Abnormal re-epithelialization and lung remodeling in idiopathic pulmonary fibrosis: the role of deltaN-p63

Products of the p63 gene, a recently described member of the p53 family, are constitutively expressed in the basal cells of human bronchi and bronchioli. The truncated isoforms of the p63 gene (deltaN-p63 proteins) counteract the apoptotic and cell cycle inhibitory functions of p53 after DNA damage, and this property is likely to be central in the cell renewal strategy of stratified epithelial tissues. To investigate the dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we immunohistochemically analyzed the expression of the transactivating and dominant-negative isoforms of the p63 gene on 16 tissue samples obtained from patients suffering from this disorder. In most IPF cases herein investigated, epithelial cells expressing deltaN-p63 were observed at sites of abnormal proliferation at the bronchiolo-alveolar junctions, characterized by epithelial hyperplasia, squamous metaplasia, bronchiolization, and abnormal p53 nuclear accumulation. Similar features were not observed in normal lung and in samples taken from other pulmonary diseases used as controls, including acute interstitial pneumonia, idiopathic bronchiolitis obliterans organizing pneumonia, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia. On the basis of these findings, we can hypothesize a new model for UIP pathogenesis, involving a deregulated development of mesenchymal-epithelial interactions and abnormal proliferation of epithelial cells at the bronchiolo-alveolar junction after cell injury. In our view, the progressive loss of alveolar tissue and lung remodeling after injury in IPF/UIP is concomitantly produced by pneumocyte loss and alveolar collapse on one hand and by progressive bronchiolar proliferation and architectural distortion on the other.     

Fibroblast activation protein: a serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis

Fibroblast activation protein (FAPalpha) is a member of the cell surface dipeptidyl peptidase (DPP) family of serine proteases. In its dimer form, FAPalpha exhibits gelatinase, collagenase, and DPP activity in vitro. Reactive fibroblasts in healing wounds and stromal fibroblasts associated with epithelial tumors express FAPalpha. Idiopathic pulmonary fibrosis (IPF) is a disease of the lung characterized by progressive fibrosis with no clear etiology or molecular marker for disease activity. Recently, it has been shown that fibroblast FAPalpha expression is induced in liver cirrhosis, with an expression pattern distinct from alpha-smooth muscle actin (alpha-SMA). In this study, we determine whether FAPalpha expression is selectively induced in areas of ongoing tissue remodeling characterized by fibroblast foci in IPF. Human lung tissue was obtained from patients with IPF, centrilobular emphysema, and normal lung. Immunohistochemical studies were performed using anti-FAPalpha antibody and antibodies against alpha-SMA and CD26 (DPPIV), another member of the DPP family. We found that FAPalpha was not expressed in normal human lung tissue or tissue with evidence of centriacinar emphysema, but was induced in all patients with IPF and With a pattern distinct from that of CD26 found primarily on hyperplastic alveolar epithelium. Specifically, FAPalpha was detected in fibroblast foci and in fibrotic interstitium and not in the interstitium of adjacent architecturally normal lung. Alveolar/airway epithelium and vascular smooth muscle did not express FAPalpha. This is the first report of FAPalpha expression in IPF and our results suggest that FAPalpha is selectively induced in fibrotic foci, but not in normal or emphysematous lung. Future studies will address whether FAPalpha may be used as a marker for disease activity in IPF.     

Incomplete expression of epithelial-mesenchymal transition markers in idiopathic pulmonary fibrosis

