Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection

Rutgers et al. Arthritis Research & Therapy 2010, 12:R114 http://arthritis-research.com/content/12/3/R114 Open Access RESEARCH ARTICLE Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection Research article Marijn Rutgers1, Daniël BF Saris1, Wouter JA Dhert1,2 and Laura B Creemers*1 Abstract Introduction: Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration. Methods: The effect of conditioned serum on PG metabolism of cultured OA cartilage explants was compared to unconditioned serum. The effect of serum conditioning on levels of interleukin-1beta (IL-1β), IL-4, IL-6, IL-10, IL-13, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG), oncostatin M (OSM), interleukin1 receptor (IL-1ra) and transforming growth factor beta (TGF-β) were measured by multiplex ELISA. As TNF-α levels were found to be increased in conditioned serum, the effect of TNF-α inhibition by etanercept on PG metabolism was studied in cartilage explants cultured in the presence of conditioned serum. Furthermore, cytokine levels in SF were measured three days after intraarticular ACS injection in OA patients to verify their retention time in the joint space. Results: PG metabolism was not different in the presence of conditioned serum compared to unconditioned serum. Levels of the anti-inflammatory cytokines IL-1ra, TGF-β, IL-10 as well as of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and OSM were increased. IL-4, IL-13 and IFN-γ levels remained similar, while OPG levels decreased. TNF-α inhibition did not influence PG metabolism in cartilage explant culture in the presence of condtioned serum. Although OPG levels were higher and TGF-β levels were clearly lower in ACS than in SF, intraarticular ACS injection in OA patients did not result in significant changes in these cytokine levels. Conclusions: ACS for treatment of osteoarthritis contains increased levels of anti-inflammatory as well as proinflammatory cytokines, in particular TNF-α, but conditioned serum does not seem to have a net direct effect on cartilage metabolism, even upon inhibition of TNF-α. The fast intraarticular clearance of cytokines in the injected ACS may explain the limited effects found previously in vivo. Introduction Osteoarthritis (OA)-associated cartilage degradation is mediated in part by cytokines and growth factors, excreted into the intraarticular environment by synoviocytes, activated immune cells, or by the articular cartilage itself [1,2]. Therapies interfering with these cytokines * Correspondence: 1 Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Full list of author information is available at the end of the article may influence disease progression and improve the patient's quality of life. A pivotal role in the progression of OA has been assigned to the pro-inflammatory cytokine interleukin1β (IL-1β), which induces a cascade of inflammatory and catabolic events including the expression of cartilage degrading matrix metalloproteinases (MMP) [3], nitric oxygen (NO) production and prostaglandin E2 (PGE2) release [4], while inhibiting proteoglycan and collagen synthesis [5,6]. The number of type-1 IL-1 receptors is © 2010 Rutgers et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rutgers et al. Arthritis Research & Therapy 2010, 12:R114 http://arthritis-research.com/content/12/3/R114 significantly increased in OA chondrocytes [7] and synovial fibroblasts [8], increasing the susceptibility for IL-1α and IL-1β mediated effects. In addition, it was suggested that in OA synovium, a relative deficit in IL-1ra-production exists [1]. As intraarticular administration of recombinant human interleukin-1 receptor antagonist has been shown to alleviate symptoms in several animal models of OA and rheumatoid arthritis [9-11], intraarticular treatment with IL-1ra was also suggested as a feasible treatment for patients with OA. One example of a disease-modifying osteoarthritisdrug (DMOAD) based on blocking the intraarticular effects of IL-1 associated with OA, is autologous conditioned serum (ACS or Orthokine®; Orthogen, Düsseldorf, Germany). Autologous conditioned serum (ACS) treatment consists of six repetitive injections of ACS over a period of 21 days. ACS is prepared from whole blood that is incubated in the presence of glass beads to initiate monocyte activation [12,13]. The resulting conditioned serum (ACS), has been shown to contain increased levels of IL-1ra as well as IL-4 and IL-10 [12]. In horses with arthroscopically induced osteochondral defects, ACS treatment demonstrated a reduction in lameness and a decrease in synovial membrane hyperplasia [14]. ACS treatment of human OA patients, however, demonstrated limited to moderate clinical effects [15,16]. Despite the fact that this approach has already been introduced in the clinic, the mechanisms by which administration of this product may result in reduction of OA symptoms is not yet fully understood [14,16,17]. Although the primary goal of ACS treatment is alleviation of OA symptoms, one of the mechanisms may be enhancement of cartilage integrity through the inhibition of inflammatory activity, in particular with respect to Il-1 signalling. In fact, the direct effect of the entire blend of known and unknown factors present in ACS on cartilage metabolism in human OA cartilage has not been described. Moreover, only limited data are available on the actual composition of the conditioned serum. Besides IL-1ra, growth factors, such as transforming growth factor beta 1 (TGF-β1), which stimulates chondrocyte proliferation [18,19], are upregulated during incubation [17]. Of several pro-inflammatory cytokines like IL-1β, tumour necrosis factor-alpha (TNF-α) [20,21] and IL-6 [22], of the last of which also anti-inflammatory effects have been described [23], it is not entirely clear if their levels remain equal or are upregulated during incubation [12,17]. As a consequence of monocyte activation during incubation of blood, antiinflammatory cytokines such as IL-13, which was shown to inhibit the production of IL-1β and enhance production of IL-1ra [24], and osteoprotegerin (OPG) [25], which protected cartilage in a murine model of surgically induced osteoarthritis from further degeneration [26 (...truncated)