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Published ahead of print on January 26, 2006, doi:10.1164/rccm.200507-1126OC
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American Journal of Respiratory and Critical Care Medicine Vol 173. pp. 1145-1154, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200507-1126OC

Original Article

Protein Profiles of Bronchoalveolar Lavage Fluid from Patients with Pulmonary Sarcoidosis

Eva Kriegova*, Christian Melle*, Vitezslav Kolek, Beata Hutyrova, Frantisek Mrazek, Annett Bleul, Roland M. du Bois, Ferdinand von Eggeling and Martin Petrek

Faculty of Medicine, Palacky University, Olomouc, Czech Republic; Core Unit Chip Application, Institute of Human Genetics and Anthropology, Friedrich Schiller University, Jena, Germany; and Royal Brompton Hospital, London, United Kingdom

Correspondence and requests for reprints should be addressed to Martin Petrek, M.D., Palacky University, Medical Faculty, I.P. Pavlova Str. 6, CZ-775 20 Olomouc, Czech Republic. E-mail: petrekm{at}fnol.cz


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Pulmonary sarcoidosis is a multisystem granulomatous disease with various clinical phenotypes. So far, there has been little information on protein patterns (PPs) of bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis and no data are available on PPs in clinical disease subtypes.

Objectives: To investigate the PP of BALF from patients with pulmonary sarcoidosis, to evaluate whether PPs reflect disease course as assessed by chest X-ray (CXR), and to compare PPs between patients with/without Löfgren's syndrome.

Methods: Surface-enhanced laser desorption/ionization–time-of-flight mass spectroscopy was applied to investigate PPs in unconcentrated BALF from 65 patients (CXR stage I, n = 32; CXR stage II, n = 22, CXR stage III, n = 11) and 23 healthy control subjects. The Mann-Whitney U test was used to detect differentially expressed protein peaks. After reversed-phase fractionation, peptide fingerprint mapping and immunodepletion were used to identify deregulated (up-regulated or down-regulated) proteins.

Results: Forty differentially expressed protein entities (2.75–185.62 kD) were detected in patients with pulmonary sarcoidosis versus control subjects (p < 0.05). Whereas 13 peaks (33%) were present across all CXR stages, 27 (67%) were specific for particular CXR stages. Comparison of PPs between CXR stage I patients with or without Löfgren's syndrome revealed 25 differentially expressed peaks. The total number of deregulated peaks and also of those associated with sarcoidosis as a whole were markedly lower in patients with Löfgren's syndrome in comparison with other sarcoid phenotypes. Human serum albumin, {alpha}1-antitrypsin, and protocadherin-2 precursor were identified from sarcoidosis-associated PP.

Conclusion: Surface-enhanced laser desorption/ionization–time-of-flight mass spectroscopy enables determination of protein patterns in sarcoid BALF and allows detection of protein patterns linked to a particular disease course.

Key Words: albumin • {alpha}1-antitrypsin • Löfgren's syndrome • protocadherin-2 • surface-enhanced laser desorption/ionization–time-of-flight mass spectroscopy

Sarcoidosis is an inflammatory disorder characterized by the accumulation of CD4+ helper T-cell type 1 lymphocytes and macrophages with subsequent granuloma formation at the site of disease, notably in the lung (1, 2). This multisystem disorder of unknown etiology has a prevalence of 63.1 cases per 100,000 in the Czech Republic (3). Spontaneous remission/stabilization of the disease occurs in approximately two-thirds of cases, but up to 20% of patients develop a chronic functional deficit and even progress to fibrosis (4). More recent studies of immunobiological mechanisms in sarcoidosis have relied mainly on mRNA expression experiments (5, 6). However, the RNA data should be complemented by protein studies (7–10). Protein-profiling analysis in sarcoidosis may provide comprehensive insight on the expression, modification, interactions, and regulation of proteins in this multifactorial disease.

