Proteomic Analysis of Peripheral Blood Mononuclear Cells: Selective Protein Processing Observed in Patients with Rheumatoid Arthritis Martin Schulz,‡ Helmut Dotzlaw,‡ Stefan Mikkat,† Martin Eggert,# and Gunther Neeck*,# BIOMEDRO Biomedical Research and Development Rostock Ltd., Robert-Koch-Strasse 9, D-18059 Rostock, Germany, Core Facility Proteomeanalysis, Medical Faculty, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany, and Rostock Clinic South, Suedring 81, D-18059 Rostock, Germany Received May 15, 2007
In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases. Keywords: Proteome • Lymphocytes • Inflammation • Autoimmune • Rheumatoid Arthritis
Introduction Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases, with a prevalence of about 1% of the world’s population. It is characterized by chronic inflammation of the joints, eventually causing irreversible joint damage that can lead to severe disability. The inflamed joints are infiltrated by a large number of activated mononuclear cells that contribute to the destruction of articular cartilage. Although the precise etiology and pathogenesis are still unresolved, it is commonly accepted that RA is a complex systemic multifactorial autoimmune disorder characterized by overexpression of inflammatory cytokines, by the occurrence of a multitude of autoreactive cells, as well as by predisposing genetic factors.1-3 The diagnosis of rheumatoid arthritis is based on the criteria of the American College of Rheumatology and relies on the expression of clinical symptoms of RA that include radiological changes as well as testing for the presence of the rheumatoid factor.4 The disease assessment is supported by the determination of indicators of inflammation such as blood sedimentation rate and the C-reactive protein (CRP). More recently the measurement of autoantibodies directed to cyclic citrullinated peptides (CCP) has emerged as a diagnostic and prognostic test in RA.5 * To whom correspondence should be addressed. Prof. Dr. Gunther Neeck, BIOMEDRO Biomedical Research and Development Rostock Ltd., RobertKoch-Strasse 9, D-18059 Rostock, Germany. Fax: +49(0)381-44015099. Phone: +49(0)381-44015000. E-mail:
[email protected]. ‡ BIOMEDRO Biomedical Research and Development Rostock Ltd. † University of Rostock. # Rostock Clinic South.
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Journal of Proteome Research 2007, 6, 3752-3759
Published on Web 08/17/2007
Over the past decade, the molecular mechanisms underlying RA have been intensively investigated, which has resulted in the identification of several proteins, gene loci, and cellular pathways associated with this disease. Various materials obtained from RA patients, experimental animal models of induced arthritis, and cell culture systems have been successfully used in these approaches.6-8 Whereas many studies used fluid or biopsy samples taken from the inflamed synovium,9,10 where the major pathologic events in RA occur, we chose to use peripheral blood mononuclear cells (PBMCs) in our investigation of RA patients. PBMCs have been shown in several investigations to represent an attractive source material in the characterization of RA patients.11-15 PBMCs are a heterogeneous cell population consisting of about 80-85% of lymphocytes that are known to be key players in inflammatory processes. They mediate and coordinate the immunological response in autoimmune diseases and are responsible for cytokine secretion in the absence of an ongoing infection. In RA, lymphocytes take part in the joint destruction as they drive the activation of monocytes and the transformation of the synovial fibroblasts into aggressive cells, both of which are capable of destroying the cartilage.2,16,17 Unlike biopsy of the tissues targeted in RA, they are easily accessible from patients as well as from control subjects, are simple to prepare, and therefore make standardization possible. Aiming at the identification of factors associated with disease-specific alterations in the immune system, we performed a comparative proteomics approach on PBMCs derived from patients suffering from RA and from healthy control individuals. Two-dimensional gels of the cells derived from 32 10.1021/pr070285f CCC: $37.00
2007 American Chemical Society
research articles
Proteomic Approach in Rheumatoid Arthritis
clinically characterized RA patients and 33 healthy blood donors were prepared, quantified, and screened for protein spots that are significantly deregulated in the patient group. Characterization of candidate spots by mass spectrometry and Western blotting together with the use of algorithm-based similarity measurements of the relative spot intensities resulted in the identification of a molecular fingerprint of circulating immunocompetent cells, that is composed of 16 protein spots and is characteristic for the RA patient study group.
