The role of enzymes and cytokines in the pathogenesis of osteoarthritis

In recent years, a great deal of research has focused on the identification of proteases responsible for the degradation of ECM of articular cartilage in osteoarthritis. According to modern ideas, an important role in the pathogenesis of osteoarthritis is played by matrix metalloproteases (MMP). Patients with osteoarthritis have an increased level of three representatives of MMP - collagenases, stromelysins and gelatinases. Collagenase is responsible for degradation of native collagen, stromelysin - collagen type IV, proteoglycans and laminin, gelatinase for gelatin degradation, collagen IV, Vh XI types, elastin. In addition, the presence of another enzyme - aggrecanase, which has the properties of MMPs and is responsible for the proteolysis of cartilaginous proteoglycan aggregates.

Three types of collagenases were identified in the articular cartilage of the person, the level of which is significantly increased in patients with osteoarthritis - collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13). The coexistence of three different types of collagenases in the articular cartilage indicates that each of them plays its own specific role. Indeed, collagenases-1 and -2 are localized mainly in the surface and upper part of the intermediate zone of the articular cartilage, whereas collagenase-3 is found in the lower part of the intermediate and deep zones. Moreover, the results of immunohistochemical study demonstrated that during the progression of osteoarthritis, the level of collagenase-3 reaches a plateau and even decreases, while the level of collagenase-1 gradually increases. There is evidence that in the case of osteoarthritis, collagenase-1 is mainly involved in the inflammatory process in articular cartilage, whereas collagenase-3 is involved in tissue remodeling. Cartilagin-3, expressed in the cartilage of patients with OA, degrades type II collagen more intensively than collagenase-1.

Of the representatives of the second group of metalloproteases, stromelysin, three-stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and stromelysin-3 (MMP-11) were also identified. Today it is known that only stromelysin-1 is involved in the pathological process in osteoarthritis. In the synovial membrane of patients with osteoarthritis, stromelysin-2 is not detected, but it was detected in a very small number in synovial fibroblasts of patients with rheumatoid arthritis. Stromelysin-3 is also found in the synovial membrane of patients with rheumatoid arthritis near the fibroblasts, especially in fibrosis zones.

In the gelatinase group, only two have been identified in the human cartilage tissue: 92 kD gelatinase (gelatinase B, or MMP-9) and gelatin at 72 kD (gelatinase A, or MMP-2); in patients with osteoarthritis, an increase in the level of 92 kD gelatinase is determined.

Not so long ago, another group of MMPs was identified that are localized on the surface of cell membranes and are called MMP membrane type (MMP-MT). To this group belong four enzymes - MMP-MT1-MMP-MT-4. MMP-MT expression is found in the human articular cartilage. Although MMP-MT-1 possesses the properties of collagenase, both MMP-MT-1 and MMP-MT-2 are able to activate gelatinase-72 kD and collagenase-3. The role of this group of MMP in the pathogenesis of OA requires refinement.

Proteinases are secreted in the form of a zymogen, which is activated by other proteinases or organic compounds of mercury. The catalytic activity of MMP depends on the presence of zinc in the active zone of the enzyme.

The biological activity of MMP is controlled by specific TIMPs. To date, three types of TIMP have been identified that are found in the human articular tissues, TIMP-1-TIMP-3. The fourth type of TIMP is identified and cloned, but it has not yet been detected in the human articular tissues. These molecules specifically bind to the active site of MMP, although some of them are able to bind the active center of pro-gelatinase 72 kD (TIMP-2, -3, -4) and 92 kD gelatinase (TIMP-1 and -3). The data indicate that with OA in the articular cartilage there is an imbalance between MMP and TIMP, which results in a relative deficiency of inhibitors, which may be partly due to an increase in the level of active MMP in the tissue. TIMP-1 and -2 are found in articular cartilage, they are synthesized by chondrocytes. With osteoarthritis in the synovial membrane and synovial fluid, only the first type of TIMP has been detected. TIMP-3 is detected exclusively in the ECM. TIMP-4 has an identical amino acid sequence with TIMP-2 and-ZIN of almost 38% -STIMP-1. In other target cells, TIMP-4 is responsible for modulating activation of 72 kD progestogenase on the cell surface, which indicates an important role as a tissue-specific regulator of ECM remodeling.

