5.1 Interleukin 1 Cytokine Cascade

The interleukin 1 (IL-1) cascade is complex with various agonists, antagonists, as well as receptors that function in biologic activity and as decoy receptors not inciting biologic activity of IL-1 signaling. The agonists consist of an intracellular form (IL-1α) and a secreted β form (IL-1 β) both of which are capable of binding the various cell surface receptors. Two forms of IL-1 receptors also exist, one that results in downstream signaling (type I IL-1 receptor) with biologic activity and the type II IL-1 receptor (cell surface and soluble form) that acts as decoy receptors and produces no downstream signal or biologic activity. Finally, a natural antagonist (IL-1 receptor antagonist and IL-IRa) is able to bind the type I receptor without resulting in downstream signaling and thus, prevent binding of an IL-1 agonist and its subsequent biologic activity. Since the discovery of the IL-IRa molecule in 1986, various methods of producing IL-IRa to combat IL-1-mediated inflammation have been explored; the applications in sports medicine will be the focus of this chapter.

5.2 Synthetic Interleukin 1 Blockade

Anakinra is a synthetic IL-IRa protein produced from Escherichia coli and while it differs from the eukaryotic protein because it lacks glycosylation, it is effective in blocking the IL-1 cascade. It has been assessed for treatment of various inflammatory disease processes including septic shock and rheumatoid arthritis through a subcutaneous (SQ) route of administration. Using an intra-articular (IA) route of administration in the treatment of osteoarthritis (OA), a safety and pilot efficacy study was performed and demonstrated a good safety profile as well as promising efficacy. ¹ A follow-up double blind, placebo-controlled study (n = 170) also concluded good safety but anakinra was not associated with improvements in OA symptoms when compared with placebo. ² The short half-life of anakinra, 4 to 6 hours following SQ administration, has been postulated as a reason for lack of efficacy in chronic OA. ³ This has been further supported in a pilot study by the reduction in pain and improved function when administered IA within the first month of anterior cruciate ligament injury. ⁴

Other synthetic proteins (rilonacept and canakinumab) with longer half-lives following SQ administration (7 and 30 days, respectively ⁵ ) have been developed for IL-1 blockade but have yet to be investigated for OA.

The production of IL-1 Ra using gene therapy has also been successfully performed in both preclinical ⁶ and clinical trials ⁷ ; however, short time of protein expression as well as immune response to the vector have slowed the clinical progress of this treatment strategy. In 2003, a novel method of upregulating autologous IL-IRa from whole blood (autologous-conditioned serum, ACS) was described ⁸ and gained clinical acceptance especially in the realm of human and veterinary sports medicine. This acceptance has been followed by the slow validation of this treatment modality in more controlled studies.

5.3 Autologous-Conditioned Serum

The processing of whole blood by incubating (typically 24 hours) at 37°C in the presence of medical grade glass beads that had been exposed to CrSO₄ followed by harvesting, filtering the serum, is known as ACS. The process was first reported to increase anti-inflammatory cytokines such as IL-lRa, IL-1, and IL-10 without significantly increasing proinflammatory cytokines (IL-1 β and tumor necrosis factor α [TNF-α]). ⁸ The latter finding has been difficult to confirm in subsequent publications; rather, a concurrent increase in proinflammatory cytokines has been observed. However, improving the anti- to proinflammatory ratio does seem to be repeatable. Assessing this ratio was not performed by the original authors from Orthogen the company that brought Orthokine (ACS product) to the market (Orthokine; Orthogen Veterinary GmbH, Dussoldorf, Germany).

It is believed that the mononuclear and macrophages within whole blood are responsible for cytokine production, including IL-IRa. ^{9 ,​ 10} Furthermore, surface area as well as surface chemistry/cell interaction is thought to play a role in the type of product produced. The exact protein composition of the product generated by this process has not been fully characterized, but the author has documented > twofold upregulation and/or downregulation of more than 35+ proteins in response to a 24-hour culture period (David D. Frisbie, unpublished data). This information has resulted in the product being described as a “soup” which can vary depending on changes in all of the constituent parts. Investigators have quantified a few specific proteins that are thought to participate in the clinical effects of ACS and include IL-IRa, interleukin 1β (IL-1 β ), interleukin 6 (IL-6), TNF-α, interleukin 10 (IL-10), fibroblast growth factor b (FGFb), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF AB), and transforming growth factor β (TGF β ). ¹¹ It should be noted in some studies nonincubated serum has been used as the control while others have used both nonincubated whole blood as well as whole blood incubated without glass beads in a glass tube, the latter most likely is more appropriate. Interestingly, the 24-hour incubation period without glass beads had an IL-IRa:IL-1 ratio that was not significantly different than one of the commercial processes and although numerically better than unincubated it was not significantly better (Fig. 5.1). The other commercial product did show significant superiority to the unincubated serum as well as numerically superior IL-IRa:IL-1 ratio. Because IL-1 is also upregulated in the ACS processing, using a ratio of IL-1 Ra to IL-1 (beneficial to detrimental) cytokines provides a “net” measure of benefit. These results appear to bring into question the benefit of the glass beads in some commercial applications; although, not enough information has been published to reach a definitive conclusion. As previously stated, the products contain numerous proteins in addition to IL-IRa which may also be responsible for the positive clinical effects and only focusing on IL-IRa would be inappropriate.

The first placebo-controlled in vivo work with ACS in a target species demonstrated symptom and disease-modifying action in experimentally induced equine OA. ¹² In this study, four treatments were administered (6 mL) once a week. A significant elevation in synovial fluid IL-IRa concentrations was seen at the time of the final treatment and was still being elevated 35 days later, which was the last measured time point. Also of interest was the significant increase in synovial fluid IL-IRa in nontreated joints; although, to a lesser degree as compared with joints directly treated with ACS. Since this publication, the usage of ACS for the treatment of equine OA has been estimated as “occasionally used” by 54% and “frequently used” by 21% of equine practitioners suggesting an acceptance in this species. ¹³