The hypothesis that epithelial-mesenchymal transition (EMT) contributes to the formation of fibroblast foci (FF), which are the histological hallmark and the site of active disease progression of Idiopathic Pulmonary Fibrosis (IPF), has not yet received a conclusive demonstration. Cells undergoing EMT lose epithelial features and acquire mesenchymal markers and morphology. Cadherin expression switch (from E to N) is one of the first events in EMT.We investigated the immunohistochemical expression of E- and N-cadherin, vimentin, fibronectin, laminin-5-γ2, α-smooth muscle actin, and fibroblast-specific protein-1 involved in EMT in 20 IPF lung biopsies, focusing on metaplastic squamous cells of bronchial basal origin, positive for laminin-5-γ2 and ΔNp63/p40, that cover FF. The results were compared with organizing pneumonia, reactive squamous cell metaplasia of bronchiolar epithelia, and squamous cell carcinoma.Bronchiolar basal metaplastic cells in IPF partially lost E-cadherin and expressed vimentin and fibronectin. Hyperplastic pneumocytes in IPF and controls coexpressed E-cadherin and N-cadherin, and were weakly positive for lam5-γ2. Reactive squamous cell metaplasia did not show any mesenchymal markers. Squamous cell carcinoma only expressed lam5-γ2.In IPF lungs, we observed two epithelial cell populations with a different expression profile of markers involved in EMT. Although neither hyperplastic pneumocytes nor bronchial basal cells showed evidence of complete EMT, only the latter seem to be specific for UIP and might have a role in its development.     

p53 gene alteration in atypical epithelial lesions and carcinoma in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is well known to be associated with lung cancer. Several atypical epithelial lesions are frequently observed in the fibrotic area in IPF patients, and they have been suspected to be related to lung carcinogenesis. Several studies have suggested that p53 protein accumulation and mutation occur in the early pathogenesis of squamous cell carcinoma of the lung, suggesting some abnormality of the p53 tumor-suppressor gene in interstitial lung diseases. To examine the cause of the high frequency of lung cancer in IPF, we examined the p53 changes in atypical epithelial lesions and carcinoma in patients with IPF by immunohistochemistry and mutational analysis. We examined 19 lung cancer patients with IPF who underwent surgical resection for lung cancer in our institute. Paraffin-embedded tissues were treated by microwave and stained with an anti-p53 antibody (RSP53) by the avidin-biotin-peroxidase complex method. Mutations in exons 5 through 8 of the p53 gene were also examined by polymerase chain reaction mediated single-strand conformation polymorphism (polymerase chain reaction-single-strand conformation polymorphism) analysis and DNA sequencing. p53 protein was immunohistochemically detected in 13 (62%) of 21 squamous cell carcinomas, 3 (60%) of 5 squamous metaplasia with atypia, 16 (54%) of 30 squamous metaplasia, and 1 (4%) of 26 other hyperplastic lesions. p53 mutation was detected in 12 (57%) of 21 squamous cell carcinomas, 2 (40%) of 5 squamous metaplasia with atypia, 7 (23%) of 30 squamous metaplasia, and 0 (0%) of 26 other hyperplastic lesions. In conclusion, there are frequent p53 gene alterations in squamous metaplasia, which is distributed in the peripheral zone of the fibrotic area in patients with IPF. The present findings might provide a clue to the molecular mechanisms underlying the high incidence of lung cancer, especially peripheral-type squamous cell carcinoma in IPF patients, and suggest that p53 gene alterations play an important role in the early stages of lung carcinogenesis in patients with IPF.     

Immunohistochemical observations of keratins,involucrin, and epithelial membrane antigen in urinary bladder carcinomas from patients infected withSchistosoma haematobium

Summary Squamous cell carcinomas of the urinary bladder and the epithelial lesions associated with infection bySchistosoma haematobium were histopathologically and immunohistochemically described for keratin proteins (TK, 41–65 kDa; KL1, 55–57 kDa; PKK1, 40, 45 and 52.5 kDa), involucrin, and epithelial membrane antigen (EMA). Normal urothelial epithelium was positive for all keratins, and showed absent or slight reactions for involucrin and EMA in superficial umbrella cells. The intestinal type of epithelium was composed of columnar cells and small basal cells; TK was positive in the basal cells, KL1 staining was positive in the columnar cells, whereas PKK1 was negative or slight in the columnar cells. Involucrin was confined to columnar cells. Squamous metaplastic epithelium showed a rather regional keratin distribution: TK was distributed in all layers, KL1 decorated upper spinous and granular layers, but PKK1 did not bind, and involucrin staining existed only in upper spinous and granular cells. Keratin expression in squamous cell carcinomas indicated heterogeneity and its stainability was dependent on the degree of keratinization: The G 1 type revealed strong reaction, the G 2 type showed a similar distribution pattern, but the staining intensity was less, and the G3 type showed irregular staining with decreased intensity. Involucrin staining was limited to keratinized cells of carcinoma as was that for EMA.     

