 IgG4+ plasma cells per high power field (HPF). Clinicopathological characteristics were compared between IgG4-SC patients with and without small bile duct involvement.

Results

Small bile duct involvement was observed in 5 (26%) of the patients with IgG4-SC. Patients with small bile duct involvement showed a higher incidence of intrahepatic biliary strictures on cholangiography (80 vs. 21%, p?=?0.038). Conversely, 4 of 7 (57%) patients with intrahepatic biliary strictures on cholangiography had histologically evident small duct involvement. The number of IgG4+ plasma cells was significantly correlated with the site of the most proximal stricture on cholangiograms (p?=?0.021). The number of IgG4+ plasma cells per HPF was significantly higher in IgG4-SC patients with intrahepatic biliary strictures than in those with PSC (13.4 vs. 0.4?cells/HPF, p?<?0.001).

Conclusions

Involvement of small bile ducts is more frequent in patients with intrahepatic biliary strictures on cholangiography, and liver needle biopsy is especially useful for these patients.     

Autoantibodies to a 140‐kd polypeptide,CADM‐140, in Japanese patients with clinically amyopathic dermatomyositis

Objective

To identify novel autoantibodies specific for dermatomyositis (DM), especially those specific for clinically amyopathic DM (C‐ADM).

Methods

Autoantibodies were analyzed by immunoprecipitation in 298 serum samples from patients with various connective tissue diseases (CTDs) or idiopathic pulmonary fibrosis (IPF). Antigen specificity of the sera was further examined by immunoblotting and indirect immunofluorescence (IF). The disease specificity and clinical features associated with the antibody of interest were determined.

Results

Eight sera recognized a polypeptide of ∼140 kd (CADM‐140 autoantigen) by immunoprecipitation and immunoblotting. Immunoreactivity was detected in the cytoplasm, and indirect IF revealed a granular or reticular pattern. Anti–CADM‐140 antibodies were detected in 8 of 42 patients with DM, but not in patients with other CTDs or IPF. Interestingly, all 8 patients with anti–CADM‐140 antibodies had C‐ADM. Among 42 patients with DM, those with anti–CADM‐140 autoantibodies had significantly more rapidly progressive interstitial lung disease (ILD) when compared with patients without anti–CADM‐140 autoantibodies (50% versus 6%; P = 0.008).

Conclusion

These results indicate that the presence of anti–CADM‐140 autoantibodies may be a novel marker for C‐ADM. Further attention should be directed to the detection of rapidly progressive ILD in those patients with anti–CADM‐140 autoantibodies.

     

Angiotensin-Converting Enzyme (ACE) Gene Polymorphisms are Associated with Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive dyspnea and worsening lung function. ACE is increased in the bronchoalveolar lavage fluid from patients with IPF, suggesting the role of ACE in the pathogenesis of IPF. We evaluated the role of single-nucleotide polymorphisms (SNPs) in the development risk of IPF.

Methods

Two-hundred twenty patients with IPF and 456 healthy subjects were included in this study. Eleven polymorphisms were selected among those reported previously. Genotype was performed by single base extension.

Results

Although absolute LD (|D′| = 1 and r ² = 1) was not present, 11 SNPs showed tight LDs. The logistic analysis of the all of 11 SNPs on the ACE genes between patients with IPF and healthy subjects were found to be related with the risk of IPF in recessive type. However, in patients with IPF diagnosed by surgical lung biopsy, only two SNP of ?5538T>C and +21288_insdel SNPs were related with the risk of IPF in co-dominant type, and there were no SNPs related with the risk of IPF in dominant type. In patients with IPF diagnosed by clinical criteria or surgical lung biopsy, four SNPs on promoter (?5538T>C, ?5508A>C, ?3927T>C, ?115T>C), one on intron (+15276A>G), one on exon (+21181G>A), and one in three prime region (+21288_insdel) were related with the risk of IPF.

Conclusions

This study showed a newly discovered SNP of ACE associated with the risk of development of IPF. ACE ?5538T>C and ?5508A>C significantly associated with risk of IPF in Korea.     

Predictive factors for the long-term use of pirfenidone in patients with fibrosing interstitial lung disease