The sarcoidosis bronchoalveolar lavage fluid (BALF) proteome has so far been analyzed exclusively by two-dimensional (2D) gel electrophoresis followed by mass spectrometry (11–16). The proteins identified from BALF were aggregated in a "BALF-2D-map" (15). Several proteins involved in oxidative, antiinflammatory processes and in the regulation of proliferation processes were found in BALF from patients with sarcoidosis (12, 13) in parallel with an increase in plasma proteins (11, 13, 14). Some of the reported proteins (e.g., Clara cell protein-16) (13) corresponded to previously obtained findings (17). By contrast, other proteins, the presence of which was expected from mRNA expression studies, were not detected by the 2D proteomic approach. This applies, for example, to chemotactic cytokines, which have already been found up-regulated in sarcoidosis in both mRNA and ELISA studies (18–24). Other proteins expected, but not yet detected in sarcoid BALF, are cytokines and their receptors, adhesion molecules, and also proteins implicated in the regulation of inflammatory processes (25).

To expand current knowledge of the sarcoidosis BALF proteome, we have, therefore, used a novel technique—surface-enhanced laser desorption/ionization–time-of-flight mass spectroscopy (SELDI-TOF MS) (26)—to determine the protein patterns in unconcentrated BALF from 65 patients with well-defined sarcoidosis in comparison with 23 control subjects. We were also interested to see whether this technique, previously used, for example, in cancer research (27–30), can reflect disease development and progression. Therefore, we have compared BALF protein patterns within chest X-ray (CXR) stages I, II, and III according to Scadding criteria (31). Because of its different clinical symptoms and also specific genetic associations, we also investigated whether the patients presenting with or without Löfgren's syndrome have a typical BALF protein pattern. We used four ProteinChip array surfaces with different adsorption conditions to capture the broad spectrum of BALF proteins. To identify the proteins associated with sarcoidosis, BALF was fractionated by reversed-phase chromatography and the fractions with proteins of interest were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by peptide fingerprint mapping and immunodepletion assay. Some of the data have been presented in abstract form (32).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
BAL was performed during fiberoptic bronchoscopy (33) in patients with pulmonary sarcoidosis (n = 65) and healthy control subjects (n = 23). The diagnosis of pulmonary sarcoidosis was determined in compliance with criteria presented in the joint statement on sarcoidosis (4): typical clinical features together with granulomas on lung biopsies and supported by the BALF cellular profile. Regarding chest radiography, patients were divided into stages (CXR stage I, n = 32; CXR stage II, n = 22; CXR stage III, n = 11). Stage I patients were further subdivided into those presenting with Löfgren's syndrome (LS, n = 14) and without LS (non-LS, n = 18). No patient received corticosteroid treatment before BAL. Detailed characteristics of enrolled patients are shown in Table 1. The control group consisted of subjects who showed no clinical signs of lung inflammation at the time of presentation and subsequently and had no identifiable lung disease in their medical history. All had normal BAL cytology, immunology, and microbiology. In this group, two patients with suspected pulmonary malignancy were included; lavage was performed in an unaffected lobe. The study was approved by the Ethics Committee of the Faculty of Medicine (Palacky University, Olomouc, Czech Republic). All subjects signed informed consent forms to allow use of an aliquot of BALF, taken for routine diagnostic purposes, for the research purposes of this study.


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  		TABLE 1. CLINICAL AND LABORATORY DATA ON SUBJECTS

 
Processing of BALF
BALF samples (20–40 ml) were filtered through gauze and centrifuged (400 x g for 10 min) to separate cells from the fluid. Aliquots (0.5– 1.0 ml) of BALF were stored at –80°C until further analysis. Exclusion criteria were visually nonclear fluid and more than 1% erythrocytes on BALF cell count.

ProteinChip Array Analysis
Unconcentrated BALF samples were analyzed on four array surfaces: Q10, CM10, IMAC Cu(II), and IMAC Ni(II), using a ProteinChip reader (Series 4000; Ciphergen Biosystems, Fremont, CA). See the online supplement for details.

