Pneumocyte injury and ubiquitin‐positive pneumocytes in interstitial lung diseases

Pneumocyte injury is a characteristic of pulmonary interstitial pneumonias (IPs). Histological markers of pneumocyte injury and inflammation include pneumocyte necrosis, erosion, hyaline membrane and fibrin exudation with subsequent intraluminal granulation tissue formation. We found that intracytoplasmic inclusions in pneumocytes are ubiquitin‐positive (Ub⁺) and that the number of Ub⁺ pneumocytes shows positive correlation with the extent of diffuse alveolar damage (DAD). To determine the role of Ub⁺ pneumocytes and inclusions in IPs, we studied their relationship with pathological and clinical features of DAD, usual interstitial pneumonia (UIP) and organizing pneumonia (OP), including airspace enlargement with fibrosis (AEF). We analysed Ub⁺ pneumocytes, inclusions, erosions and intraluminal granulation tissue in relation to pneumocyte injury. The numbers of immunohistochemically identified Ub⁺ inclusions in each IP were higher than the number of inclusions detected by light microscopy. The inclusions detected by Ub⁺ immunostaining were identical to the inclusions observed by light microscopy. UIP and DAD had many Ub⁺ inclusions, while OP and AEF had fewer Ub⁺ inclusions. These results suggest that the extent of Ub⁺ inclusions reflects the severity of pneumocyte injury among IPs. Thus, Ub⁺ inclusions are a histological marker of pneumocyte injury that may be helpful in determining the severity and prognosis of IPs.     

Time course of bleomycin-induced lung fibrosis

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

ICAM-1, ICAM-2 and ICAM-3 in the Sera of Patients with Idiopathic Pulmonary Fibrosis

In order to test the serum levels of ICAM-1, ICAM-2 and ICAM-3 in patients with idiopathic pulmonary fibrosis (IPF), twenty patients with IPF and eleven with secondary interstitial fibrosis (SIF), as well as forty healthy volunteers (HV) were studied. Serum intracellular adhesion molecules (ICAM) 1, 2 and 3 were assessed by ELISA. Functional respiratory tests, which included spirometry and lung diffusing capacity were simultaneously performed. Median values of serum ICAM-1 and ICAM-2 were higher in the patients' than in the healthy volunteers' (HV) group: IPF group: 946.60 ng/ml and 400.14 ng/ml; SIF group: 901.58 ng/ml and 378.27 ng/ml; HV group: 308.40 ng/ml and 217.55 ng/ml, respectively (p < 0.05). ICAM-3 serum levels were equal between the three groups. ICAM-2 negatively correlated to DLCO values. (p < 0.005). It can be concluded that ICAM 1 and 2 are elevated in the sera of patients with pulmonary fibrosis. ICAM-2 might be associated with a more impaired clinical status.     

Smoking‐related changes in the background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course

Aims: To assess the pathological findings in lobectomy specimens, to correlate them with smoking history and postoperative course and to compare the findings with those in smoking‐related interstitial lung disease. Methods and results: Patients who had undergone lobectomy for lung cancer were reviewed. Subjects included 230 non‐smokers and 587 smokers, of whom 572 had a known smoking index (SI). They were classified into mild, moderate and heavy smokers. Centrilobular emphysema (CLE), respiratory bronchiolitis, airspace enlargement with fibrosis (AEF), the presence of foci resembling usual interstitial pneumonia pattern (UIP/P) and the rate of postoperative respiratory failure were assessed. The incidence of AEF was 6.5% in mild smokers, and 17.7% in moderate smokers (P < 0.01) with lower lobe predominance. There were significant correlations (P < 0.01) between AEF and CLE and AEF and UIP/P. The rate of respiratory failure after lobectomy was 6%, and 10% in patients having UIP/P with or without AEF, but was not seen in patients with AEF alone (P < 0.01). Conclusions: AEF is an important smoking‐related change in the lung that appears to correlate with the smoking history, and its distinction from UIP/P may be important.     

