PERSPECTIVE
A Cytokine Reborn?
Endothelin-1 in Pulmonary Inflammation and Fibrosis

Priit Teder and Paul W. Noble

Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut

Since the first identification of a vasoconstrictor activity present in endothelial cell supernatants (1) and the subsequent isolation of endothelin (ET)-1 in 1988 (2), there has been tremendous interest in the biology of endothelins. In addition to ET-1, further studies have demonstrated the existence of two other endothelins, ET-2 and ET-3, which differ from ET-1 with two and six amino acid residues, respectively (3). The family of endothelins has been an object of intensive research for scientists from many disciplines. The majority of research has focused on the cardiovascular system (for review see References 4 and 5), but there has also been a significant accumulation of data indicating the mediator role of endothelins in a variety of lung disorders (for further review see References 6 and 7). However, despite intense investigation and the identification of ET-1 expression under a number of pathologic conditions related to the lungs, such as pulmonary vascular disease, asthma, and pulmonary fibrosis, a direct link between ET-1 activity and a disease state has been elusive. This Perspective will focus on aspects of endothelin biology that pertain to the pulmonary system.

Endothelins are synthesized from precursors known as preproendothelins (ppET), comprising 212 amino acid residues. The large precursors undergo an intermediate cleavage by endopeptidases to form the 38-amino-acid biologically inactive proendothelins, also called big endothelins. Endothelin-converting enzymes are membrane-bound metalloendopeptidases that further cleave proendothelins. The biologically active endothelins are 21-amino-acid peptides, with two disulfide bridges joining cysteins in positions 1-15 and 3-11 in the N-terminal half, and a cluster of hydrophobic amino acid residues at the C-terminal end of the structure. The structure of the N-terminal domain determines the affinity to the receptor, while the C-terminal domain contains the binding site of the peptide to the receptor (for review see Reference 8).

Most tissues contain more ET-1 than ET-2 or ET-3, with the highest levels of ET-1 found in lung. ET-1 is secreted by endothelial cells (2, 9), epithelial cells (10, 11), alveolar macrophages (12, 13), polymorphonuclear leukocytes (14), and fibroblasts (15). Release of endothelins is regulated at the level of gene expression and peptide synthesis because cells do not store endothelins. The expression of the gene is induced by a variety of factors including thrombin, angiotensin II, adrenalin, cytokines, and growth factors. The calcium-dependent protein kinase C (PKC) is involved in this stimulation, and the expression of ET-1 is reduced in the presence of PKC inhibitors. The most potent physiologic factor in regulating ET-1 production and release from endothelial cells seems to be blood flow. An increase in blood flow elicits vasodilatation via activation of the shear stress receptors of endothelial cells that, in turn, produce and release nitric oxide and decrease the production and release of ET-1. The multitude of stimuli that influence the release of ET-1 have made the precise biologic significance of ET-1 elusive. Approximately 80% of the synthesized amount of ET-1 is secreted into the basolateral compartment toward the surrounding smooth-muscle cells and interstitium where it acts in an autocrine and paracrine manner. The concentration detected in vascular tissue is approximately 100 times higher than that in plasma, with the vast majority receptor bound and only minute amounts remaining in a free form. ET-1 has a half-life of approximately 4 to 7 min in the blood because of quick binding to tissues and rapid metabolization by a specific endothelin-degrading enzyme (for further references see Reference 8).

Two endothelin-specific receptors have been cloned in humans: some selective for isoforms ET-1 and ET-2 (ET_A) (16), the other nonisoform selective (ET_B) (17). Homology between these receptors is 60%, and both have two subtypes. The affinity of the three isopeptides for the ET_A receptor can be described as ET-1 > ET-2 >> ET-3. In humans, the ET-1 affinity for this receptor is 1,000-fold higher compared to ET-3 (18). ET_B receptors have similar affinity to all isoforms, as the receptor recognizes the identical C-terminal end. Both receptors are associated with vasoconstriction when localized on smooth-muscle cells, whereas ET_B mediates vasodilatation when commonly localized on endothelium. The third receptor (ET_C) is isolated in Xenopus laevis and has the highest affinity to ET-3 (19).

