| Angiogenesis, apoptosis and re-epithelialization at the foci of recent injury in usual interstitial pneumonia and bronchiolitis obliterans organizing pneumonia | ||
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Apoptosis is a biochemically regulated and active cell death program whereby individual cells die without injuring neighboring cells or causing inflammatory reaction. During apoptosis, the dying cell separates from its neighbors and undergoes a period of membrane blebbing, condensation of cytoplasm and increase in cell density. Simultaneously the nuclear chromatin becomes compact, segregates and forms sharply delineated masses along the nuclear envelope. The nucleus splits into discrete fragments and finally, the cell splits into a cluster of membrane bound apoptotic bodies, each containing a variety of organelles. Apoptotic bodies are ingested by nearby cells and macrophages before they cause an inflammatory reaction (Kerr et al. 1972, Kerr et al. 1994). The morphologically visible process of apoptosis takes a few hours, and the majority of the time is spent on the degradation within the phagocytic cells (Wyllie 1997a). Apoptosis contrasts with necrosis, which is a passive and accidental form of cell death. In necrosis the cell swells, the cell membrane is disrupted, and the nuclear and cytosolic structures are demolished, causing an inflammatory reaction in the neighboring cells (Kerr et al. 1972, Wyllie 1997a).
Apoptosis takes place in a variety of biologically significant situations including embryogenesis, organogenesis and the maintenance of homeostasis as well as normal function of the immune system (reviewed by Cohen et al. 1992, Wyllie 1997a, and Cummings et al. 1997). Abnormal regulation of apoptosis has been implicated in the onset and progression of diseases both in the form of inhibited and excessive apoptosis (reviewed by Thompson 1995 and Antonsson 2001). In the lung, as in other organs, apoptosis plays a critical role in organogenesis and alveolarization by reducing the number of fibroblasts and type II epithelial cells (Schittny et al. 1998). The normal resolution of inflammation in the lung occurs also through the regulated removal by apoptosis of unwanted cells such as granulocytes without the release of damaging histotoxins. Controlled and localized apoptosis is a prominent feature in the resolution of pneumonia (reviewed by Haslett 1999). According to current knowledge, abnormal regulation of apoptosis also plays a role in the pathogenesis of asthma and COPD (Kasahara et al. 2000, Dorscheid et al. 2001, Melis et al. 2002).
Apoptosis can be induced by a variety of physiologic, damage-related and therapy-associated agents (reviewed by Thompson 1995, and Wyllie 1997b). Two major apoptosis pathways have been identified, namely the death receptor pathway (also called extrinsic pathway) and the mitochondrial (intrinsic) pathway. The death receptor pathway involves at least five transmembrane receptors belonging to the TNF (tumor necrosis factor)/NGF (nerve growth factor) -receptor superfamily (reviewed by Timmer et al. 2002). Fas is a type I cell surface protein belonging to the TNF/NGF receptor family (Itoh et al. 1991). FasL is a type II membrane protein that belongs to the TNF family and is expressed predominantly in activated T lymphocytes and in tissues including small intestines, kidney, testis, and lung (Suda et al. 1993). The mitochondrial pathway is mediated by mitochondrial membrane permeabilization and the release of cytochrome c (reviewed by Antonsson 2001). In both of these pathways, the final result is the activation of the so-called caspase cascade, which leads to proteolysis of structural and regulatory proteins and cell death, bcl-2 family proteins and p53 regulating apoptosis (reviewed by Antonsson 2001; Timmer et al. 2002). p53 is a nuclear protein important in cell cycle regulation and homeostasis. p53 is up-regulated in response to DNA damage and functions either by inhibiting cellular division through G1 arrest or by facilitating apoptosis. p21Waf1/Cip1 is a cyclin-dependent kinase inhibitor that is induced by p53 and can activate both G1 and G2 cell cycle arrests (reviewed by Bálint & Vousden 2001)
The bcl-2 family is a group of apoptosis-regulating genes which are able to inhibit or promote apoptosis. At least 20 bcl-2 family members have been identified in mammalian cells (Table 3). All members posses at least one of the four bcl-2 homology domains (BH1-BH4). Current data suggest that there are two potentially independent mechanisms for promoting cell death. One mechanism is based on dimerization, which is essential for the function of pro-apoptotic BH3 subfamily and provides an important mechanism for controlling the activity of bcl-2 and bax. The other, an intrinsic, heterodimerization-independent function is probably related to the ability of these proteins to insert into membranes (reviewed by Antonsson 2001).