Statistical Analysis
After normalization based on total ion current and calibration of data, peaks with a mass tolerance of 0.3% and a signal-to-noise ratio of 10 were combined as clusters, using the CiphergenExpress 3.0 program (Ciphergen Biosystems). The p values for mean peak intensities at a given molecular mass-to-charge ratio (m/z) were calculated by nonparametric Mann-Whitney U test (two-way comparisons), using the CiphergenExpress 3.0 program. Protein peaks with p values less than 0.05 were considered significantly deregulated (up-regulated or down-regulated) when the study groups were compared. No adjustments have been made for multiple comparisons. For details, see the online supplement.

Protein Separation by Reversed-Phase Chromatography and Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis
Concentrated BALF (Centricon-10; Millipore Corp., Bedford, MA) was fractionated by reversed-phase chromatography (BioSepra RPC Polybeads; Ciphergen Biosystems) as described previously (34). For details, see the online supplement.

In-Gel Digestion and Immunodepletion Assay
Bands corresponding to the approximate molecular mass of proteins from the sarcoidosis-associated protein profile were excised from the stained gel and digested with trypsin as previously described (35). The generated fragment masses were used to identify proteins by searching a publicly available database (http://129.85.19.192/profound_bin/WebProFound.exe), and the identity of the best candidates was confirmed by immunodepletion (36). For details, see the online supplement.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of BALF Protein Expression Pattern in Sarcoidosis
To investigate whether protein expression profiles differ between patients with sarcoidosis and control subjects, unconcentrated BALF samples were analyzed on four array surfaces with distinct binding properties, using SELDI-TOF MS technology. Forty differentially expressed protein entities with molecular m/z ratios between 2.75 and 185.62 kD were found in the BALF of patients with sarcoidosis in comparison with control subjects (Table 2, part A). Of these 40 peaks further referred to as "sarcoidosis associated," 26 (65%) protein peaks were significantly up-regulated in sarcoidosis and 14 (35%) were found to be down-regulated in sarcoidosis in comparison with control subjects. The number of deregulated protein peaks represents about 10% of all protein clusters detected on the surfaces used. The majority of protein entities were detected only on one array surface; two of them (5%) were adsorbed on two different surfaces. The corresponding p values for detected protein peaks are shown in Table E1 of the online supplement (part A). A representative example of obtained normalized ProteinChip array profiles for sarcoidosis samples and corresponding control samples is shown in Figure 1. The distribution of the peak intensities of detected protein entities is illustrated by a representative example in Figure 2.


Figure 1
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  		Figure 1. Representative example of normalized ProteinChip array profiles for sarcoidosis samples and corresponding control samples (chip type, CM10; low range, 10.0–15.0 kD). In this range, two up-regulated protein peaks were detected in sarcoidosis versus control samples (m/z: 11.73 and 11.87 kD).

 

Figure 2
Figure 2
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  		Figure 2. Distribution of the intensities of peaks at 15.35 kD (A) and 59.22 kD (B) on immobilized metal affinity capture ProteinChip array activated with Cu2+ ions: a representative example of down-regulated and up-regulated protein entities in sarcoidosis as a whole (S) versus control subjects (C) and in particular in various chest X-ray (CXR) stages of sarcoidosis (S-I, S-II, and S-III) versus control subjects. The lines represent median values in each group. *p < 0.05; **p < 0.005; ***p < 0.0005; n.s. = not significant.

 

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  		TABLE 2. PROTEIN PEAKS DIFFERENTIALLY EXPRESSED BETWEEN PATIENTS WITH SARCOIDOSIS AND CONTROL SUBJECTS