Effects of injury and repair of the pulmonary endothelium on lung metastasis after bleomycin

Acute endothelial injury induced by bleomycin has been shown to enhance the localization and metastasis of circulating tumour cells. In the present study we wished to determine whether increased metastases to the lung is related to the degree of endothelial damage as indicated by morphology and protein leakage to alveoli and whether the progression to repair with pulmonary fibrosis also effects metastatic tumour growth. C57b1/6 mice were injected with a single intravenous dose of bleomycin (120 mg/kg). After 5 days, severe enothelial injury was demonstrated by morphology and by increased levels of protein in lung lavage fluid. When [131I]-iododeoxyuridine labeled syngeneic fibrosarcoma cells were injected intravenously at this time, a 9-fold increase in their localization was detected 24 h later in bleomycin-treated lungs compared with saline controls. By electron microscopy tumour cells were observed at sites of denuded vascular basement membrane. There was also a significant increase in the number of gross metastases which developed subsequently and in the percentage of lung occupied by tumour in the bleomycin group. Animals examined 10 days after bleomycin showed less endothelial damage and a smaller increase in tumour cell localization and metastases. At 21 days, when endothelial structure and alveolar protein levels had returned to normal, and at 6 weeks, when there was focal fibrosis, no increase in tumour cell localization or metastases was found. It is concluded that damage to the pulmonary endothelium is a key factor in enhancing the trapping of circulating tumour cells and increasing metastatic tumour growth after bleomycin.     

Postmitotic differentiation of rat lung fibroblasts: induction by bleomycin and effect on prolyl 4-hydroxylase

The cytostatic drug bleomycin (BLM) induces pulmonary fibrosis as its main side effect. Fibroblasts in fibrotic foci are the main cellular source for extracellular matrix accumulation that typifies fibrosis. In vitro studies demonstrated the ability of cytotoxic drugs to induce terminal differentiation of fibroblasts. These postmitotic cells are very active in regard to production of collagens. The present study was addressed to investigate the potential of BLM to induce terminal differentiation of rat lung fibroblasts in vitro and the consequences for collagen production and for the expression and activity of the collagen modifying enzyme prolyl 4-hydroxylase (P4H). The BLM effects were compared with those of mitomycin C (MMC), another cytotoxic agent with known potential for initiation of postmitotic differentiation of fibrobasts.

BLM induced postmitotic differentiation of rat lung fibroblasts. The capacity of the cells to form clones was diminished by BLM or MMC in a concentration dependent manner. Both drugs initiated the formation of an increasing number of postmitotic cell clones. The postmitotic differentiation was accompanied by an increase in total collagen production by the cells. Administration of BLM to cultures of lung fibroblasts at concentrations of 1 or 10 mU/ml resulted in an increase of the collagen amount to about the 1.5-fold and 1.6-fold of controls, respectively. Treatment of fibroblasts with MMC elevated the collagen level to about the 2-fold. P4H activity and P4H mRNA levels in cells exposed to BLM or MMC were found to be increased.

We conclude that terminally differentiated fibroblasts might be part of the heterogeneous population of fibroblast-like cells in fibrotic foci responsible for the increased production of collagen during the fibrotic phase of the development of pulmonary fibrosis.     

肿瘤浸润淋巴细胞与IL-2联合诱导Lewis肺癌凋亡的研究

目的：研究肿瘤浸润淋巴细胞（ＴＩＬ）与ＩＬ－２联合体内诱导凋亡产生抗瘤作用的机理。为应用ＴＩＬ治疗肺癌提供依据。方法：从Ｌｗｉｓ肺癌（ＬＬＣ）瘤体中分离提了参含１０００Ｕ／ｍｌＩＬ－２的完全培养基中培养后，与ＬＬＣ细胞中１０：１接种于Ｃ５７ＢＬ／６小鼠腋下，每日１０００Ｕ／ｍｌＩＬ－２瘤内注射，并测量肿瘤体积，于接种后第１８天处死小鼠。结果：发现ＴＩＬ加ＩＬ－２有明显地抑制ＬＬＣ生长的作用，ＨＥ感     

气管内应用白介素2重组腺病毒免疫治疗肺转移癌

目的：研究气管内应用白介素2（IL－2）重组腺病毒（IL－2 gene recombinant adenovirus,AdIL-2)对实验性肺转移癌的治疗作用。方法：①小鼠气管内滴入AdIL-2,ELISA法测定局部及外周血IL－2及相关因子的水平。②C57BL／6小鼠尾静脉注射Lewis肺癌3LL细胞建立肺转移癌模型，观察气管内应用AdIL-2对实验性肺转移癌的治疗作用，包括肺转移结节、动物存活期、生存率变化等，并比较自然杀伤活性（Natural killer,NK)和特异性细胞毒（Cytotoxic T lymphocytes,CTL)活性的变化。结果：①气管应用AdIL-2后肺灌洗液中测到IL－2且高于外周血水平，对照组肺灌洗液及外周血中的水平明显低于实验组（P＜0．01）。②气管内滴入AdIL-2对实验性肺转移癌有治疗作用，可使肺转移结节减少、动物的生存期延长及存活率升高，同时对NK、CTL活性有增强作用（P＜0．01）。结论：气管内应用IL－2重组腺病毒可有效表达并对实验性肺转移癌有治疗作用，为肺部肿瘤的治疗提供了一种新方法。     