There are many approaches to analyze apoptosis, but none can exceed morphologic examination. Nuclear shrinkage and budding, loss of cell shape, and eventual cytoplasmic blebbing are hallmarks of an apoptotic cell. Although these are features of late-stage apoptotic cells, they are the gold standard in the recognition of apoptotic morphology (Kerr et al. 1972, Kerr et al. 1994). Additional methods include the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay for identification of DNA fragmentation, and methods based on detection of apoptosis-related proteins or cell membrane changes. These techniques also identify later stages of apoptosis, and their detection abilities vary considerably (reviewed by Soini et al. 1998, and Stadelmann & Lassmann 2001). Combining a plain morphological evaluation of apoptosis with the 3´-end labeling method forms a reliable tool for analysis of apoptosis (Soini et al. 1998).
Polunovsky et al. (1993) showed that bronchoalveolar lavage fluid obtained from patients suffering from adult respiratory distress syndrome (ARDS) induced cell death of fibroblasts and endothelial cells. The mode of cell death for endothelial cells was apoptosis. Fibroblast death was distinct from necrosis, but also differed from typical apoptosis (Polunovsky et al. 1993). Since then research on apoptosis in pulmonary fibrosis has focused on apoptosis of epithelial cells in order to understand the mutual interaction between mesenchymal and epithelial cells. It has been shown that altered fibroblasts isolated from fibrotic human or rat lung release soluble factors capable of inducing cell death and net loss of alveolar epithelial cells (Uhal et al. 1995). These factors have later been identified as angiotensin peptides (Wang et al. 1999). Apoptosis has been shown to be the major pathway responsible for the resolution of type II pneumocytes in acute lung injury (Bardales et al. 1996, Guinee et al. 1996). In acute lung injury, apoptosis has also been observed in interstitial and endothelial cells, but to lesser extent than in epithelial cells (Guinee et al. 1996).
Similarly to acute lung injury, increased apoptosis of alveolar epithelial cells has been observed in advanced fibrotic lung of idiopathic UIP (Kuwano et al. 1996, Uhal et al. 1998, Barbas-Filho et al. 2000). Apoptosis of epithelial cells occurs both in areas of BM injury in proximity to underlying myofibroblasts (Uhal et al. 1998) and in normal alveoli (Barbas-Filho et al. 2000). p21 and p53 have been observed to be upregulated in bronchial and alveolar epithelial cells in patients with idiopathic UIP (Kuwano et al. 1996). There is also evidence that apoptosis of epithelial cells is at least partly mediated by Fas-Fas ligand pathway. By immunohistochemistry Fas has been detected in bronchiolar and alveolar epithelial cells and FasL in lymphocytes and granulocytes (Kazufumi et al. 1997, Kuwano et al. 1999). Kuwano et al. (2000) also observed that the levels of soluble FasL in BAL were increased in patients with idiopathic UIP, while there was an elevation of soluble Fas in patients with BOOP, suggesting a pathogenetic role for epithelial cell apoptosis and activated T-cells in both diseases. Recently it was reported that the immunoreactivity for the Fas-associated death domain protein as well as for caspase-1 and caspase-3 were significantly increased in alveolar epithelial cells of idiopathic UIP compared with normal controls (Maeyama et al. 2001).