BackgroundPirfenidone is an anti-fibrotic agent approved for idiopathic pulmonary fibrosis (IPF), and long-term treatment data and the effect of continuation after disease progression have been reported. The efficacy and safety of pirfenidone in fibrosing interstitial lung disease (ILD) patients without IPF have been recently reported in clinical trials; therefore, the benefits of long-term treatment are also expected. This study aims to analyze the long-term treatment data of pirfenidone and clarify the predictive factors for long-term use of pirfenidone in non-IPF patients.MethodsWe retrospectively reviewed the records of consecutive fibrosing ILD patients who started using pirfenidone between 2008 and 2014.ResultsOf the 266 fibrosing ILD patients, 167 patients had IPF, and 99 had non-IPF. Despite the non-significant differences in body size and pulmonary function between IPF and non-IPF patients, the non-IPF patients had better overall survival than the IPF patients (median 4.06 years vs. 2.09 years, p < 0.0001). In addition, the non-IPF patients had a significantly longer time to treatment discontinuation than the IPF patients (median 2.20 years vs. 1.20 years, p = 0.002). Multivariate logistic regression analysis for ≥2 years of use of pirfenidone showed that the percent predicted forced vital capacity (%FVC) and age were predictive factors common to both IPF and non-IPF patients.ConclusionsOur results indicate that non-IPF patients can continue using pirfenidone for longer durations than IPF patients. Initiation of pirfenidone for fibrosing ILD patients with higher %FVC and younger age would lead to long-term use of pirfenidone.     

Expression of Matrix Metalloproteinase-1 in Alveolar Macrophages,Type II Pneumocytes,and Airways in Smokers: Relationship to Lung Function and Emphysema

Background

An imbalance between proteolytic enzymes and their inhibitors is thought to be involved in the pathogenesis of chronic obstructive pulmonary disease. Matrix metalloproteinase-1, also known as interstitial collagenase, has been implicated as a potentially important proteinase in the genesis of chronic obstructive pulmonary disease and, more specifically, emphysema.

Methods

We performed quantitative immunohistochemical assessment of matrix metalloproteinase-1 expression in the resected lung of 20 smokers/ex-smokers who had varying severity of airflow obstruction and emphysema and compared this with the lungs of 5 nonsmokers. Emphysema was measured using a morphometric measure of the lungs’ surface area/volume ratio and with qualitative and quantitative computed tomography (CT) measures of emphysema.

Results

There were significantly more matrix metalloproteinase-1-expressing alveolar macrophages and type II pneumocytes as well as a greater percentage of small airways that stained positively for matrix metalloproteinase-1 in the lungs of smokers than in those of nonsmokers (p < 0.0001, p < 0.0001, and p = 0.0003, respectively). The extent of staining of type II pneumocytes and airways for matrix metalloproteinase-1 was significantly related to the extent of smoking (p = 0.012 and p = 0.013, respectively). In addition, the extent of matrix metalloproteinase-1 staining of alveolar macrophages was related to the lung surface area/volume ratio and to qualitative estimates of emphysema on CT.

Conclusion

These findings suggest that cigarette smoking increases expression of matrix metalloproteinase-1 in alveolar macrophages as well as in alveolar and small airway epithelial cells. Smokers who develop emphysema have increased alveolar macrophage expression of matrix metalloproteinase-1.     

A Possible Role of Galectin-9 in the Pulmonary Fibrosis of Patients with Interstitial Pneumonia

Background

Galectin-9 (Gal-9) is a β-galactoside-binding protein that induces biological reactions, such as cell activation, chemoattraction, and apoptosis. We evaluated the role of Gal-9 in the pathogenesis of interstitial pneumonia (IP).

Methods

Gal-9 levels in the bronchoalveolar lavage fluid (BALF) of patients with IP associated with collagen vascular disease (CVD-IP) and with idiopathic interstitial pneumonias (IIPs), including idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (idiopathic NSIP), were estimated by enzyme-linked immunosorbent assay. Gal-9 expression in the lungs of these patients was evaluated using immunohistochemistry. The effect of Gal-9 on the growth and apoptosis of human lung fibroblast cells was assessed in vitro.

Results

Gal-9 levels in the BALF were significantly higher in patients with CVD-IP than in patients with IIPs. Gal-9 levels significantly correlated with both the total cell count and the absolute number of lymphocytes in the BALF of patients with IIPs and CVD-IP. Strong reactivity with anti-Gal-9 antibody was observed in the cytoplasm of alveolar macrophages, lymphocytes, and type II pneumocytes in the lungs of patients with IP. Gal-9 expression in those cells was more remarkable in patients with CVD-IP than in patients with IPF and idiopathic NSIP. Gal-9 suppressed the growth of human lung fibroblast cells in a dose- dependent manner. Gal-9 induced apoptosis of human lung fibroblast cells in a dose-dependent manner.

Conclusions

Our findings suggest that the expression level of Gal-9 in the lung is increased in patients with CVD-IP and that Gal-9 may have a protective role against pulmonary fibrosis of these patients.     

Effect of Cigarette Smoke Extract on Insulin Transport in Alveolar Epithelial Cell Line A549

Background

The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells.

Methods

We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined.

Results

When A549 cells were treated with 30???g/ml of CSE for 48?h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats.

Conclusions

Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.     

How common is sleep-disordered breathing in patients with idiopathic pulmonary fibrosis?