Titration of non-replicating adenovirus as a vector for transducing active TGF-β1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta₁ (TGF‐β₁) through a replication‐deficient recombinant adenovirus vector instilled into the lungs ( Sime et al. 1997 ). We have shown that this vector induces IPF in fibrogenic‐resistant tumour necrosis factor alpha‐receptor knockout (TNF‐αRKO) mice ( Liu et al. 2001 ). The object of our studies is to understand how peptide growth factors, such as TGF‐β₁, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF‐β₁ (AVTGFβ₁) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF‐β₁. The findings here show that 10⁶ plaque‐forming units (pfu) of AVTGFβ₁, provide essentially a ‘no‐effect’ dose, but even this amount of TGF‐β₁ causes a significant increase in whole‐lung collagen by day 28 after treatment. In contrast, 10⁸ and 10⁹ pfu cause severe IPF in 4 days, whereas 10⁷ and 5 × 10⁷ are intermediate for all parameters studied, i.e. TGF‐β protein, inflammatory cells, cell proliferation, pro‐α 1(I) collagen gene expression and whole‐lung collagen accumulation, and expression of growth factors such as TGF‐β₁, TNF‐α and PDGF‐A and ‐B. Interestingly enough, TGF‐β₁, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF‐β₁ biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

Titration of non-replicating adenovirus as a vector for transducing active TGF-beta1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta1 (TGF-beta1) through a replication-deficient recombinant adenovirus vector instilled into the lungs (Sime et al. 1997). We have shown that this vector induces IPF in fibrogenic-resistant tumour necrosis factor alpha-receptor knockout (TNF-alphaRKO) mice (Liu et al. 2001). The object of our studies is to understand how peptide growth factors, such as TGF-beta1, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF-beta1 (AVTGFbeta1) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF-beta1. The findings here show that 10(6) plaque-forming units (pfu) of AVTGFbeta1, provide essentially a 'no-effect' dose, but even this amount of TGF-beta1 causes a significant increase in whole-lung collagen by day 28 after treatment. In contrast, 10(8) and 10(9) pfu cause severe IPF in 4 days, whereas 10(7) and 5 x 10(7) are intermediate for all parameters studied, i.e. TGF-beta protein, inflammatory cells, cell proliferation, pro-alpha 1(I) collagen gene expression and whole-lung collagen accumulation, and expression of growth factors such as TGF-beta1, TNF-alpha and PDGF-A and -B. Interestingly enough, TGF-beta1, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF-beta1 biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

NF-κB在大鼠急性肺损伤模型肺组织中的表达及N-乙酰半胱氨酸的影响

目的 :检测NF κB在LPS诱导的急性肺损伤 (ALI)肺组织中的表达 ,以及N 乙酰半胱氨酸 (NAC)对ALI的抑制作用。方法 :采用免疫组化染色 (ABC法 )和Westernblot,检测NF κB在急性肺损伤大鼠气道和肺组织中的表达 ,以及NAC干预后活性NF κB表达的变化。结果 :正常对照组大鼠气道黏膜上皮和肺间质中 ,仅见少量散在的NF κB核阳性细胞 ;而LPS诱导ALI后 ,气道黏膜、肺间质、肺泡腔及血管内皮细胞中NF κB核阳性的细胞明显增多 (P <0 .0 1)。NF κB核阳性反应细胞主要为气道黏膜上皮细胞、浸润的炎症细胞、肺泡上皮细胞和血管内皮细胞。NAC治疗组NF κB核阳性细胞较LPS诱导的ALI组及对照组均明显减少 (P <0 .0 1)。Westernblot的结果显示 ,LPS诱导的ALI后不同时间点 ,NF κB的表达不同 ,于急性肺损伤 3h达高峰。各时间点NF κB的表达均较正常对照组高。结论 :LPS诱发的大鼠急性肺损伤的气道和肺组织内NF κB的表达增加 ,肺组织内的多数细胞参与了NF κB的激活。NAC可通过抑制NF κB的激活减轻急性肺损伤的炎症程度     

Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of beta-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear beta-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (DeltaN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear beta-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the beta-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/beta-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue.     