 
Comparison of BALF Protein Expression Profiles among Distinct Chest X-Ray Stages of Patients with Sarcoidosis versus Control Subjects
To explore whether protein peaks are differentially expressed in particular CXR stages (I, II, and III) of sarcoidosis, our patient group was subdivided according to CXR stage. The obtained m/z values and signal intensities for each CXR stage were compared separately with those of control subjects. The analysis revealed that 13 of 40 sarcoidosis-associated protein peaks (m/z: 4.53, 5.85, 11.91, 14.18, 15.35, 33.35, 44.43, 66.64, 89.77, 100.99, 122.98, 133.98, and 170.64 kD) were significantly deregulated in all studied patients at CXR stages I, II, and III versus control subjects (Table 2, part B). For detailed p values, see Table E1 (part B). A representative example of the distribution of the peak intensities of detected protein entities in particular CXR stages is shown in Figure 2. Figure 3 illustrates the number of protein peaks that are specific to the particular CXR stages and provides an indication of the proportion of specific entities within each stage compared with the global sarcoidosis group.


Figure 3
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  		Figure 3. Protein entities found to be present in particular patient subgroups. Gray areas indicate the proportion of protein entities within a given CXR stage compared with the sarcoidosis-associated protein pattern.

 
In addition to the protein masses found across all CXR stages, several other protein peaks characteristic of distinct CXR stages were detected. Thirty-six significantly deregulated protein peaks were obtained in CXR stage I in comparison with normal subjects. Of these, a majority of the peaks (32, 80%) were associated with sarcoidosis as a whole (Table 2, part B). Three additional protein peaks (m/z: 11.70, 39.29, and 59.62 kD) were up-regulated and one was down-regulated (m/z: 97.34 kD) in this CXR stage in comparison with control subjects. For corresponding p values, see Table E1 (part B).

Forty-one protein peaks were characteristic of CXR stage II in comparison with control subjects (Table E1, part B). Twenty-nine (71%) of these were associated with sarcoidosis as a whole (Table 2, part B); 11 were up-regulated (m/z: 4.83, 6.42, 6.43, 6.65, 6.98, 8.22, 9.19, 11.35, 13.75, 44.70, and 196.31 kD) and one was down-regulated (m/z: 97.34 kD) specifically in this CXR stage when compared with control subjects (Table E1, part B).

Analysis of the protein pattern in CXR stage III compared with control subjects revealed 33 protein peaks. Of these, 22 (67%) protein peaks were associated with sarcoidosis as a whole (Table 2, part B). An additional 11 protein peaks were specific for this CXR stage; eight peaks were up-regulated (m/z: 3.37, 3.43, 4.70, 7.85, 8.02, 8.21, 39.29, and 39.94 kD) and three (m/z: 8.22, 10.84, and 22.12 kD) were down-regulated versus control subjects. For details, see Table E1 (part B).

Differential Protein Expression Profile of BALF from Patients with Sarcoidosis CXR Stage I Presenting with or without LS
To assess whether the distinct clinical symptomatology and disease course in LS are reflected in a typical protein pattern, we have analyzed the protein expression pattern in BALF of patients with CXR stage I presenting with or without LS.

There were 37 protein peaks deregulated in the non-LS subgroup compared with control subjects; of these, 18 (49%) were significantly down-regulated in non-LS patients versus control subjects (Table 3; for details, see Table E2). Twenty-five (68%) detected peaks were associated with sarcoidosis as a whole (Table 2, part C).


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  		TABLE 3. PROTEIN PEAKS DIFFERENTIALLY EXPRESSED BETWEEN PATIENTS PRESENTING WITH OR WITHOUT LÖFGREN'S SYNDROME, IN COMPARISON WITH CONTROL SUBJECTS

 
The specific LS protein pattern consisted of 26 significantly deregulated protein peaks in comparison with healthy control subjects (Table 3; for details, see Table E2). Apart from four (15%) protein peaks, the remainder were up-regulated in the LS subgroup when compared with controls. Thirteen (50%) peaks were found to be a part of a sarcoidosis-associated protein pattern (Table 2, part C).

Twelve identical protein entities were significantly deregulated in both LS and non-LS patient subgroups in comparison with control subjects. These 12 identical entities represent 46% (26) of all peaks identified in BALF from patients with LS and only 32% (37) of peaks from patients without LS.

