Soluble Macrophage Biomarkers Indicate Inflammatory Phenotypes in Patients With Knee Osteoarthritis

With the advent of biologic therapies for rheumatic diseases, there is a growing armamentarium of effective therapeutics targeting specific inflammatory mediators. With the growing consensus that osteoarthritis (OA) is an inflammatory condition (1,2), many of these new therapies might be brought to bear on OA if a means could be developed to readily identify individuals with inflammatory disease phenotypes. One means of identifying inflammatory phenotypes in vivo, etarfolatide (EC20) imaging, has recently become available (3). This imaging technique is dependent on detecting the selective expression of folate receptor (FR) on activated, but not resting, macrophages (3–5). In a recent study, we showed that the quantity of FRβ+ macrophages in joints, based on EC20 imaging, correlated with radiographic knee OA severity and symptoms (6). Although it is a powerful means of phenotyping patients, EC20 entails radiation exposure; it would therefore be desirable to identify soluble biomarkers correlating with EC20 imaging that could be used adjunctively or independently of imaging techniques to identify patient subsets. The goal of this study was therefore to identify soluble biomarkers of activated macrophages that could provide a means of identifying inflammatory OA phenotypes in a clinical setting through analysis of biologic specimens (blood or synovial fluid [SF]). We hypothesize that soluble macrophage markers, shed into the systemic circulation upon local macrophage activation in the joint, correlate with the level of joint tissue inflammation as quantified by EC20 imaging. We also hypothesize that macrophage markers may be used as predictors of OA progression. We focused on 2 macrophage markers, CD163 and CD14. Both CD163 (7) and CD14 (8) have been observed to be coexpressed with FRβ on tumor-associated macrophages (9). Both of these macrophage markers exist in soluble form and can be measured in serum/plasma and SF. CD163 cell surface expression is limited to the monocyte/macrophage lineage (10) and is activated by antiinflammatory signals such as glucocorticoids (11) and certain cytokines (interleukin-6 [IL-6] and IL-10) (12,13). CD163 cell surface and gene expression is down-regulated by proinflammatory mediators such as lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), and interferon-γ (IFNγ) (12). In 1 study, CD163+FRβ+ mononuclear cell numbers were increased in OA synovial tissue compared to rheumatoid arthritis (RA) synovial tissue (14). Several signals, including phorbol 12-myristate 13-acetate (PMA) and LPS, induce metalloprotease-dependent cleavage of ectodomain of CD163, which subsequently results in the shedding of soluble CD163 from the surface of macrophages (10). CD14 is found on various cell types, predominantly monocytes and macrophages (15). CD14 serves as a receptor for the bacterial LPS–LPS binding protein complex (16). The CD14–LPS–LPS binding protein complex then binds to cell surface Toll-like receptor 4 (TLR-4)–myeloid differentiation protein 2 complexes (17), leading to activation of innate host defense mechanisms, including production of the inflammatory mediators TNFα, IL-1, IL-6, IL-8, IL-10, and IL-12 (18). Like CD163, various stimuli (PMA, IFNγ, and LPS) induce shedding of soluble CD14 via membrane-associated serine proteases (19). The soluble forms of CD163 and CD14 can therefore be considered a reflection of the inflammatory activation status of macrophages.