IL-10抗内毒素气管内滴注致急性肺损伤作用的实验研究关

目的:探讨中性粒细胞(PMN)在急性肺损伤(ALI)发生中的作用及IL-10对ALI的拮抗作用。方法: 用LPS(100 μg/只)或LPS+IL-10(1 μg/只)向SD大鼠气管内滴注复制ALI模型,检测支气管肺泡灌洗液(BALF)中PMN数目、蛋白质及丙二醛(MDA)含量,并进行组织学观察。结果: LPS气管内滴注可引起BALF中PMN数目明显增加,伴有蛋白质及MDA含量的增高,光镜观察显示肺组织间隙弥漫性炎细胞浸润。LPS+IL-10组则BALF中PMN数目、蛋白质及MDA含量显著低于LPS组,肺组织中PMN浸润程度也明显轻。结论: PMN在ALI发病中具有重要作用。IL-10能够拮抗LPS所致ALI的发生。     

类风湿关节炎并发间质性肺疾病患者血清蛋白质指纹图谱的临床价值分析

目的:应用表面加强激光解吸电离飞行时间质谱筛选出类风湿关节炎并发间质性肺疾病(RA-ILD)患者血清中的差异蛋白,并对其临床价值进行初步分析。方法:用WCX2(弱阳离子交换芯片)蛋白芯片结合表面增强激光解吸电离飞行时间质谱(Surface enhanced laser desorption/ionization time of flight mass spectrum,SELDI-TOF-MS)检测19例RA-ILD患者,15例单纯RA、未合并间质性肺疾病患者,13例相对健康的对照组患者的血清样本,应用Biomarker Wizard软件、Biomarker Pattern软件以及SPSS13.0对测得的数据进行处理,初步构建分类树模型并随机选取部分病例进行验证。结果:患者血清中共检测出142个蛋白峰,经方差分析,发现了4个蛋白质峰差异有统计学意义,质荷比分别为3382.59、3453.39、11886.0、5825.48,Biomarker Pattern软件对所检测的所有数据分析后,发现以质荷比11689.4、2266.49、1020.22、4392.28、5074.38、2764.69为最适合建立分类树模型,灵敏度为92.31%(12/13),特异度为91.18%(31/34)。随机选取RA-ILD、RA、相对健康的对照组患者的血清样本各10例进行模型验证,灵敏度为90%(9/10),特异度为90%(18/20)。结论:SELDI-TOF-MS技术可筛选出RA-ILD患者血清中差异性表达蛋白,作为生物标志物,其诊断敏感度高,特异度好,对其测定具有较好的临床应用前景。     

Evaluation of soluble IL-6 receptor concentration in serum and epithelial lining fluid from patients with interstitial lung diseases.

We measured soluble IL-6 receptor (sIL-6R) levels in serum and bronchoalveolar lavage fluids (BALF) from patients with interstitial pneumonia of unknown etiology (IP) (n = 17), sarcoidosis (n = 8) and normal control subjects (n = 10), to investigate its role in pulmonary diseases. Soluble IL-6R was determined by an ELISA. The volume of epithelial lining fluid (ELF) in BALF was estimated using an urea method. We found that levels of sIL-6R in serum, BALF, and ELF from patients with IP or sarcoidosis were significantly higher than those from normal subjects. Furthermore, levels of sIL-6R in BALF or ELF were significantly correlated with those of albumin, indicating that sIL-6R, together with albumin, may enter ELF as a result of the increased permeability caused by pulmonary inflammation. Thus most of the sIL-6R in ELF would be from serum, and relatively small amounts of it might be produced locally. However, sIL-6R levels in ELF, but neither serum nor BALF, were significantly correlated with levels of C-reactive protein in patients with IP. These results suggest that both systemic and local production of sIL-6R are increased, and raised sIL-6R is involved in the modulation of systemic and local inflammatory responses in patients with IP and sarcoidosis.     

