Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and various internal organs. Interstitial lung disease (ILD) and its complications represent the most prominent causes of death in SSc. Alveolitis develops in up to 80% of SSc patients, and progression to end-stage fibrosis occurs in about 15% [1]. Unfortunately, factors that predict progression and poor prognosis are missing. Cellular differentiation of bronchoalveolar lavage (BAL) cells is often used to define alveolitis. In addition, neutrophilic alveolitis has been suggested to predict the progression of fibrosing alveolitis [2]. In a recent multicentric study including 141 patients, BAL neutrophilia was associated with early and overall mortality, but the effect on overall mortality was lost when disease severity was taken into account [3]. The authors concluded that BAL findings add only limited prognostic information in SSc-related interstitial lung disease in addition to HR-CT scans and lung-function parameters (LFP) [3, 4]. Nevertheless, the authors argued that other markers might reflect disease progress and the pathogenic mechanisms present in SSc-ILD.

The role of chemokines and cytokines as markers reflecting disease severity and predicting outcome in SSc-related lung disease has not been studied extensively. Chemokines are important regulators of cell migration and the recruitment of leukocytes to specific tissue sites [5]. Among them, monocyte chemoattractant protein-1 (MCP-1 or CCL2) and macrophage inflammatory protein-1β (MIP1β or CCL4) may play a role in SSc, as the overexpression of these chemokines has been detected in SSc-related lung disease [6, 7]. In addition to chemokines, cytokines such as IL-6 or TGF-β also can mediate different pathogenic processes in systemic sclerosis. Polymorphisms of several cytokines found to be associated with SSc and involved in the regulation of fibrosis support their role in SSc pathogenesis [8, 9]. Therefore, both chemokines and cytokines could play a role in the pathogenesis of SSc-ILD and as targets of future therapies [10].

In the present investigation, we have determined levels of cytokines and chemokines in BAL fluids (BALF) in an early SSc cohort. Furthermore, we analyzed controls with ILD due to other diseases to identify key cytokines specifically involved in the pathogenesis of SSc-related lung disease. Furthermore, in a cross-sectional study, the correlation of cytokine and chemokine levels with signs of lung fibrosis was studied. Finally, by follow-up investigations of the clinical data, lung function, and HR-CT scores, the predictive value of cytokines and chemokines was evaluated. We have identified key cytokines that appear to be associated with lung fibrosis and that may predict worsening of ILD in SSc patients.

In the present work, we studied cytokine and chemokine levels in BALF to identify key players in the disease process and potential therapeutic targets of SSc-related lung disease. Systemic sclerosis is a rare disease, and most studies analyzing BALF cytokines have included only few patients with SSc. Our analysis is one of the largest studies addressing soluble mediators in BALF associated with SScs. Furthermore, in contrast to other investigations that have used ultrafiltration or other methods to concentrate BALF cytokines and chemokines for ELISA testing [18–21], we used a highly sensitive Bioplex assay, allowing the detection of cytokines without any BALF manipulation that could influence the stability of cytokines. By using this technique, we observed abnormalities in a broad range of cytokines and chemokines, probably reflecting the complexity of the underlying disease processes present in SSc. Cytokines/chemokines produced by lymphocytes (for example, IL-4, IL-2) and monocytes/macrophages (CCL2, CCL4, TNF-α, IL-8, IL-6), as well as other cell types, were shown to be increased, indicating activation of different cell types in SSc. In controls with ILD due to other diseases, fewer abnormalities were observed; however; some cytokines/chemokines, such as CCL2 and CCL3, were increased in idiopathic ILD as well as in SSc-associated ILD, indicating an important, but nonspecific contribution of these chemokines in lung fibrosis. As tested here for SSc patients, CCL2 concentrations were found to be correlated with lung-function parameters and HR-CT score. The importance of CCL2 as a key mediator for lung fibrosis also is supported by data from animal models showing a reduction and prevention of bleomycin-induced lung fibrosis by anti-CCL2 monoclonal antibodies or by pharmacologic blockade, respectively [22, 23]. CCL2 also mediates profibrotic effects in SSc through the release of IL-4 from T cells, and IL-4 also was found to correlate with lung-function parameters in our study [24]. Taken together, our data support the role of CCL2/3 as targets for future therapies.

Additional cytokines and chemokines, such as IL-8, also could be important, as IL-8 gene polymorphisms are associated with an increased risk of SSc [25]. IL-8 also is expressed by scleroderma fibroblasts and by alveolar macrophages [26–28], and increased IL-8 levels in BALF and serum of SSc patients have been described by others [18, 29]. By reducing multivariate logistic regression analysis on BALF cytokines, high IL-8 levels also were predictive of a poor prognosis. IL-8 is a potent chemoattractant for neutrophils, and the correlation of IL-8 levels with LFTs suggests a possible role of this cytokine in ILD pathogenesis, as suggested by others [29]. Therefore, IL-8 could also serve as a potential therapeutic target.

In comparison with alveolitis due to other diseases, SSc-related alveolitis was characterized by higher levels of IL-7, suggesting disease-specific pathogenic processes. IL-7 was originally described as a potent proliferative stimulus of pro-B and pre-B cells from bone marrow [30] and as a promoter of the growth and expansion of mature effector T cells [31]. It is expressed by stromal medullar cells, epithelial cells, and macrophages [32] and exhibits both fibrotic and antifibrotic effects, probably underlined here by the missing correlations between IL-7 levels and LFTs or HR-CT scores detectable for other cytokines. IL-7 transgenic mice showed increased levels of the profibrotic cytokines IL-4 and IL-13 [33]. Here, higher IL-4 levels also were detected in SSc-associated alveolitis. The antifibrotic effect of IL-7 is reflected by an improvement of bleomycin-induced pulmonary fibrosis through IL-7 [34]. This effect could explain the better prognosis of SSc-associated alveolitis compared with that of idiopathic ILD [35].