Background and aim

The frequency of obstructive sleep apnea–hypopnea syndrome (OSAHS) in patients with idiopathic pulmonary fibrosis (IPF) remains controversial. The aim of this study was to assess the frequency of OSAHS in newly diagnosed IPF patients and to identify possible correlations with body mass index and pulmonary function testing parameters.

Materials and methods

Thirty-four newly diagnosed IPF patients were included. All subjects underwent attended overnight PSG. None of the included subjects was under any of the currently available IPF treatments or nocturnal supplemental oxygen therapy.

Results

Total apnea–hypopnea index (AHI) was <5, 5–15, and ≥15/h of sleep in 14 (41%), 15 (44%), and five patients (15%), respectively. REM AHI was statistically significant correlated with TLC [Total lung capacity] (p?=?0.03, r?=??0.38). Diffusing capacity of the lung for carbon monoxide was correlated with mean oxygen saturation during sleep (p?=?0.02, r?=?0.39).

Conclusions

Sleep-disordered breathing seems frequent, although remains usually under diagnosed in IPF patients. A decrease in TLC, reflecting the severity of pulmonary restriction, might predispose IPF patients in SDB, especially during the vulnerable REM sleep period.     

Identification of Citrullinated Hsp90 Isoforms as Novel Autoantigens in Rheumatoid Arthritis–Associated Interstitial Lung Disease

Objective

Subsets of patients with rheumatoid arthritis (RA) develop extraarticular complications that include interstitial lung disease (ILD). Because standardized algorithms for identification of RA patients at risk of developing clinically significant ILD are lacking, the purpose of this study was to elucidate unique serologic markers of RA‐associated ILD (RA‐ILD).

Methods

Sera from RA patients with ILD and from RA patients without ILD were used to immunoprecipitate citrullinated and uncitrullinated proteins derived from K562 cell extracts. Mass spectrometry was performed to facilitate identification of citrullinated proteins differentially immunoprecipitated by RA‐ILD patient sera. These candidate proteins were then used as substrate antigens in custom enzyme‐linked immunosorbent assays (ELISAs) for high‐throughput screening of sera obtained from cohorts of patients with RA, RA‐ILD mixed connective tissue disease (MCTD), or idiopathic pulmonary fibrosis (IPF).

Results

Differential immunoprecipitation and subsequent mass spectrometric sequencing identified citrullinated Hsp90α and citrullinated Hsp90β as candidate autoantigens in patients with RA‐ILD. ELISAs incorporating uncitrullinated and citrullinated isoforms of Hsp90 as substrate antigens demonstrated that sera from patients with RA‐ILD preferentially recognized citrullinated versions of Hsp90 with moderate sensitivity (range 20–30%) and great specificity (>95%) relative to sera derived from patients with RA alone (without ILD), patients with MCTD, or patients with IPF.

Conclusion

These studies demonstrate the utility of a novel autoantigen discovery method based on differential immunoprecipitation of citrullinated protein extracts. Application of these techniques identified citrullinated versions of Hsp90α and Hsp90β as autoantibody targets distinguishing RA‐ILD from RA without ILD, MCTD, and IPF, suggesting that anti–citrullinated Hsp90α/β autoantibodies may serve as effective biomarkers for the potentially devastating pulmonary manifestations of RA‐ILD.

     

Sera from anti–Jo‐1–positive patients with polymyositis and interstitial lung disease induce expression of intercellular adhesion molecule 1 in human lung endothelial cells

Objective

To investigate whether sera or purified IgG from patients with polymyositis (PM) and patients with dermatomyositis (DM), with or without interstitial lung disease (ILD), can activate endothelial cells (ECs).

Methods

Patients' sera were selected based on the presence or absence of anti–Jo‐1, anti‐SSA, or anti–U1 small nuclear RNP autoantibodies. The presence of autoantibodies was determined by line blot assays. Cultured human microvascular ECs derived from lung tissue (HMVEC‐L) were incubated with sera or purified IgG from 22 patients with PM, 7 patients with DM, and 10 healthy individuals as controls. Assessment of intercellular adhesion molecule 1 (ICAM‐1) expression was conducted by immunofluorescence (n = 22) and by cell‐based enzyme‐linked immunosorbent assay (ELISA) (n = 20). Serum levels of soluble ICAM‐1 (sICAM‐1) were determined by ELISA.

Results

Sera from PM patients with ILD who were positive for anti–Jo‐1 autoantibodies had a significantly stronger effect on the expression of ICAM‐1 by HMVEC‐L in comparison with sera from healthy controls and patients with other autoantibodies. Purified IgG did not induce ICAM‐1 expression. Higher serum levels of sICAM‐1 were found in patients with myositis compared with healthy controls.

Conclusion

EC activation with ICAM‐1 expression could contribute to the multiorgan involvement, including the development of myositis and ILD, in patients carrying anti–Jo‐1 autoantibodies. The EC‐activating factors are not the autoantibodies themselves, but might be systemic factors associated with these autoantibodies.