Pulmonary fibrosis is a common outcome of interstitial lung diseases, which form an etiologically heterogeneous group of diffuse inflammatory disorders affecting the pulmonary parenchyma (for review see Reference 20). Despite a myriad of potential mechanisms of lung injury, a common pathway is injury to the structures of the alveolar wall. In response to lung injury, there is an influx of inflammatory cells, release of mediators, recruitment of mesenchymal cells, and turnover of the extracellular matrix (ECM). The inflammatory phase is initiated by epithelial and/or endothelial injury, followed by an invasion of inflammatory cells to the alveolar interstitium. Released mediators facilitate recruitment of neutrophils and blood monocytes to the site of injury. Neutrophil infiltration declines after the first few days of tissue injury, and the number of alveolar macrophages, as well as lymphocytes, increases over time. During this period the spectrum of mediators in the alveolar interstitium changes markedly. Monocyte invasion, adherence, and in situ proliferation during the inflammatory phase result in the activation and release of excessive amounts of bioactive substances, such as reactive oxygen species, prostaglandins, leukotrienes, chemotactic factors, proteases, and growth factors. Among the factors secreted that have been shown to play an important role in mediating fibrosis are transforming growth factor-beta (TGF-) and tumor necrosis factor-alpha (TNF-) (21, 22). Since ECM is essential for proper growth and repair of injured tissue, reorganization of ECM structure may interfere with this repair process. During the inflammatory phase of lung injury the glycosaminoglycan hyaluronan forms an initial matrix but is quickly replaced by a collagen type I and III, as well as a proteoglycan matrix. Eventually, a fibrotic repair phase with lost normal architecture of the alveolocapillary unit and interstitial thickening occurs, leading to reduced lung volume and impaired gas exchange.

ET-1 was initially characterized as a potent smooth-muscle spasmogen (for review see Reference 23), but accumulating evidence is pointing out the ET-1 role as a proinflammatory cytokine. ET-1 is known to prime neutrophils (24), stimulate neutrophils to release elastase (25), activate mast cells (26), stimulate monocytes to produce variety of cytokines such as interleukin (IL)-1, IL-6, and IL-8, TNF-, TGF-, and granulocyte-macrophage colony-stimulating factor (for additional references, see Reference 27). ET-1 has been shown to induce fibronectin gene expression and fibronectin release from human bronchial epithelial cells (28). Fibronectin is an important ECM component, as well as being a chemotactic factor for fibroblasts. Furthermore, there is evidence that ET-1 can function as a profibrotic cytokine by stimulating fibroblast chemotaxis and proliferation (13, 29, 30) and procollagen production (31, 32). ET-1 has been shown to have mitogenic activity for smooth-muscle cells, myocytes, and fibroblasts. In addition, ET-1 may act in synergy with platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, TGF-, insulin, and others to potentiate cellular transformation or replication. ET-1 and ET-2 are equipotent in promoting DNA synthesis, whereas ET-3 is less active (for further references see Reference 33).

Localization of ET-1 under physiologic and pathologic conditions has been well studied. In normal subjects, occasional ET-1 staining occurs in pulmonary endothelium and neuroendocrine cells, as well as in airway and vascular smooth-muscle cells. Epithelial cells do not normally express ET-1. In contrast, patients with idiopathic pulmonary fibrosis (IPF) have increased ppET-1 messenger RNA expression and intense immunoreactivity in airway epithelial cells, proliferating type II pneumocytes, and endothelial and inflammatory cells. Endothelin-converting enzyme-1 immunostaining colocalizes to proendothelin-1 and ET-1 immunostaining and is correlated to disease activity (34, 35). ET-1 can be detected in the circulation of normal subjects (36) and is elevated in the plasma of patients with pulmonary hypertension, scleroderma, and IPF (for supplementary references see Reference 37). In patients with pulmonary fibrosis associated with systemic sclerosis, bronchoalveolar lavage fluid contains 5-fold greater ET-1 levels than those of controls (30) and is elevated in patients with asthma (38).