Alveolar epithelial cells in idiopathic pulmonary fibrosis display upregulation of TRAIL,DR4 and DR5 expression with simultaneous preferential over-expression of pro-apoptotic marker p53

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease of unknown aetiology with no current cure. The pathogenesis of IPF remains unclear but repeated alveolar epithelial cell (AEC) injuries and subsequent apoptosis are believed to be among the initiating/ongoing triggers. However, the precise mechanism of apoptotic induction is hitherto elusive. In this study, we investigated expression of a panel of pro-apoptotic and cell cycle regulatory proteins in 21 IPF and 19 control lung tissue samples. We reveal significant upregulation of the apoptosis-inducing ligand TRAIL and its cognate receptors DR4 and DR5 in AEC within active lesions of IPF lungs. This upregulation was accompanied by pro-apoptotic protein p53 overexpression. In contrast, myofibroblasts within the fibroblastic foci of IPF lungs exhibited high TRAIL, DR4 and DR5 expression but negligible p53 expression. Similarly, p53 expression was absent or negligible in IPF and control alveolar macrophages and lymphocytes. No significant differences in TRAIL expression were noted in these cell types between IPF and control lungs. However, DR4 and DR5 upregulation was detected in IPF alveolar macrophages and lymphocytes. The marker of cellular senescence p21^WAF1 was upregulated within affected AEC in IPF lungs. Cell cycle regulatory proteins Cyclin D1 and SOCS3 were significantly enhanced in AEC within the remodelled fibrotic areas of IPF lungs but expression was negligible in myofibroblasts. Taken together these findings suggest that, within the remodelled fibrotic areas of IPF, AEC can display markers associated with proliferation, senescence, and apoptotosis, where TRAIL could drive the apoptotic response. Clear understanding of disease processes and identification of therapeutic targets will direct us to develop effective therapies for IPF.     

Calcifying odontogenic cyst immunohistochemical detection of keratin and involucrin in cyst wall

Summary Calcifying odontogenic cysts (COC) were immunohistochemically described using different keratin proteins and involucrin as well as histopathology. The cystic lining epithelium was composed of calcifying, keratinizing, squamous, and columnar epithelial cells, and included calcified masses of irregular shape and various size as well as ghost cells. Calcifying epithelium gave negative or only trace staining for keratins detected with low molecular keratin (PKK1), but were regularly positive with high molecular keratin (KL1) and polyclonal antibody for keratin (TK). They were occasionally positive for involucrin. The cells located in the periphery of the calcified masses had a particular abundance of high molecular weight and total keratins (KL1 and TK). Calcified bodies and ghost cells were devoid of any immunoreactivity. Squamous epithelium was relatively similar to that of normal squamous cell epithelium in the oral mucosa. It were most commonly found in columnar cystic epithelial cells which displayed intense staining with all immunoreagents. It is postulated that such epithelial cells may have a strong potentiality to transform into ghost cells or to undergo metaplasia. They may develop altered synthesis of homogenous acellular materials and finally become transformed into calcifying epithelium containing dystrophic calcified masses.     

Microsatellite instability in transforming growth factor-beta 1 type II receptor gene in alveolar lining epithelial cells of idiopathic pulmonary fibrosis

It has been reported that transforming growth factor (TGF)-beta, which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the growth-inhibitory effects of TGF-beta, we searched for mutations in the microsatellite of the TGF-beta receptor type II (T beta RII) gene. To detect a deletion in the polyadenine tract in exon 3 of the T beta RII gene, cells were isolated by microdissection from lung sections of IPF patients, and DNA was extracted from these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low T beta RII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite instability in the T beta RII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this genetic disorder might play a partial role in the pathologic changes of IPF.     

