This article appears in the following Rheumatology issue: Update in systemic sclerosis [View the issue table of contents]
Histopathology and bronchoalveolar lavage
1Division of Rheumatology & Immunology, Department of Medicine, Medical University of South Carolina, Charleston, SC, USA and 2Interstitial Lung Disease Unit, Royal Brompton Hospital, London, UK.
Correspondence to: R. M. Silver, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 912, Charleston SC 29425, USA. E-mail: silverr{at}musc.edu
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Although neither lung biopsy nor bronchoalveolar lavage (BAL) is recommended for routine clinical use in patients with SSc, studies employing lung biopsy material and BAL fluid (BALF) have provided insight into the pathogenesis of scleroderma-associated interstitial lung disease (SSc-ILD). Most often, SSc-ILD is classified as a non-specific interstitial pneumonia, with abundant myofibroblasts and evidence of both epithelial cell and endothelial cell injury. Recently, SSc-ILD fibroblasts have been shown to express reduced levels of the caveolin-1 protein which, in turn, may lead to activation of the signalling molecules associated with increased collagen production and overexpression of
-smooth muscle cell actin (-SMA). BALF often contains increased numbers of inflammatory cells as well as myofibroblasts expressing -SMA. Analysis of BALF suggests an imbalance between pro-fibrotic and anti-fibrotic factors, e.g. an overabundance of TGF-β, connective tissue growth factor (CTGF), PDGF, leucotriene B4, etc. and in some cases a deficiency of hepatocyte growth factor, 15-hydroxyeicosatetraenoic acid (15-HETE), lipoxin A, etc. Until the pathogenesis is fully understood, lung biopsy and BAL will remain useful research tools to better understand the inflammatory and fibrosing processes that underlie SSc-ILD.
KEY WORDS: Scleroderma, Interstitial lung disease, Lung biopsy, Lung histopathology, Myofibroblast, Bronchoalveolar lavage, Cytokines, Growth factors
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Introduction |
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Interstitial lung disease (ILD) is a frequent complication of scleroderma (SSc), occurring in up to 90% of patients evaluated by sensitive diagnostic techniques, e.g. CT. Both the degree and the course of lung fibrosis may be variable, but as many as 30% of SSc-related deaths are attributable to pulmonary fibrosis. Severe and rapidly progressive SSc-associated ILD (SSc-ILD) is more apt to occur in patients who have diffuse cutaneous SSc; other factors conferring an increased risk of severe restrictive lung disease are gender (males > females), race (African-Americans > Caucasians) and auto-antibody profile (Scl-70 antibody > other SSc-associated autoantibodies). As a major cause of morbidity and mortality, SSc-ILD must be better understood if we are to make the sort of progress that has occurred in treating scleroderma renal crisis, and that it appears we may be making in treating scleroderma-associated pulmonary arterial hypertension (SSc-PAH). Two techniques—lung biopsy and bronchoalveolar lavage (BAL)—have provided important insights into the pathogenesis of SSc-ILD, and the major findings of each are discussed subsequently.
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Lung biopsy studies have demonstrated that the majority of SSc-ILD patients fall within the classification of non-specific interstitial pneumonia (NSIP), or less frequently usual interstitial pneumonia (UIP) and other types of ILD. In the largest reported series of SSc lung biopsies [1], 78% of cases were classifiable as an NSIP pattern, with a fibrotic subset being present more often than a cellular subset. Outcome, however, appears to be related to baseline disease severity and to physiological measures, e.g. diffusion capacity for carbon monoxide (DLCO), but not to histopathological classification; therefore, surgical lung biopsy no longer has a routine clinical role when high resolution computed tomography (HRCT) appearances are typical [1].
A study utilizing lung tissue from a small number of SSc-ILD patients and normal subjects suggested that SSc-ILD may be divided into stages according to the extent of histopathological changes, and that such stages are variable within the lungs of a given patient [2]. An early stage is characterized by the presence of abundant numbers of myofibroblasts expressing -smooth muscle cell actin (-SMA), as well as numerous capillaries with endothelial cells expressing von Willebrand factor (vWF) and platelet/endothelial cell adhesion molecule (PECAM-1)/CD31. As lung fibrosis progresses to a more advanced stage, myofibroblasts and endothelial cells are significantly less apparent.
Microvascular injury is omnipresent in SSc, and the capillary microvascular changes seen in early stages of SSc-ILD may represent an attempted repair process. Indeed, BAL and histological studies demonstrate evidence of microvascular and tissue injury with coagulation pathway activation generating fibrin and thrombin in the lungs of SSc-ILD patients [3]. In addition to its important role in coagulation, thrombin's cellular effects are intimately involved in promoting myofibroblast differentiation, endothelial cell activation, extracellular matrix (ECM) deposition and the induction of profibrotic factors, including TGF-β [4]. Endothelial cell injury generates release of thrombin, which leads resident fibroblasts to differentiate to a myofibroblast phenotype. Additionally, there is evidence for epithelial cell injury, which also may increase the number of myofibroblasts through a process known as epithelial–mesenchymal transition (EMT) [5]. TGF-β and ET-1 have each been shown to induce alveolar EMT [6]. Myofibroblasts may also arise from activation of circulating fibrocytes that home to foci of inflammation under the influence of various chemokines over-expressed in SSc-ILD. Cells with a myofibroblast phenotype, i.e. expressing -SMA with contractility and actively engaging in ECM synthesis, are present in the fibroblastic foci and probably arise as a result of tissue injury to both endothelial cells and epithelial cells.