Another mechanism for controlling the biological activity of MMP is their physiological activation. It is believed that enzymes from the family of serine and cysteine proteases, such as AP / plasmin and cathepsin B, respectively, are physiological activators of MMP. In the articular cartilage of patients with osteoarthritis, an elevated level of urokinase (UAP) and plasmin was detected.

Despite the fact that several types of cathepsins are found in the joint tissues, cathepsin-B is considered the most likely activator of MMP in cartilage. In the tissues of the human joint, physiological inhibitors of serine and cysteine proteases were detected. The activity of the inhibitor AP-1 (IAP-1), as well as cysteine proteases, is reduced in patients with osteoarthritis. Similarly to MMP / TIMP, it is the imbalance between serine and cysteine proteases and their inhibitors that can explain the increased MMP activity in the articular cartilage of patients with osteoarthritis. In addition, MMPs are able to activate each other. For example, stromelysin-1 activates collagenase-1, collagenase-3 and gelatinase 92 kD; Collagenase-3 activates 92 kD gelatinase; MMP-MT activates collagenase-3, and gelatinase-72 kD potentiates this activation; MMP-MT also activates 72 kD gelatinase. Cytokines can be divided into three groups - destructive (pro-inflammatory), regulatory (including anti-inflammatory) and anabolic (growth factors).

Types of cytokines (according to van den Berg WB et al)

Destructive	Interleukin-1 TNF-a Leukemic Inhibitory Factor Interleukin-17
Regulatory	Interleukin-4 Interleukin-10 Interleukin-13 Enzyme Inhibitors
Anabolic	Msulin-like growth factors TGF-b Bone morphogenetic proteins Morphogenetic proteins derived from cartilage

Destructive cytokines, in particular IL-1, induce an increase in the release of proteases and inhibit the synthesis of proteoglycans and collagens by chondrocytes. Regulatory cytokines, in particular IL-4 and -10, inhibit the production of IL-1, increase the production of IL-1 receptor antagonist (IL-1 RA), and reduce the level of NO-synthase in chondrocytes. Thus, IL-4 counteracts IL-1 in three ways: 1) reduces production, prevents its effects, 2) increases the production of the main "scavenger" IL-1RA and 3) reduces the production of the main secondary "messenger" NO. In addition, IL-4 reduces enzymatic tissue degradation. In vivo conditions, the optimal therapeutic effect is achieved with the combination of IL-4 and IL-10. Anabolic factors such as TGF-p and IGF-1 do not really interfere with the production or action of IL-1, but exhibit the opposite activity, for example, stimulate the synthesis of proteoglycans and collagen, inhibit protease activity, and TGF- (3 also inhibits the release of enzymes and stimulates their inhibitors.

Proinflammatory cytokines are responsible for the increased synthesis and expression of MMP in the joint tissues. They are synthesized in the synovial membrane, and then diffuse into the articular cartilage through the synovial fluid. Proinflammatory cytokines activate chondrocytes, which in turn are also capable of producing pro-inflammatory cytokines. In the joints affected by osteoarthrosis, the role of the effector of inflammation is played mainly by the cells of the synovial membrane. It is the synovitis of the macrophage type that secrete proteases and inflammatory mediators. Among them, in the pathogenesis of osteoarthritis, IL-f, TNF-a, IL-6, leukemic inhibitory factor (LIF) and IL-17 are involved to the greatest extent.

