Introduction
The analysis of the bronchoalveolar lavage fluid (BALF) proteome can potentially provide important information about changes in protein expression and secretion during the course of pulmonary disorders. At the moment, more than 100 human proteins or protein isoforms have been identified in human BALF by using two-dimensional gel electrophoresis (2-DE), including both proteins released locally in the lung by inflammatory or epithelial cells and proteins derived from serum by diffusion across the capillary–alveolar barrier [
1,
2]. Previous studies showed that the protein composition of BALF was altered in sarcoidosis, idiopathic pulmonary fibrosis, allergic asthma, and chronic obstructive pulmonary disease [
3‐
5]. Furthermore, Rottoli and colleagues [
6] recently demonstrated that systemic sclerosis patients with pulmonary fibrosis (SSc
Fib+) showed a BALF profile that was intermediate between that of patients with sarcoidosis and idiopatic pulmonary fibrosis, despite some particularities.
Systemic sclerosis (SSc) is an autoimmune disorder of unknown origin, characterized by an excessive deposition of collagen and other extracellular matrix proteins on the skin as well as multiple internal organs, alterations in microvasculature, and humoral and cellular abnormalities [
7]. Clinically, the disease is very heterogeneous, ranging from limited skin involvement with limited systemic alterations to clinical pictures with diffuse skin sclerosis and severe visceral involvement with fibrosis, microvascular abnormalities and mononuclear cell infiltration of the gastrointestinal tract, lungs, heart, and kidneys. Pulmonary involvement has emerged as potentially the most serious visceral lesion in SSc. Alveolitis is more commonly seen in SSc with diffuse skin sclerosis, particularly in the presence of serum anti-topoisomerase I (anti-Scl70) antibodies, but only 15% of patients eventually reach severe end-stage lung fibrosis [
8].
Given the complexity of the pathogenesis of lung fibrosis and the lack of biological markers that can reliably predict which patients will evolve the disease, great interest has been shown in these issues, also comparing cohorts of SSc patients with or without lung involvement. The ability to identify the proteins that are differently expressed in the lower airways of SScFib+ patients from those in SScFib- patients might provide new insight into the disease pathogenesis and into the identification of possible protein biomarkers of the disease's progression. With this aim, we used the 2-DE analysis to assess and compare the protein profile of BALFs from a group of SScFib+ and SScFib- patients, respectively.
Discussion
The pathogenesis of SSc is extremely complex, and no single unifying hypothesis or pathogenetic mechanism can explain all the aspects of this disease. It is therefore widely accepted that functional alterations of several cell effectors, including fibroblasts, endothelial cells, and T and B lymphocytes, are intimately involved in the development of the clinical and pathological manifestations of SSc [
7,
18,
19]. Abnormalities in lung function occur in about 70% of SSc patients and currently represent the main cause of mortality in this population [
7]. However, the factors that drive the severe and progressive visceral fibrosis remain mostly undetermined. Unlike the recent pathogenetic hypothesis for idiopathic interstitial lung fibrosis, which assigns only a marginal role to inflammation, fibrogenesis in SSc-related lung involvement is generally thought to be the result of a complex series of interstitial immunoinflammatory reactions [
20]. The persistent and unregulated activation of genes that encode various collagen and other extracellular matrix proteins is regarded as a crucial pathogenetic event [
21,
22]. Furthermore, an alteration in the production of pro-inflammatory and/or pro-fibrotic cytokines and chemokines, growth factors (in particular transforming growth factor-β), and signaling molecules also seems to have a relevant role in fibrogenesis [
23,
24]. The identification of proteins that are differently expressed in the airways of SSc
Fib+ patients compared with patients with no signs or symptoms of lung involvement may cast light on the pathogenetic mechanism of the disease and may thereby indicate protein biomarkers that are useful for the diagnosis of lung involvement, for the monitoring of SSc progression, and for the identification of possible new therapeutic targets. In this context, the use of 2-DE and the mapping of BALF seem highly interesting.
