The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

The complement system as a part of innate immunity is one of the first lines of defence against invading pathogens. Apart from this basic, evolutionarily conserved function, it also plays a role in scavenging of cellular debris and immune complexes, opsonisation and guidance of adaptive immunity by anaphylatoxins and stimulation of B and T lymphocytes [1]. Importantly, complement is an aggressive system capable of targeting even its own cells and tissues when improperly regulated by endogenous inhibitors and/or autoantibodies, or because of gain-of-function mutations in complement components [1, 2]. Such autoreactivity was shown to be an important constituent of pathological mechanisms of inflammatory and autoimmune diseases, including rheumatoid arthritis (RA) [3]. Cartilage degradation products released in the course of joint degenerative diseases form a separate class of potent complement modulators [4‐7].

Joint injury is a major risk factor for the development of post-traumatic osteoarthritis (OA) [8]. Mechanical injury to the joint leads to haemarthrosis, death of chondrocytes and bone cells [9, 10], as well as damage to the tissue extracellular matrix (ECM) directly or mediated by chondrocytes and synoviocytes via increased expression and activation of matrix-degrading enzymes stimulated by proinflammatory cytokines [11]. Furthermore, complement activation has been implicated in the connective tissue repair and inflammatory response initiated by trauma [12, 13]. These observations led us to hypothesize that mechanical joint injury may be an initial trigger of local inflammation, including activation of complement, and that subsequent ECM erosion of injured joints may reinforce complement activation in a positive feedback manner. Monitoring of the complement activation profile after injury could give information potentially important for the design of therapeutic strategies.

We therefore investigated complement activation in OA and the time-dependent activation after knee injuries. RA and pyrophosphate arthritis (PPA) samples were used as positive controls for complement activation. We measured complement activation products characteristic of classical and/or lectin (C4d) and alternative (C3bBP) pathways, as well as the soluble terminal complement complex (sTCC), in the synovial fluid from different patient diagnostic groups (Fig. 1).

Using synovial fluid from subjects in different diagnostic groups, we show that C4d, C3bBbP and sTCC concentrations were elevated not only in our positive controls RA and PPA but also in OA knees and in knee injury compared with levels in the healthy reference group knees. In injured knees, this increase was immediate, seen in knees aspirated on the day of injury, compared with lower levels in knees aspirated up to weeks after injury. Compared with levels of reference knees, knees aspirated several years after injury only showed elevated levels of C4d.

Complement C4d fragment (a proteolytic fragment of C4 with unknown biological function) is an early-stage marker for the classical and lectin pathways, C3bBbP (a C3 convertase) is an early stage marker for the alternative pathway, and sTCC (also known as membrane attack complex [MAC]) is a late-stage marker for all three pathways [1]. In autoimmune diseases such as RA, all three pathways are activated [3]. Analysis of synovial fluid by mass spectrometry and ELISA has shown that levels of complement components are elevated in patients with OA compared with levels found in healthy individuals [25‐27]. Further, patients with OA had increased synovial fluid concentrations of C3a, an anaphylatoxin (i.e., stimulator of inflammation) that can be generated from all three pathways, and of sTCC compared with levels in healthy control subjects [27]. Our results confirm that patients with OA have increased concentrations of sTCC, and they extend previous findings by showing that the alternative pathway, together with the classical and/or lectin complement pathways, was activated in the synovial fluid of patients with OA and after knee injury.

Complement factors present in the synovial fluid after knee trauma may originate from intra-articular bleeding (i.e., haemarthrosis from the ligaments and/or from the synovium) or may be produced by synovial cells (leucocytes and synoviocytes) and/or chondrocytes [27‐31]. Joint bleeding is very common after acute knee injury, and 96 of the 98 recent injury samples with available MRI data had haemarthrosis in our study. At the moment of knee trauma, impact forces can also result in BMLs and OC fractures visualized by MRI. On the basis of previous evidence [24], we anticipated that the presence of OC fractures could be used as a proxy for a strong impact trauma and that disruption of the cortical bone may be an indicator of more severe joint trauma. Complement is involved in fracture healing, where it affects osteoblasts and osteoclasts. These bone cells also produce C3 and C5, and type I collagen is capable of activating platelets, forming sTCC and upregulating receptors for C3a and C5a [32, 33]. At the fracture edge of matrix cracks, the chondrocyte death rate is very high, and damage-associated molecular patterns released from dying cells (i.e., cellular debris such as mitochondria, histones, DNA) contributes to an activation of the innate system [10, 32, 34] and directly triggers C1q [35]. Further, factor VII-acting serine protease, an activator of the coagulation system that is activated by DNA and histones, together with plasmin and clotting factors (e.g., thrombin and factor Xa) can generate C3a/C3b and C5a/C5b [32, 36]. Together, this might explain why patients with knee injury with OC fractures who have major cell osteocyte and chondrocyte death and intra-articular bleeding also have increased complement factor activation of C4d and sTCC compared with patients without such fractures, as shown in our present study.