Biologically active substances that stimulate articular cartilage degradation in osteoarthritis

• Interleukin-1
• Interleukin-3
• Interleukin-4
• TNF-a
• Colony-stimulating factors: macrophage (monocytic) and granulocyte-macrophage
• Substance P
• PGE ₂
• Activators of plasminogen (tissue and urokinase types) and plasmin
• Metalloproteases (collagenases, ellastases, stromelysins)
• Cathepsins A and B
• Trilsin
• Bacterial lipopolysaccharides
• Phospholipase Ag

The literature data indicate that IL-ip and, possibly, TNF-a, are the main mediators of the destruction of articular tissues in osteoarthritis. However, it is still not known whether they operate independently of each other or there is a functional hierarchy between them. On models of osteoarthrosis in animals, it has been shown that blockade of IL-1 effectively prevents the destruction of articular cartilage, whereas blockade of TNF-α leads only to a decrease in inflammation in the tissues of the joint. In the synovial membrane, synovial fluid and cartilage of patients, increased concentrations of both cytokines were detected. In chondrocytes they are able to increase the synthesis of not only proteases (mainly MMP and AP), but also minor collagens, for example, I and III types, and also to reduce the synthesis of collagen types II and IX and proteoglycans. These cytokines also stimulate active oxygen species and inflammatory mediators such as PGE ₂. The result of such macromolecular changes in articular cartilage with osteoarthritis is the inefficiency of repair processes, which leads to further degradation of the cartilage.

The above-named pro-inflammatory cytokines modulate the processes of inhibition / activation of MMP in osteoarthritis. For example, an imbalance between the levels of TIMP-1 and MMP in cartilage in osteoarthritis may be mediated by IL-ip, as an in vitro study demonstrated that an increase in IL-1 beta concentration leads to a decrease in TIMP-1 concentration and an increase in MMP synthesis by chondrocytes. Synthesis of AP is also modulated by IL-1beta. Stimulation of articular cartilage chondrocytes in vitro using IL-1 causes a dose-dependent increase in AP synthesis and a sharp decrease in the synthesis of IAP-1. The ability of IL-1 to reduce the synthesis of IAP-1 and stimulate the synthesis of AP is a powerful mechanism for the generation of plasmin and the activation of MMP. In addition, plasmin is not only an enzyme that activates other enzymes, it also takes part in the process of degradation of cartilage by direct proteolysis.

IL-ip is synthesized as an inactive precursor with a mass of 31 kD (pre-IL-ip), and, after cleavage of the signal peptide, is converted to an active cytokine with a mass of 17.5 kDa. In joint tissues, including the synovial membrane, synovial fluid and articular cartilage, IL-ip is found in active form, and in vivo studies the ability of the synovial membrane in osteoarthritis to secrete this cytokine is demonstrated. Some serine proteases are able to convert pre-IL-ip into its bioactive form. In mammals, such properties are found only in one protease, which belongs to the family of cysteine aspartate-specific enzymes and is called IL-1p-converting enzyme (ICP, or caspase-1). This enzyme is able to specifically convert pre-IL-ip into a biologically active "mature" IL-ip with a mass of 17.5 kD. IKF is a proenzyme with a molecular mass of 45 kD (p45), which is localized in the cell membrane. After proteolytic cleavage of the proenzyme p45 to form two subunits, known as p10 and p20, which are characterized by enzymatic activity.

TNF-a is also synthesized as a membrane-bound precursor with a mass of 26 kD; by proteolytic cleavage, it is released from the cell as an active soluble form with a mass of 17 kD. Proteolytic cleavage is carried out by the TNF-a-converting enzyme (TNF-KF), which belongs to the family of adamalysins. AR Amin and co-authors (1997) found increased expression of TNF-CF mRNA in the articular cartilage of patients with osteoarthritis.

The biological activation of chondrocytes and synovitocytes IL-1 and TNF-a is mediated by binding to specific receptors on the surface of cells - IL-R and TNF-R. For each cytokine, two types of receptors have been identified: IL-IP of types I and II and TNF-P I (p55) and II (p75) types. For the transmission of signals in the cells of the joint tissues, IL-1PI and p55 respond. IL-1P type I has a slightly higher affinity for IL-1beta than for IL-1a; IL-1P type II - on the contrary, has a greater affinity for IL-1a than for IL-ip. It remains unclear whether IL-IP II type II can mediate IL-1 signals or it serves only to competitively inhibit IL-1 binding to IL-1PI type. In chondroits and synovial fibroblasts in patients with osteoarthritis, a large number of IL-1PI and p55 are found, which in turn explains the high sensitivity of these cells to stimulation with appropriate cytokines. This process leads both to an increase in the secretion of proteolytic enzymes, and to the destruction of articular cartilage.