Instillation of intratracheal bleomycin is a frequently used animal model of lung injury and fibrosis. The model is characterized by an initial alveolitis consisting mainly of neutrophils with pathologic changes of diffuse alveolar damage with peak injury around day 7. The inflammatory phase remits, and collagen production gradually increases to maximal levels at days 21 to 30. Increases in the number and intensity of ET-1-stained macrophages and epithelial cells compared with those found in normal rat lung have been observed after bleomycin treatment (39, 40). This finding is consistent with studies on lung tissue from patients with pulmonary fibrosis (34, 37). In a time course study, a rapid 3-fold increase in lung ET-1 content was found peaking at day 7. An ET-1 increase precedes the onset of fibrosis, colocalizing with developing fibrotic lesions (39). Antagonists of endothelin receptor function have been used to prevent the development of pulmonary fibrosis with conflicting results. ET receptor antagonists have been shown to inhibit the effects of ET-1 on lung fibroblast proliferation (30) and collagen synthesis in vitro (32). In addition, other studies have shown that ET receptor antagonists are effective in attenuating ECM production in vivo (40, 41). However, Mutsaers and colleagues provide evidence that continuous administration of nonselective ET_A and ET_B, as well as selective ET_A receptor antagonists, did not prevent collagen accumulation in the bleomycin model (42). In summary, circumstantial evidence for a role of ET-1 in lung fibrosis has accumulated, but whether this is a causative or bystander role remains unknown.

In the current issue of the American Journal of Respiratory Cell and Molecular Biology, Hocher and colleagues extend their previous published work on ET-1 transgenic mice, where they had demonstrated an interesting kidney phenotype (43). The human ET-1 gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the kidney, lung, and brain. Immunohistochemistry revealed that ET-1 expression was detectable within all parts of the lung, including blood vessels, alveoli, the bronchial wall, and interstitia. Strongest expression was in the bronchial wall. The surprising result from this study was that no significant abnormalities were observed in the pulmonary vasculature. Specifically, there was no evidence of pulmonary hypertension. Furthermore, no difference was seen in endothelin receptor density and binding activity, vascular morphometric parameters, as well as in the right ventricular pressure between ET-1 transgenic mice and control littermates. Instead, ET-1 transgenic mice developed progressive accumulation of ECM proteins in the perivascular and peribronchial space, as well as an extensive accumulation of mononuclear cells. Analysis of hydroxyproline content demonstrated an increase in collagen accumulation. Further elucidation of other matrix components such as hyaluronan and proteoglycans requires further study. A second interesting observation was the accumulation of inflammatory cells. Analysis of the mononuclear cell infiltration revealed a preponderance of CD4-positive cells. This raises the intriguing possibility that ET-1 might recruit a subset of T cells that promotes fibrosis. The role of T-cell subsets in the pathophysiology of fibrosing lung diseases is an area of intense investigation. Further studies to elucidate the cytokine profile (i.e., T helper (Th)2 versus Th1-type cytokines) are indicated and may uncover a previously unanticipated role for ET-1 in mediating lung disorders. This present study, however, exemplifies the power of the transgenic approach to identifying biologic roles for soluble mediators. A number of interesting in vitro observations suggested diverse roles for ET-1, but this important contribution by Hocher and colleagues will undoubtedly refocus studies on further understanding the role of endothelins in inflammatory and fibrosing lung disorders.

View larger version (35K): [in this window] [in a new window]   Figure 1.   Schematic representation of regulators of endothelin-1 (ET-1) production, cellular source, and biologic effects. LDL, low-density lipoprotein; ANP, atrial natriuretic peptide; AA, amino acids; ECE, endothelin-converting enzyme; PAF, platelet activating factor.

	Footnotes

Address correspondence to: Paul W. Noble, M.D., Yale University School of Medicine, Department of Internal Medicine, Pulmonary and Critical Care Section, 333 Cedar St., LCI 105, New Haven, CT 06520-8057. E-mail: paul.noble{at}yale.edu

(Received in original form May 19, 2000).

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