Does a primary host defense abnormality involving monocytes-macrophages underlie the pathogenesis of lung disease in cystic fibrosis?

The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is chronic pulmonary disease. Pulmonary disease in CF is characterized in part by: (a) obstruction of the bronchi and bronchioles by inspissated secretions (mucus is hypersecreted and may also be abnormal), (b) recurrent or persistent bacterial infections, and (c) a chronic inflammatory state. We propose herein that much of the pathophysiology of lung disease in CF stems from a genetically inherited metabolic defect in monocyte-macrophages (M-MØ), and we review evidence which indicates that CF M-MØ are innately metabolically abnormal. Once activated by various stimuli, CF M-MØ become metabolically hyperactive and hypersecretory as evidenced by the production of excessive levels of a variety of mediators which could have definite roles in both the initiation of pulmonary obstruction and the accelerated development of a chronic inflammatory response in CF. Evidence is also reviewed which indicates that other CF M-MØ functions crucial to the afferent and efferent phases of the immune response to bacterial infections in the lung may be adversely affected. Mechanisms proposed to explain the abnormal production of mediators by CF M-MØ are discussed, and it is concluded that hyperproduction of mediators by CF M-MØ and their metabolic hyperactivity probably result from a defect in autoregulation. The nature of the metabolic defect in CF M-MØ indicates that CF should be classified as a “new” primary host defense abnormality or alternatively as a “new” primary immune deficiency disease.     

TREM-2对小鼠肺成纤维细胞凋亡的影响

目的:初步探讨髓样细胞触发受体-2(triggering receptors expressed on myeloid cells-2,TREM-2)过表达对小鼠肺成纤维细胞凋亡的影响.方法:将小鼠肺成纤维细胞分成脂多糖( lipopolysaccharide,LPS)组、LPS+转染试剂组、LPS+空质粒组和LPS+...     

Early IL-2 treatment of mice with Pseudomonas aeruginosa pneumonia induced PMN-dominating response and reduced lung pathology

IL-2 is a pro-inflammatory and a Th1 inducing cytokine, which is important for activation of the cell-mediated immunity. IL-2 in serum and sputum has been observed to be reduced in cystic fibrosis (CF) patients. The present IL-2 treatment study of Pseudomonas aeruginosa (PA) lung infected mice was performed in order to evaluate the effect of IL-2 supplement. One hundred-and-twenty female BALB/c mice were divided into three groups: 1) IL-2 treatment/infection (TIG), 2) non-treatment/infection (NTIG), and 3) IL-2 treatment/non-infection (TNIG). The mice were challenged intra-tracheally with PA (PAO579) embedded in seaweed alginate to resemble the biofilm mode of growth. At day 0 and 1, the treatment groups received IL-2 s.c. Mice were killed on day 1 or 2, and cytokine production, lung pathology, and quantitative lung bacteriology were estimated. IL-2 treatment of infected mice reduced the number of mice with signs of macroscopic lung pathology at day 2 (p < 0.05). The reduced macroscopic pathology was paralleled by a reduced IL-1β and TNF-α. In contrast, an increased PMN response at day 2 was observed in the IL-2 treated mice (p < 0.01). This was preceded by a significantly higher degree of microscopic inflammation at day 1 (p < 0.02). The IL-12 levels decreased in both groups of infected mice at day 2 (p < 0.01), however, significantly more in the IL-2 treated mice (p < 0.02). In accordance, but surprisingly, IFN-γ was significantly reduced in the IL-2 treated PA infected group at day 2 (p < 0.05). The present results indicate that early IL-2 treatment prolongs the PMN response but also reduces pro-inflammatory IL-1β and TNF-α and macroscopic signs of pathology.     

Effect of recombinant interleukin-5, interleukin-3, and eotaxin on apoptosis in eosinophilic granulocytes

We studied in vitro effects of recombinant interleukin-5, interleukin-3, and eotaxin on programmed death of eosinophils from healthy donors and patients with non-Hodgkin’s lymphomas associated with severe blood eosinophilia. Interleukin-5 and eotaxin produced the most potent antiapoptotic effect on eosinophils from healthy donors. In patients with non-Hodgkin’s lymphomas, spontaneous apoptosis in eosinophilic leukocytes was low and remained unchanged during incubation with recombinant proteins. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 370–373, April, 2007