Importantly, the comparison of protein patterns in BALF from patients with or without LS revealed 25 protein peaks, the expression of which differed between these groups (Table 3; for details, see Table E2). Of these, only four (16%) were up-regulated in patients without LS, and 21 (84%) were up-regulated in the patients presenting with LS.

Identification of Differentially Expressed Proteins
For protein identification, three linked procedures were performed: reversed-phase fractionation, SDS-PAGE separation, and peptide fingerprint mapping. The reversed-phase fraction with 30% acetonitrile contained the proteins associated with the sarcoidosis-associated protein profile, and this fraction was separated by SDS-PAGE. Peptide mapping of the excised bands from SDS–polyacrylamide gels revealed the following proteins as the best candidates of database searches: protocadherin-2 precursor (m/z: 100.99 kD on the Q10 surface), human serum albumin (m/z: 33.33 and 66.64 kD on CM10), and {alpha}1-antitrypsin (AAT; m/z: 44.43 kD on CM10). To prove that protocadherin-2 precursor, human serum albumin, and AAT match to differentially expressed signals found by ProteinChip analysis, immunodepletion assays were performed with unconcentrated BALF as starting material. Analyses of the supernatants from the immunodepletion assays by ProteinChip arrays showed significant reduction of the signals corresponding to the peaks of interest. In the negative controls without the specific antibody the peaks were clearly detectable (Figure 4; see also Figures E1 and E2).


Figure 4
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  		Figure 4. Identification of protocadherin-2: normalized ProteinChip array profiles of a representative immunodepletion assay. Unconcentrated bronchoalveolar lavage fluid was used as starting material for an immunodepletion assay with anti–protocadherin-2 antibody (top). The negative control was performed without specific antibody to protocadherin-2 (bottom). The peak (m/z: 100.99 kD on Q10 surface) corresponding to protocadherin-2 precursor is marked with an asterisk. For details, see IN-GEL DIGESTION AND IMMUNODEPLETION ASSAY.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This first report on the application of SELDI-TOF MS for the analysis of protein expression patterns in unconcentrated BALF revealed that 40 differentially expressed protein peaks are associated with sarcoidosis in comparison with healthy control subjects. Of these 40 peaks, 13 were present across all CXR stages and may, therefore, represent the protein entities directly involved in pathogenesis of the disease (disease essential, i.e., susceptibility proteins). Furthermore, specific protein peaks typical of individual radiologic stages of sarcoidosis were detected and these may have a role as phenotype modifiers. Our data also suggest that the BALF protein pattern of patients with CXR stage I sarcoidosis is strongly dependent on the presence of LS. The BALF protein pattern in patients presenting with LS was quite different not only from the pattern in patients without LS, but also from the sarcoidosis-associated protein pattern. Finally, peptide mapping confirmed by immunodepletion revealed the identity of three of the proteins detected by SELDI-TOF MS: in addition to human serum albumin and AAT, a purely novel molecule (protocadherin-2 precursor) was identified as a component of the sarcoidosis-associated protein profile.

The clinical outcome of patients suffering from sarcoidosis is variable and ranges from acute self-resolving granulomatous responses to a more chronic form with progression to fibrosis (4). Interestingly, as many as 40 protein entities, the expression of which differed significantly between sarcoidosis as a whole and healthy control subjects, were detected. Of the sarcoidosis-associated protein pattern, more than 30% of the protein peaks were present across all radiologic stages studied. This finding supports the concept that there is a uniform basic event in the earlier phases of sarcoidosis, characterized by CD4+ T-cell accumulation and macrophage stimulation (4, 25, 37). We propose that even more protein entities from the sarcoidosis-associated protein pattern are deregulated in individual CXR stages I, II, and III, but in our study they did not reached statistical significance because of subgroup size.