Previously, we showed that patients with recent knee injury have signs of cartilage degradation, as reflected by an increased release into synovial fluid over several weeks of proteolytic products of aggrecan, type II collagen and COMP [14‐17, 19, 23, 37, 38]. Many of the ECM proteins and their fragments, such as aggrecan, chondroadherin, fibromodulin and osteoadherin, activate the classical complement pathway [6, 7, 27, 39], while others, such as COMP and type II collagen, activate the alternative pathway [5, 40]. However, several ECM proteins also inhibit complement: Proline/arginine-rich end leucine-rich repeat protein (PRELP) and the NC4 domain of type IX collagen directly inhibit formation of MAC [41, 42]; biglycan, decorin and COMP inhibit the classical pathway via binding of C1q [5, 43]; and PRELP inhibits the alternative pathway [41]. The ECM proteins and their fragments thus seem to have dual effects on complement, either activating or inhibiting it. This duality could in part explain why in the synovial fluid from the recent injury group no correlations were found between the cartilage protein fragments and the components of the different complement pathways.

Compared with individuals with healthy knees, TNF-α concentrations in synovial fluid are increased in patients with OA and also in patients with recent (weeks) as well as old (years) knee injuries [14, 38, 44‐46]. Interestingly, in this study, the strongest correlation for the recent injury group samples (0–83 days after injury) was between synovial fluid concentrations of C4d and TNF-α (r _s = 0.547), and the levels of C4d were also found to be elevated many years after injury. This resembles what was found for patients with ACL injury, who had elevated synovial fluid levels of TNF-α 5 years after injury [38].

In injured knee joints, proinflammatory cytokines are produced by chondrocytes and synoviocytes and by infiltrated leucocytes, resulting in the production and activation of matrix metalloproteases (MMPs) and aggrecanases [11, 47, 48]. As a parallel route, complement components such as sublytic concentrations of MAC stimulate chondrocytes to produce MMPs and aggrecanases [27], and complement anaphylatoxins (C3a and C5a) stimulate granulocytes, macrophages, and B and T cells to produce proinflammatory cytokines [3]. Further, it has been shown that C1s (a protease of the classical pathway) can activate proMMP-9, and C1s can also cleave types I, II and IV collagens [49, 50]. This indicates that there are several possible routes for the induction of extracellular proteases in these patients.

In patients with a recent knee injury (0–5 weeks), the release into the synovial fluid of proinflammatory cytokines IL-1β, IL-6 and TNF-α precedes the release of cartilage degradation products of aggrecan, COMP and type II collagen [14, 19, 23, 38]. In the same patients in the present study, we found no correlation between the complement factors C4d, C3bBbP and sTCC and cartilage degradation products of aggrecan, type II collagen and COMP; however, the complement factor concentrations correlated positively with proinflammatory cytokine levels of IL-1β, IL-6 and TNF-α. This supports the concept that complement together with coagulation represents an alternative route to activation of cytokine pathways, and together these routes drive the catabolic process seen as cartilage degradation after joint injury and most likely later in OA.

This study has certain limitations. First, the lack of dilution linearity for the C3bBbP and sTCC assays using synovial fluid samples complicated the assessments, and only relative comparison could be made by using the same dilution (1:20) for all samples. Second, because the subjects in the OA, RA and PPA groups were older than the subjects in the reference group, we cannot exclude the possibility that the increased levels of the complement factors found in these patient groups could be due partly to differences in age. Third, the knee injury group was cross-sectional, so the design precludes drawing firm conclusions of trends in time because no repeated sampling was done within individual patients. Hence, our suggestions regarding differences in complement activation over time need to be confirmed in longitudinal studies. Finally, we measured only the soluble components of C4d, C3bBbP and sTCC found in synovial fluid, and these also ought to be analysed as cell-bound (e.g., chondrocytes, synoviocytes) components for the different diagnostic groups.

We show that alternative and classical and/or lectin complement pathways are activated in the synovial fluid of subjects with knee injury and in patients with OA. After an injury to the knee, the complement factors C4d, C3bBbP and sTCC are instantly activated and are associated with proinflammatory cytokines, while C4d and sTCC are associated with OC fractures.

This and previous reports have suggested that complement is activated in OA [27]. Knee injury is a well-known and strong risk factor for rapid knee OA development, but the driving mechanisms are not well understood. The sudden activation of complement after knee injury found in this study, especially among those with more severe trauma, most likely represents one of several important pathways involved in the onset of this complex disease. Therefore, more clinical studies using longitudinal cohorts are needed to elucidate these pathological pathways to determine if or when to use complement inhibitors as a treatment strategy for OA.