Our results demonstrated that quantitative and qualitative differences can be found between patients with SSc-associated lung fibrosis and patients with SSc with no signs or symptoms of lung involvement. At least 24 well-defined spots showed significant and reproducible variations in relative abundance between the two patient groups. Some of these spots corresponded to previously described BALF proteins and protein fragments, namely α1-acid glycoprotein, GSTP, Cu–Zn SOD, haptoglobin-α chain, and calgranulin B, whereas 4 of the proteins found had not previously been described in published two-dimensional BALF maps, namely cytohesin-2, calumenin, cystatin SN, and human mtDNA TOP1 fragment. In SScFib+ patients, GSTP and cystatin SN were below the detection limit, whereas Cu–Zn SOD was downregulated. In contrast, α1-acid glycoprotein, calumenin, cytohesin-2, haptoglobin-α chain, and calgranulin B were significantly upregulated, and the expression of mtDNA TOP1 was observed only in SScFib+ patients.
On the basis of these preliminary results, we can infer that 2-DE analysis of BALF can provide useful insight into the understanding of mechanisms involved in lung fibrogenesis through the identification of proteins that are deregulated in SSc-associated lung fibrosis. However, our results, as well as those of previous studies, clearly indicated that 2-DE analysis did not provide an exhaustive identification of all proteins that can be present in BALF, especially those of low molecular mass such as cytokines, chemokines, growth factors, and signaling molecules. Because these molecules are thought to be relevant to the pathogenesis of SSc-associated lung fibrosis, a combination of 2-DE analysis and proteomic approaches that specifically address the identification of these factors is necessary for the assessment of the whole protein pattern of BALF. Despite these considerations, most of the proteins we found differently regulated between SScFib+ patients and SScFib- patients could have a role in the development of lung fibrosis, whereby some of them are involved in the mechanisms that protect against lung injury or inflammation whereas others are involved in the mechanisms that drive fibroproliferation.
Oxidative stress is believed to be important in the pathogenesis of lung damage and in the development of lung fibrosis [
25]. Among various enzymatic and non-enzymatic mechanisms that protect cells and tissues from oxidants [
26,
27], glutathione transferases [
28] and SODs [
29] are among such mechanisms that are thought to have a key protective role, especially in the lung. High levels of extracellular SODs are thought to have a protective role for lung matrix [
29]. Recently, Tourkina and colleagues [
30] demonstrated a deficiency in GSTP1 in scleroderma lung fibroblasts. The observation that GSTP and Cu–Zn SOD levels are severely downregulated in BALF from SSc
Fib+ patients is in line with these findings, and confirms the relevance of these molecules as a defense tool for the lung against oxidant-induced damage and/or fibroproliferation.
Moreover, in BALF from SSc
Fib+ patients, we did not detect levels of cystatin SN, which was, in contrast, well represented in SSc
Fib- patients. Cystatin SN is a secreted protein, which belongs to SD-type or type-2 cystatins [
31], which are potent inhibitors of CA clan mammalian cysteine peptidases. Intracellularly, these enzymes participate in normal protein turnover, in antigen and protein processing, and in apoptosis, whereas extracellularly they are involved in the inhibition of the proteolytic cascade [
32]. The direct inhibition of endogenous and exogenous cysteine peptidases is the only function described so far for cystatin SN, suggesting that its function is to protect tissues against injury induced by these enzymes. However, recent results obtained with other type-2 cystatins seem to suggest a broader spectrum of activities, including the upregulation of IL-6 by human gingival fibroblasts, interferon-γ expression in CD4
+ T cells [
33,
34], and the involvement in the differentiation and maturation of human dendritic cells [
35].
Furthermore, we found an upregulation of at least six proteins in BALF from SScFib+ patients: haptoglobin-α chain, α1-acid glycoprotein, calumenin, cytohesin-2, Cal B, and mtDNA TOP1.
Haptoglobin and α1-acid glycoprotein are positive acute-phase proteins, whose plasma concentrations increase during inflammation [
36]. The increased levels of both proteins in BALF from SSc
Fib+ patients is likely to originate from the passive diffusion from plasma, as a result of the increased permeability of the air–blood barrier during the course of fibrosing alveolitis.