We hypothesized that the BALF proteome may undergo changes during disease development and progression. We have therefore studied protein patterns in CXR stages I to III. Radiologic assessment of lung involvement was chosen because of its precise definition (4, 31) and clinical importance in terms of outcome prediction (38). CXR stage I is limited to hilar lymphadenopathy, and an inflammatory reaction probably plays the predominant role in this stage. In our study, the most dynamic changes in BALF protein content were, however, observed in patients with CXR stage II. This stage is characterized not only by lymphadenopathy but also by lung parenchyma involvement. We therefore suggest that the lung proteome in CXR stage II involves not only proteins of the inflammation response, but also locally produced proteins involved in the parenchymal changes. CXR stage III represents a more advanced stage with likely evolution of lung scarring. We propose that proteins specific for CXR stage III may play a role in extracellular matrix deposition. The most severe stage, CXR stage IV, is characterized by fibrotic changes and could not be investigated in our study because of the low number of available BALF samples.

We were also interested in exploring whether patients presenting with LS, which is characterized by the presence of erythema nodosum, fever, bilateral lymphadenopathy, and arthralgia (4), have a different BALF protein profile than the patients without this acute form of sarcoidosis. Apart from the notable clinical differences, patients with LS share specific genetic associations—for example, an extended MHC haplotype encompassing alleles in HLA-DQ, HLA-DR, CCR2, and TNF (39–42). We therefore speculated that the BALF proteome may reflect these differences. Indeed, we were able to detect 25 protein entities the expression of which significantly differed between BALF from patients with and without LS. Surprisingly, the protein pattern of BALF from patients with LS was characterized by up-regulation of the majority (85%) of the dysregulated protein entities when compared with control subjects and also with patients with sarcoidosis without LS. However, in the patients without LS, the proportion of up-regulated and down-regulated protein peaks was equal when compared with control subjects (50 vs. 50%). Interestingly, the total number of deregulated peaks and also of those associated with sarcoidosis as a whole was markedly lower (by 40%) in patients with LS in comparison with other sarcoid phenotypes. The detection of additional protein peaks in BALF from patients presenting with LS allows the speculation that these protein entities may promote an effective resolution of disease consistent with the better prognosis of patients with LS. Here we provide the first evidence that BALF from patients presenting with LS has a different protein profile in comparison with patients without LS.

Although the BAL procedure has been used for more than 30 yr for diagnostic purposes in diffuse lung diseases (43, 44), information about the protein content of BALF is still limited at least in part because of methodologic problems in sample processing. Dilution of proteins by the lavage procedure, high salt content, and/or low concentrations of some proteins require desalting and concentration, leading to the significant loss of BALF proteins (45). Further, the complex binding of some proteins to albumin and the masking of some proteins by highly abundant albumin isomers complicate BALF analysis (45). The ProteinChip technology seems, however, to be suitable for analysis of the BALF proteome (46). Concentration of BALF samples for SELDI-TOF MS analysis is not required because of its femtomole sensitivity and the salts are washed out after selective retention of proteins on the ProteinChip array. Another benefit of SELDI-TOF MS is the fact that overrepresented proteins of BALF (47) do not disturb the protein patterns obtained. In addition, this technique has been shown repeatedly to be reproducible and capable of identifying quantitative differences between samples (48–50).

From a methodologic point of view, no basic rules have been formulated on how to perform a proteomic study and proteome scientists have various options in experimental design, including statistical analysis and interpretation (51). This applies in particular to statistics, where just as in other "-omics" studies, large amounts of data are obtained. One may decide to apply a strict Bonferroni correction adjustment (52) or to leave the data as they stand and refrain from any adjustment (53). In this study we have, for the first time, partially characterized the sarcoid proteome by SELDI-TOF MS. We therefore decided not to adjust our data in line with other proteomic studies of a similarly exploratory character (46, 54, 55). We are aware that there may be an inherent potential for overinterpretation, but we believe this was outweighed by our reporting a full spectrum of candidates for future, more detailed investigations. The decision not to apply any correction factor was further supported on realizing that, because of the existence of a myriad of proteins typically present in biological samples such as BALF, the exact value of a correction factor is difficult to define.

