Proteomics in rheumatology

Figure 1. (A) Normal synovial joint and (B) rheumatoid arthritis joint.

Reprinted with permission from Macmillan Publishers Ltd: Nature Drug Discovery, © 2003 [1].

Figure 2. Autoantigen detection in rheumatology by 2D-immunoblotting of synovial tissue proteins.

Total 2D image of synovial tissue protein extract (12.5% Laemmeli, pH 4–7, silver staining) (left). Reincubation of 2D-blots with rheumatoid arthritis serum (B) of membranes previously probed with control serum (A). New immunoreactive spots appear at the acidic side of the spot train (right).

Reproduced with permission from Whiley-VCH Verlag GmbH & Co. KGaA: Proteomics – Clinical Application, © 2007 [20].

Rheumatic diseases

Rheumatology is a very broad discipline comprising many different, complex and related diseases primarily affecting the joints, especially freely movable joints known as synovial joints (Figure 1A)[1]. The general public mostly associates rheumatic diseases with one type of rheumatic joint disease, namely arthritis. When elderly people experience pain in the joints, they often refer to it as being arthritis. Indeed, arthritis actually means joint (arthro) inflammation (-itis). However, in reality, there are many different types of rheumatic diseases that are not only related to the joint, but which can also affect kidneys, lungs, eyes, the skin and the CNS, not only in elderly persons but in people of all ages.

Without going into too much detail regarding the different sorts of rheumatic diseases, we will briefly elaborate on joint-associated pathologies. Rheumatic joint diseases are largely divided into noninflammatory and inflammatory pathologies. In inflammatory rheumatic diseases, of which rheumatoid arthritis (RA) and spondyloarthropathy (SpA) are two frequent forms, the primary target of inflammation is the synovial tissue. This synovial tissue lines the joint cavity and is responsible for the joint homeostasis. During the course of the disease, the phenotype of the synovial tissue changes into an activated, proliferative, invasive tissue. This uncontrolled process of tissue growth leads to narrowing of the joint space and subsequent increased efflux of synovial fluid, resulting in swollen joints. The synovial hyperplasia, also due to infiltrating lymphocytes and monocytes, is accompanied by the production of certain destructive enzymes, mainly matrix metalloproteinases. This eventually leads to cartilage and bone loss, especially in RA (Figure 1B)[1]. As for SpA, the synovitis results in less joint destruction; it is characterized by bone formation in addition to bone erosions [2].

Osteoarthritis (OA), often referred to as degenerative arthritis, is a noninflammatory rheumatic disease characterized by the absence of neutrophils in the synovial fluid and absence of systemic manifestations of inflammation. The primary target in OA is the articular cartilage, which finally degenerates owing to an imbalance between anabolic and catabolic processes in the chondrocyte [3]. Cartilage destruction is associated with changes in the adjacent bone (e.g., osteophytes: bone growth at the joint margins), ultimately resulting in joint failure.

In a discipline as broad and complex as rheumatology, proteomics is of great help in finding protein markers. Good rheumatologic protein markers should able to discriminate between different rheumatic disease subtypes like RA and SpA. Using 2D gel electrophoresis to separate cytosolic proteins from inflamed synovial tissue, our group was able to discriminate between RA and SpA, by hierarchical clustering based on the protein expression profiles [4]. Several potential protein markers were identified by us and other groups, allowing differentiation between rheumatic diseases. An example of a known biomarker discriminating between OA and RA is calgranulin-A. In the field of rheumatology, this protein is better known by its gene identification S100A8 or MRP8. The expression of this protein is specific for cells of myeloid origin such as granulocytes, monocytes and macrophages. It is specifically released during the interaction of monocytes with the inflammatory activated endothelium [5]. This marker has been identified in several proteome analyses [4,6,7]. However, rather than considering this protein as a marker for RA, it should be regarded as a marker for inflammation, since other inflammatory arthritides show higher expression levels of MRP8 [6,8].

Although several attempts have been made and some interesting proteins have been identified, the integration of potential biomarkers resulting from rheumatic proteomics analyses in differential rheumatologic diagnosis is not yet established. Today, differential diagnosis is made based on clinical observations in combination with serological analyses and histological features of the synovium. Although, this diagnostic toolbox is already substantial, clinical symptoms can develop rather atypical and the course of the disease can be very unpredictable. For example, in RA there are a few clear-cut differences between early and late stages of RA and, even on histological level, RA cannot be distinguished from other types of synovitis [9]. Also, inflammation in the synovial tissue may occur before the development of clinical symptoms or even persist despite remission of clinical signs.

Rheumatic biomarkers

Proteome studies in rheumatology are focused around two topics: protein target identification by differential screening of biological fluids (serum and synovial fluid) or rheumatic tissues (synovial tissue, cartilage and chondrocytes) on the one hand and proteomic studies for identification and characterization of autoantigens on the other. To illustrate the growing interest of proteomics in this field, in the beginning of 2005 we conducted a search using ‘arthritis’ and ‘proteomics’ as keywords and found 22 hits, now we have over 100 papers describing different proteome techniques applied to different diseases that are comprised in the field of rheumatology.

Rheumatoid arthritis and OA are still the most investigated rheumatic pathologies today. In OA there are studies on cartilage and chondrocytes where proteome analysis is conducted using 2D gel electrophoresis in combination with mass spectrometry (MS) [10–12]. Cartilage is a very attractive tissue, as it contains only a single cell type. At the same time, this single cell type comprises only 1% of the total volume, while the main components of cartilage are highly abundant extracellular matrix proteins, such as proteoglycans and collagens. Chondrocytes have to be isolated from cartilage and kept in specific conditions for days. These cells are not cultured in monolayers, but in alginate beads, allowing the chondrocyte to recuperate from the harsh isolation procedure upon which they will eventually reach a more or less normal anabolic balance without differentiation or proliferation [13]. A new extracellular matrix will be produced, making them resemble their in vivo state as well as possible.