Role of the mucosal integrin alpha(E)(CD103)beta(7) in tissue-restricted cytotoxicity

The effectiveness of lung transplantation is marred by the relatively high incidence of rejection. The lung normally contains a large population of lymphocytes in contact with the airway epithelium, a proportion of which expresses the mucosal integrin, alpha(E)(CD103)beta(7). This integrin is not a homing receptor, but is thought to retain lymphocytes at the epithelial surface. Following transplantation, a population of 'tissue-restricted' cytotoxic T cells (CTL) have been identified which have the ability to lyse epithelial cells, but not major histocompatibility complex (MHC)-identical splenic cells. We tested the hypothesis that expression of the mucosal integrin confers the ability of CTL to target and destroy e-cadherin expressing targets. Immunohistochemical and flow cytometric analyses were used to demonstrate the relevance of this model to human lung. Allo-activated CTL were generated in mixed leucocyte reactions and CD103 expression up-regulated by the addition of transforming growth factor (TGF)-beta. The functional effect of CD103 expression was investigated in (51)Cr-release assays using e-cadherin-expressing transfectant targets. Human lung epithelial cells express e-cadherin and one-third of intraepithelial lymphocytes (IEL) expressed CD103. Allo-activated and bronchoalveolar lavage (BAL) lymphocytes express more CD103 than those in blood. Transfection of e-cadherin into murine fibroblasts conferred susceptibility to lysis by alpha(E)beta(7)-expressing CTL which could be blocked by specific monoclonal antibodies to CD103 and e-cadherin. CD103 functions to conjugate CTL effectors to e-cadherin-expressing targets and thereby facilitates cellular cytotoxicity. E-cadherin is expressed prominently by epithelial cells in the lung, enabling CTL to target them for destruction.     

Reduced expression of cyclooxygenase (COX) in idiopathic pulmonary fibrosis and sarcoidosis

AIMS: To test the hypothesis that cyclooxygenase (COX)-1 or COX-2 expression is defective in lungs in idiopathic pulmonary fibrosis (IPF) and to characterize the cellular distribution. IPF is a progressive inflammatory lung disorder with an adverse prognosis. Previous work has shown that prostaglandin E2 (PGE2) regulates collagen deposition and fibroblast proliferation and a defect in COX regulation may contribute to the fibrosis that occurs in IPF. METHODS: Immunohistochemistry was utilized to determine COX immunoreactivity in lung sections from 25 IPF, six sarcoidosis and 14 control subjects. RESULTS: COX-1 and COX-2 expression in bronchiolar epithelial cells was significantly lower in IPF and sarcoidosis than in controls. No significant difference was found in COX-2 expression between macrophages in IPF and control sections, but COX-2 was reduced in macrophages in sarcoidosis compared with controls. CONCLUSIONS: These studies confirm COX-2 loss in bronchial epithelial cells but not macrophages in IPF, and show for the first time reduced constitutive COX-1 expression in epithelial cells and macrophages. Similar abnormalities were observed in sarcoidosis.     

肺癌中P63与P53、E-cadherin、Ki-67表达的比较

目的　比较 p6 3及 p5 3、E cadherin(E cad)、Ki 6 7在肺癌中的表达 ,以了解在不同组织类型肺癌发生发展过程中 ,p6 3与抑癌基因 (p5 3)突变、上皮分化标志基因 (E cad)失活及细胞增殖标志基因 (Ki 6 7)激活有无相关性。方法　采用免疫组化S P法分别检测 6 1例原发性肺癌中 p6 3、p5 3、E cad和Ki 6 7的表达情况。 结果　p6 3在肺鳞癌中阳性率为 10 0 0 % ,而在其他组织类型肺癌中 p6 3基本不表达 ,差异有显著性 (P <0 0 5 ) ;在不同分化程度的肺鳞癌中 p6 3、p5 3的表达差异有显著性 (P<0 0 5 ) ,E cad、Ki 6 7的表达差异无显著性 (P >0 0 5 ) ;E cad的表达在小细胞肺癌与肺鳞癌和肺腺癌之间差异有显著性 (P <0 0 5 ) ;Ki 6 7的表达在各种组织类型肺癌之间差异有显著性 (P <0 0 5 ) ;在不同分化程度鳞癌中 p6 3与E cad的表达呈负相关(P <0 0 5 )。结论　p6 3可作为鳞状上皮源性肿瘤标记物 ,是判断鳞状细胞癌的增殖和分化有意义的指标 ,并可作为鉴别分化差的鳞癌和腺癌、小细胞癌的指标。