Materials and Methods Selection of Blood Donors. Patients diagnosed with RA according to the classification criteria of the American College of Rheumatology (ACR)4 and that were suffering from acute medical symptoms at the time of sample donation were selected for the study. Additional entry criteria included provision of informed consent. The procedure was approved by the local ethics committee. Patients were tested for the presence of rheumatoid factor (RF), and the disease activity score 28 (DAS28)18 was calculated. The disease duration (years since first RA diagnosis), as well as the RA stage, that is a classification of the radiological changes according to Steinbrocker’s system for grading RA on plain radiographs,19 was also determined. The patient group consisted of 32 individuals with 11 male and 21 female patients ranging from 37 to 87 years of age. As a control group, 9 male and 24 female healthy blood donors ranging from 22 to 59 years of age were selected. The serum levels of C-reactive protein (CRP) and interleukin-6 (IL-6), as well as the titers of anti-cyclic citrullinated peptide antibodies (anti-CCP), were determined in clinical measurements. With the exception of 3 patients (P26, P27, and P29, see Table 1), who had received 20 mg of prednisolone 1 to 2 days prior to blood donation, the patients had not been treated with glucocorticoids or disease modifying anti-rheumatic drugs for at least 6 months prior to blood donation. The study group includes 15 individuals (7 RA patients and 8 healthy controls) from a previous study14 which were completely reanalyzed under the conditions used in the present study. Sample Processing. The blood samples obtained from patient and control subjects were prepared and analyzed as described previously.14 Briefly, PBMCs were isolated by density gradient centrifugation, and proteins were extracted in 300 mM sucrose, 10 mM Tris/Cl, pH 8, 2 mM MgCl2, and 0.5% Triton X-100 and then subjected to two-dimensional (2-D) gel electrophoresis. Precipitated proteins (200 µg) were dissolved in appropriate sample buffer and focused in the first dimension on 24 cm immobilized pH 4-7 gradient IPG strips (GE Healthcare) followed by separation in the second dimension on 12.5% SDS-PAA gels. Duplicate gels were prepared for each sample. Silver-stained gels (PlusOne Protein Silver Staining Kit, GE Healthcare) were scanned using a UMAX PowerLook III scanner, and digital images in 16-bit grayscale format at a resolution of 300 dpi were saved. Analyses of 2-D Gel Images. Gel images were loaded into Proteomweaver 3.0 (Definiens, Munich, Germany) and split into patient and control groups. The software automatically detects protein spots and matches detected spots between a virtually unlimited number of gels, making the comparative analyses of large numbers of 2-D gels practical. The quantity of a spot is expressed as a fraction of the total intensity of all spots on the gel followed by a normalization procedure that computes a normalization factor between all matched pairs of gels in the analyses. This factor is the median of the quotient of the
Table 1. Characteristics of the RA Patients antiCCPc CRPd IL-6e age duration RA rheumatoid [RU/ [mg/ [pg/ no. sex [years] [years] stage DAS28a factorb mL] L] mL] P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32
f f f f f m f f f f f f m m m f f m m f f m f m f f m m f f f m
62 58 53 62 62 71 57 40 52 37 71 73 42 52 62 72 67 58 67 69 51 50 66 64 65 63 87 55 66 59 78 55
18 24 20 14 0 0 26 5 8 8 25 0 11 18 10 0 25 10 11 21 8 0 13 2 1 0 0 5 5 0 2 0
II II IV IV II I IV II III III II I III IV III-IV I II 0 I I II-III I III I II I I II III I-II I I
5.95 6.78 3.40 7.10 5.73 7.01 3.91 7.10 6.40 6.70 4.93 5.71 5.40 6.50 7.14 7.05 5.28 5.51 4.70 5.69 7.60 3.68 6.69 6.16 5.55 N.D. N.D. N.D. N.D. N.D. N.D. N.D.
RA+ RARA+ RA+ RARARA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RA+ RARA+ RA+ RARA+ RARA-
0.0 0.0 65.9 64.4 0.0 0.0 89.5 55.8 47.7 92.8 0.0 0.0 79.4 68.1 89.2 97.7 0.0 91.8 89.2 79.4 88.1 0.0 46.5 74.3 0.0 N.D. N.D. N.D. N.D. N.D. N.D. N.D.
90.1 25.2