On the contrary, when analyzing synovial tissue derived from inflammatory arthritides, one is confronted with a very complex environment of several types of cells (e.g., synovial fibroblasts and inflammatory immune cells). The synovial fibroblasts can easily be isolated and grown in monolayers in vitro; however, it is impossible to mimic the complex in vivo inflammatory situation. In addition, when wanting to work with the inflamed tissue as a whole, one has to consider that synovial biopsy specimens are small and several biopsies will be needed to obtain enough protein material to perform a good classic proteome analysis [4].

There is an apparent lack of gel-free rheumatologic proteomic approaches using isotope-coded affinity tags. Gel-based and gel-free proteomic approaches are known to be complementary and results from both methods could provide a more complete picture [14]. However, when working with in vivo material that is scarce and not readily available, as is the case with affected tissue in rheumatic joint diseases, a sensitive gel-free quantitative procedure such as isobaric tags for relative and absolute quantification could be the method of choice. As proteomics in rheumatology is a late-bloomer in contrast with other pathologies, such as cancer, we do believe that in the years to come, gel-free quantitative techniques will also be applied to find protein biomarkers and key players in rheumatic pathologies.

Although, we encourage the use of gel-free proteomics techniques, gel-based assays will always have their place in rheumatology proteomics. The majority of the rheumatic diseases are known as autoimmune diseases and identification of autoantigens playing a role in these pathologies is extremely important. Autoantigens are mostly identified by separating tissue proteins by 2D gel electrophoresis followed by western blotting and incubation of patients’ sera.

A variety of potential autoantibodies have been described in patients with RA [15]. Among them, antibodies against citrulline-containing proteins have shown to be highly specific for RA (specificity: 96–98%) [16,17]. During this post-translational modification, arginine residues are enzymatically converted into citrulline residues [18]. Although antibodies against these modified proteins are highly specific for RA, the presence of the citrullinated proteins themselves is not able to discriminate between inflammatory arthritides as these modified proteins are also found in the inflamed SpA synovium [19]. The power of 2D gel electrophores lies in its high-resolution separation of proteins. Indeed, synovial immunoreactive proteins appear as spot trains on 2D gels (Figure 2)[20]. In our study, proteins in these trains consisted of citrullinated fibrinogen-β and the proteins that showed the highest antibody specificity arose at the acidic site of these spot trains. Reincubation with RA serum of membranes probed with control serum, revealed the appearance of additional spots at the acidic side of the spot train [20]. In a similar approach, citrullinated α-enolase has been identified as an abundantly expressed autoantigen in synovial tissue of RA patients [21].

Antibodies against these citrullinated autoantigens are regarded as serological markers. Indeed, in a recent study, a multivariate analysis of several known biomarkers was conducted and antibodies against citrullinated proteins had the highest classification power for the diagnosis of established RA [22]. To fully characterize these serological markers it is very important to be able to identify the in vivo epitope against which the antibodies react. For example, in the case of citrullination, it is still unknown where the citrullinated residues are located in the proteins present in the inflamed joint. This has been attempted by testing antibody reactivity in multiwell assays in which large collections of commercially available citrullinated and noncitrullinated peptides (spanning possible citrullinated epitopes from known citrullinated proteins) are used, in order to screen for dominant epitopes.

Although a dominant epitope can be identified in this way, the question remains if this specific citrullinated residue is actually present in the joint. Proteome analyses combined with MS could elucidate the in vivo citrullinome in the joint of several rheumatic diseases and bring us one step closer to understanding how these post-translational modifications are implicated in the pathology.

Rheumatic proteomics

As stated previously, proteomics is gaining interest in the field of rheumatology. There is a diversity of proteome analysis techniques that can be combined with different sample sources leading to many study design options. Relating to the proteome analysis techniques, gel-based approaches have been the main method of choice; however, SELDI analysis has also been used as a proteome analysis tool for rheumatic diseases. These techniques are used to study a variety of samples: serum or plasma, peripheral blood mononuclear cells and in the case of rheumatic joint diseases: synovial fluid, synovial tissue, synovial fibroblasts, chondrocytes and articular cartilage. Owing to this diversity, results of proteomic studies in rheumatology, although each unique in their design and equally important, are somewhat scattered. It would be a big step forward, if these groups or their results could somehow connect with each other. As relevant biological material is scarce, especially due to the successful treatment of patients with biologicals like anti-TNF, several centers could or perhaps should work together to be able to leave a definite proteomic footprint behind in the field of rheumatology.

In conclusion, we feel that there is a defined way on how proteomics could be of further assistance in the field of rheumatology: gel-free analyses of tissue biopsy samples could be the method of choice, because of the limited amount of tissue available and the possibility to analyze less abundant proteins. On the other hand, classic techniques like 2D gel electrophoresis will still have an impact on the research in the field of rheumatology because of the importance of post-translational modifications.

A discipline as broad and complex as rheumatology will need the whole spectrum of proteome techniques to study specific characteristics of rheumatic pathologies, and today maybe we could say that only the surface has been scratched.

Acknowledgments

We thank all our colleagues from the Department of Rheumatology at Ghent University Hospital for their longstanding collaboration with our laboratory, their help and stimulating discussions. We apologize for omissions of important work due to restrictions.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.