Calumenin is a member of the CREC family [
37] that is secreted into the medium of cultured cells including fibroblasts [
38] and early melanosomes [
39]. Intracellularly, calumenin has a chaperone function in several Ca
2+-regulated processes, but extracellularly its function is not yet fully understood. Vorum and colleagues found that calumenin binds to the serum amyloid P component, suggesting that it is involved in the formation of amyloid deposits in different tissues [
40]. Coppinger and colleagues recently described the presence of calumenin in platelets and its release on platelet activation, as well as the protein's presence in human atherosclerotic lesions [
41], therefore suggesting a possible role of this protein in leading to vascular injury through the promotion of platelet adhesion, fibrinogenesis, and intravascular thrombus deposition. Taking into account that a crucial pathogenetic step in SSc involves injury to microvasculature [
19,
20], it is possible to infer that calumenin could have a role in taking such an injury to lung level.
Cytohesin-2 is a member of a family of cytoplasmic signaling proteins [
42], which may move from the cytosol to the plasma membrane after cell stimulation [
43]. Although its biological function is mostly unknown, a recent study demonstrated that cytohesin-2 is an activator of the mitogen-activated protein (MAP) kinase signaling pathway [
44]. Because MAP kinases are activator signals for lung fibroblasts [
23], we suggest that cytohesin-2 might participate in the development and/or progression of lung fibrosis in SSc patients through the activation of the MAP kinase signaling pathways in lung fibroblasts. This hypothesis needs to be verified with further experimental observations.
Calgranulin B is an S100 protein that is unique together with Cal A in its myeloid-specific expression profile and in its abundance in neutrophils [
45]. Increased levels of Cal B are present in the bronchial secretion of patients with chronic inflammatory disease, and these levels may induce the production of IL-8 by airway epithelial cells [
46]. Furthermore, Viemann and colleagues demonstrated that the expression of Cal B and Cal A correlates with the inflammatory activity in systemic vasculitis, thereby confirming the role of these proteins in inflammatory reactions of endothelia [
47]. Although the general consensus is that the Cal B function depends mainly on heterodimer formation (Cal B–Cal A), several studies have demonstrated that the monomer is also a potent stimulator of neutrophils and is involved in the recruitment of leukocytes to the site of inflammation through the regulation of adhesion and the extravasion of neutrophils [
45,
48]. Furthermore, recombinant Cal B and its homodimer stimulate the proliferation of rat fibroblasts, suggesting that this protein might also function as a mitogen for fibroblasts in chronic inflammation [
49]. On the basis of these observations, Cal B could have a role in SSc-associated lung fibrosis through several pathways, including amplifying the inflammatory process by inducing the extravasion of neutrophils and the production of IL-8, inducing an inflammatory reaction in the endothelia, and/or stimulating lung fibroblast proliferation.
Finally, we found that a fragment of mtDNA TOP1 was detectable only in BALF from patients with interstitial lung fibrosis, including in two SSc
Fib+ patients who were negative for serum anti-Scl70 antibodies. This protein fragment was never observed in BALF from SSc
Fib- patients, including in two patients with positive anti-Scl70 antibodies. A common 13-exon motif and a similar amino-acid sequence characterize genes for mitochondrial and nuclear DNA TOP1, except for the N-terminal domain, which in mtDNA TOP1 contains a mitochondrial localization sequence instead of the nuclear localization signal [
50]. Although we demonstrated that the presence of detectable levels of mtDNA TOP1 was a constant and particular feature of SSc
Fib+ patients, we did not fully ascertain the antigenic role of this protein fragment and its relation with the development of lung fibrosis. Because autoantibodies against DNA TOP1 are strongly correlated with interstitial pulmonary involvement in SSc [
51], further studies are warranted to assess the role and the frequency of this antigenic target in BALF.
Authors' contributions
FAM generated and planned the project, supervised the work and wrote the manuscript. BAM and SR contributed equally to perform the research, analyzed critically data and participated in the preparation of the manuscript. PI assisted in designing the experiments and conducted 2-DE and immunoblotting. MM was involved in data analysis and performed statistical analysis. CL and CV selected patients and revised all clinical charts. PE was involved in the coordination of the study and in the discussion and revision of the manuscript. MF participated in the study design, supervised the work, read the bronchoalveolar lavage and helped to draft the manuscript. MC participated in the study design and its coordination and participated in preparation of the manuscript. All authors read and approved the final manuscript.