In vitro studies of lung fibroblasts grown from lung biopsy specimens reveal important differences among cells from normal and SSc-ILD patients, including an overabundance of myofibroblasts in SSc-ILD tissue [7]. Myofibroblasts from SSc-ILD patients synthesize increased quantities of type I collagen. Recently, another important difference between normal and SSc lung fibroblasts has been noted: SSc lung fibroblasts express significantly lower levels of caveolin-1 when compared with normal lung fibroblasts [8]. Caveolin-1 is the principal coat protein of caveolae and serves as a scaffold for signalling molecules. Caveolin depletion, as seen in SSc-ILD [8] and in IPF [9], activates signalling molecules and is associated with over-expression of -SMA, collagen and tenascin-C, i.e. the SSc fibroblast phenotype [8]. The importance of such observations is underscored by experiments that demonstrate that enhancing caveolin-1 levels (using an adenoviral vector) or enhancing caveolin-1 activity (using systemic administration of a caveolin scaffolding domain peptide) reverses the scleroderma fibrotic phenotype in vitro, and attenuates the in vivo fibrotic response in the lungs of mice treated with bleomycin [9, 10].
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Over the past three decades, the technique of BAL has yielded new insights and important information regarding the pathogenesis of SSc-ILD. While the clinical utility of BAL for staging the activity of SSc-ILD or for predicting response to therapy remains controversial and is not recommended for routine clinical use (see subsequently), BAL has proven to be a very useful tool to obtain cells and soluble mediators from the lower respiratory tract for research studies. Early work demonstrated the presence of increased numbers of cells in the lower respiratory tract, especially neutrophils, eosinophils and activated alveolar macrophages [11]. Alveolar macrophages were shown to synthesize increased amounts of growth factors and cytokines, e.g. fibronectin and TNF-
. Studies also demonstrated alterations in cellular immunity: patients at risk for decline in pulmonary function were reported to have an increased number of CD8+ T lymphocytes in BAL fluid (BALF), and these cells exhibit a type 2 phenotype with a pro-fibrotic pattern of gene expression [12]. The overall increase in lower respiratory tract inflammatory cells may lead to overproduction of oxidant species with an impaired oxidant/antioxidant ratio, in turn leading to tissue injury [13].
SSc-ILD patients express numerous factors in BALF that might potentially affect the inflammatory and fibrosing processes underlying SSc-ILD. Cytokines, chemokines, growth factors, coagulation factors and eicosanoids have all been shown to be present more often and in higher quantities in SSc-ILD patients than in normal subjects. In addition to fibronectin, fibrin, thrombin and TNF- mentioned earlier, BAL cells and/or BALFs express higher than normal amounts of PDGF, TGF-β, IL-1, IL-8, macrophage inflammatory protein (MIP)-1, IL-10, MCP-1 and chemokine (C-C motif) ligand 18 (CCL18). Pro-inflammatory and pro-fibrotic eicosanoids, e.g. leucotriene B(4) [LTB(4)], are also elevated in BALF and do not appear to be balanced by equal amounts of anti-inflammatory/anti-fibrotic eicosanoids, e.g. 15-hydroxyeicosatetraenoic acid (15-HETE) or lipoxin A (LXA) [14].
Recent work has demonstrated that in some SSc-ILD patients there exists a relative deficiency of an anti-fibrotic factor, hepatocyte growth factor (HGF) [15]. Especially in African-American patients, known to have an increased risk for severe disease, there appears to be a reduced amount of HGF. HGF inhibits fibrosis by blocking the effects of connective tissue growth factor (CTGF) shown to be overexpressed by SSc fibroblasts. In addition to the relative deficiency in HGF noted in African-American SSc-ILD patients, such patients also appear to have a defect in activation of an anti-fibrotic signalling pathway mediated by HGF [15]. These findings may explain, in part, the greater disease severity and worse prognosis of SSc-ILD observed in such patients.
Another important contribution of BAL has been the ability to culture and sub-passage cells for in vitro studies. Cells grown from BALF express the myofibroblast phenotype, e.g. expression of -SMA and synthesis of increased amounts of collagen, proteoglycans and other ECM proteins [7, 16]. Outgrowth of BAL myofibroblasts is more apt to occur in patients with active ILD, e.g. those patients with increased numbers of neutrophils and eosinophils in their BAL fluid. Cultured myofibroblasts, which may arise from a variety of sources (see earlier), are likely to be key players in the pathogenesis of SSc-ILD; therefore, BAL has proven to be an important tool for studies of the cellular events important in the process of lung fibrosis.
Whether or not BAL has the same clinical value as its research value is controversial. Early studies suggested that certain characteristics of SSc BALF, e.g. neutrophils or eosinophils, were predictive of outcome [17]. Other studies suggested that a normal BAL cell differential was associated with stable pulmonary function, whereas untreated patients with BAL neutrophilia were more likely to show a decline in pulmonary function [18]. Recent larger studies, however, suggest that BALF cellularity is neither predictive of response to cyclophosphamide [19], nor is the cellularity likely to change following cyclophosphamide therapy [20]. This differs from earlier reports suggesting an improvement in BAL cellularity following cyclophosphamide therapy [21]. For now, it may be said that BAL remains a valuable research tool for studying the pathogenesis of SSc-ILD; its clinical utility remains in doubt, and BAL is not recommended for routine evaluation and management of patients suspected of having SSc-ILD.
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The authors wish to acknowledge support from NIAMS and NHLBI of the NIH (R.M.S.) and from the UK Raynaud's and Scleroderma Association (A.U.W.).
Supplement: This paper forms part of the supplement entitled ‘Update in systemic sclerosis’. This supplement was supported by an unrestricted grant from Encysive.
Disclosure statement: The authors have declared no conflicts of interest.
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