SELDI-TOF MS enabled us to analyze BALF from 65 patients and 23 control subjects. In contrast, previously published reports using 2D gel electrophoresis analyzed substantially smaller groups consisting of 6 to 15 patients with sarcoidosis and of 3 to 11 control subjects (11–16). Finally, these investigators studied patients with sarcoidosis with different clinical phenotypes and diverse control groups: healthy subjects (12, 13), patients with idiopathic pulmonary fibrosis (12, 14, 16), and patients with systemic sclerosis (16). The majority of differentially expressed proteins identified in these studies (11–16) were not the same; and this was probably because of the clinical heterogeneity of relatively small patient groups and different control groups, and also because of differences in sample preparation and separation.

Of the sarcoidosis-associated protein profile characterized in this study, the identity of three proteins was revealed by peptide mapping and confirmed by immunodepletion analysis. Human serum albumin was the first abundant protein, which was present in increased amounts in all CXR stages and in patients both with and without LS. Higher albumin levels in the lungs of our patients likely result from chronic leakage due to increased vascular permeability (56). It is, however, unlikely that the increase in albumin in our patients was caused by its acute influx during bronchoscopy because an identical lavage procedure was used for both patient and control groups. Our observation of elevated albumin levels in BALF from patients with sarcoidosis may have wider implications. Albumin levels may be associated with local inflammation and, therefore, use of albumin as a coefficient for normalization of protein content in BALF may not be legitimate; this conclusion is in agreement with other reports (56–58).

AAT, the regulator of protease–antiprotease balance (59), was the second protein identified from our sarcoidosis-associated protein profile and this protein was down-regulated in sarcoidosis as a whole. However, when our patients were subdivided according to the presence of LS, AAT was down-regulated only in patients without LS; it was unchanged in patients with LS. We therefore propose that unchanged, that is, physiologic levels of AAT in patients with LS are associated with early spontaneous resolution. This interpretation is in agreement with the opinion on the role of AAT as a concentration-dependent modulator of the inflammatory response (60–62). Whereas key mediators of sarcoid inflammation (tumor necrosis factor-{alpha}, interleukin 6, monocyte chemoattractant protein-1, etc.) are inhibited by normal levels of AAT (61), production of these cytokines is potentiated at its lower levels (62). Down-regulation of AAT may, therefore, stimulate expression of tumor necrosis factor-{alpha} and other proinflammatory mediators and thus contribute to sustained inflammation in patients without LS.

Protocadherin-2 precursor, the third protein identified in our experiments, was up-regulated in sarcoidosis as a whole and also across all studied phenotypes. In general, protocadherins belong to the cadherin superfamily of cell adhesion molecules and their functions are still poorly understood (63). It has been suggested that, besides its weak cell adhesion properties, protocadherin-2 may, similarly to other protocadherins, possess additional biological activities such as suppression of tumor growth (64, 65). However, to date, no data have been available on a plausible role of protocadherin-2 precursor in lung either in health and disease and, therefore, the importance of its up-regulation in sarcoidosis needs to be clarified further.

In conclusion, the data in this article extend the concept of differential protein expression in sarcoidosis and its clinical phenotypes. Because, despite extensive investigation of sarcoidosis, a clear picture of the pathogenesis of different disease patterns has yet to emerge, characterization of the sarcoid proteome in BALF by SELDI-TOF MS technology may help to identify molecules involved in different subsets of this disease.


   Acknowledgments
 
The authors thank the staff of the Bronchoscopy Division, Department of Respiratory Medicine, Faculty Hospital Olomouc, for their help. They thank Ms. A. Vevodova and Z. Navratilova for technical assistance. Protocadherin-2 antibody was a gift from Dr. H. Omi and Dr. M. Kitagawa, Japan.


   FOOTNOTES
 
Supported by the Grant Agency of the Czech Republic (no. 310/05/2614) and the German Federal Ministry of Education and Research (BMBF).

* These investigators contributed equally to this work. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200507-1126OC on January 26, 2006

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 21, 2005; accepted in final form January 17, 2006


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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