However, as suggested by our study, the activation of T cells by IL-7 could be important for SSc-ILD. In line with this, and in addition to the clinical associations of IL-4, IL-2 concentrations correlate with predicted DLCO levels. Furthermore, high IL-2 levels together with high TNF-α levels were the best predictors for progressive/end-stage ILD. Increased levels of the IL-2 receptor are proposed as a marker of disease activity in SSc [36] and SSc-associated ILD [37]. In line with this, blockade of the IL-2 receptor activation ameliorated bleomycin lung fibrosis [38].

In another study, anti-CD3 therapy also diminished bleomycin-induced fibrosis [39]. Therefore, T-cell therapy could provide a useful target for further therapies.

Because most of the fibroblast characteristics obtained from SSc patients are reproduced in normal fibroblasts after stimulation with TGF-β1, TGF-β1 was stated as a key cytokine in SSc-associated fibrosis (reviewed in [40]). Here, BALF TGF-β1 levels were below the detection level. In contrast, serum TGF-β1 levels were detectable by using the same assay but did not provide any correlations with LFTs or HR-CT scores and revealed no predictive capacity (Figure 2). The low levels of potential profibrotic cytokines found in our study do not exclude autocrine or paracrine effects, as suggested by others to play a role in SSc [41]. However; several cytokines analyzed here and showing increased concentrations exhibit inhibitory effects on TGF-β1 expression (for example, IL-7 and TNF-α), indicating a contribution of TGF-β1-independent mechanisms in SSc-ILD proposed by others, such as autoantibodies, Th2 cytokines, growth factors, and several other cytokines/chemokines [42, 43]. As detected with our first analysis including 17 different cytokines, profibrotic cytokines such as IL-13 or IL-17 also revealed very low or undetectable BALF concentrations and no differences from controls or a relation to ILD. Levels of IL-17 correlated with BAL lymphocytosis but not with clinical parameters in SSc (data not shown). Because BAL lymphocytosis is associated with stable lung function over time [44], IL-17 does not seem to play a major role in SSc-ILD. Despite the possible important role of lymphocyte activation and cytokine release in SSc-ILD, accumulation of lymphocytes in BAL was not predictive of disease progression.

Instead, and supporting studies from several groups, we found neutrophilic alveolitis as one of the strongest predictors for progressive disease. The role of neutrophilic alveolitis and BAL analysis as predictors of progressive disease was recently discussed [45]. Several observational studies, including a total of 190 SSc patients, indicated that the presence of BAL alveolitis, and especially of neutrophilic alveolitis, was associated with deterioration of lung-function tests in patients that did not receive immunosuppressive treatments (summarized in [45]).

In contrast, a recent analysis of 66 placebo-treated patients from the Scleroderma Lung Study did not show any relation between the presence of baseline BAL granulocytosis and changes in lung function [4]. As recently outlined, a previous study was not sufficiently powered to allow subgroup stratification [45]. Furthermore, undetected infections, technical issues such as the instilled volume of saline, the site from which BAL was performed, different cut-offs used to define alveolitis, or comorbidity such as reflux can influence BAL cellularity (summarized in [45]). This could lead to different results, as reported in other studies [3, 4]. Here, in this single-center study, we used a standardized procedure, and we have adjusted the cytokine concentrations for BAL recovery. By this procedure, TNF-α, a cytokine with the capacity to increase the migration of neutrophils, was found to be one of the best predictors for poor prognosis. This cytokine was found to correlate with the absolute number and frequency of BAL neutrophils (Table 3). In line with this, our study supports the predictive value of neutrophilic alveolitis, which could be different from granulocytic alveolitis, because eosinophils did not reveal any predictive capacity in SSc. However, our study is not powered to provide conclusive information about the value of BAL to predict disease progress. Nevertheless, cytokines found to be predictive, even in our small patient sample, have promise of a high prognostic potential. Their role in a multivariate setting in addition, to known prognostic factors, must be assured with higher case numbers. Further studies are needed to address this question.

Major limitations of the study are the fact that the majority of patients received immunosuppressive therapies at the time of BAL. Therefore, it cannot be excluded that immunosuppressants could influence BALF cytokines and, subsequently, cytologic and clinical correlations. However, in the few patients investigated twice or thrice, no significant BAL changes were observed despite immunosuppressive therapies. Another limitation of the study is the low number of patients and the heterogeneity of the control group.

In conclusion, we identified several abnormalities in the cytokine and chemokine patterns in BALF of SSc patients, suggesting an important role of these mediators in the pathogenesis of ILD. According to our results, CCL2, IL-7, and probably IL-8 and IL-4 appear to be the most-promising candidates for a targeted therapy in SSc-associated ILD. Furthermore, T-cell targeted therapy could be a promising therapeutic intervention. The data also suggest the usefulness of BALF analyses as an early predictor of progression of SSc-related ILD.

High BALF cytokine and chemokine levels are associated with severe ILD in SSc and are associated with deterioration of ILD. Cytokines and chemokines could have a role in the disease pathogenesis of ILD. Analyses of BALF chemokine and cytokine levels can probably provide therapeutic targets in SSc-associated ILD.