It is not excluded the participation of IL-6 in the pathological process in osteoarthritis. This assumption is based on the following observations:

• IL-6 increases the number of inflammatory cells in the synovial membrane,
• IL-6 stimulates the proliferation of chondrocytes,
• IL-6 enhances the effects of IL-1 in increasing the synthesis of MMP and inhibiting the synthesis of proteoglycans.

However, IL-6 is able to induce the production of TIMP, but it does not affect the production of MMP, therefore it is believed that this particular cytokine takes part in deterring proteolytic degradation of articular cartilage, which is performed by the feedback mechanism.

Another representative of the IL-6 family is the LIF-cytokine, which is produced by chondrocytes obtained from patients with osteoarthritis, in response to stimulation of proinflammatory cytokines IL-ip and TNF-a. LIF stimulates the resorption of proteoglycans of cartilage, as well as the synthesis of MMP and NO production. The role of this cytokine in osteoarthritis is not fully understood.

IL-17 is a 20-30 kD homodimer having IL-1-like action, but much less pronounced. IL-17 stimulates the synthesis and isolation of a number of pro-inflammatory cytokines, including IL-ip, TNF-a, IL-6, and MMP in target cells, for example, in human macrophages. In addition, IL-17 stimulates NO production with chondrocytes. Like the LIF, the role of IL-17 in the pathogenesis of OA has been little studied.

Inorganic free radical NO plays an important role in the degradation of articular cartilage with OA. Chondrocytes obtained from patients with osteoarthritis produce more NO as spontaneously as well as after stimulation with pro-inflammatory cytokines in comparison with normal cells. A high NO content is found in the synovial fluid and serum of patients with osteoarthritis - the result of increased expression and synthesis of induced NO synthase (hNOC), an enzyme responsible for NO production. Recently, the DNA of chondrocyte-specific hNOC was cloned, the amino acid sequence of the enzyme was determined. The amino acid sequence indicates a 50% identity and 70% similarity to hNOC specific for the endothelium and neural tissue.

NO inhibits the synthesis of macromolecules of ECM of articular cartilage and stimulates the synthesis of MMP. Moreover, an increase in NO production is accompanied by a decrease in the synthesis of the antagonist IL-IP (IL-1RA) by chondrocytes. Thus, an increase in the level of IL-1 and a decrease in IL-1 RA leads to hyperstimulation of NO of chondrocytes, which in turn leads to increased degradation of the cartilage matrix. There have been reports of a therapeutic effect in vivo of a selective hNOC inhibitor on the progression of experimental osteoarthritis.

Natural cytokine inhibitors can directly inhibit the binding of cytokines to the receptors of cell membranes, reducing their pro-inflammatory activity. Natural inhibitors of cytokines can be divided into three classes according to their mode of action.

The first class of inhibitors include receptor antagonists, which prevent binding of the ligand to its receptor by competition for the binding site. To date, such an inhibitor has been found only for IL-1, the above-mentioned competitive inhibitor of the IL-1 / ILIP IL-1 PA system. IL-1 RA blocks many of the effects that are observed in the joint tissues in osteoarthritis, including synthesis of prostaglandins by synovial cells, production of collagenase by chondrocytes, and degradation of CM in articular cartilage.

IL-1PA is detected in various forms - one soluble (rIL-1PA) and two intercellular (μIL-lPAI and μIL-1APAP). The affinity of the soluble form of IL-1RA is 5 times that of intercellular forms. Despite the intensive scientific search, the function of the latter remains unknown. Experiments in vitro showed that in order to inhibit the activity of IL-1bet, the concentration of IL-1PA, 10-100 times higher than normal, is required, in vivo, a thousandfold increase in the concentration of IL-1PA is required. This fact can partially explain the relative deficiency of IL-1 RA and the excess IL-1 in the synovia of patients with osteoarthritis.

The second class of natural inhibitors of cytokines is represented by soluble receptors of cytokines. An example of such inhibitors in humans related to the pathogenesis of osteoarthrosis are pIL-1P and pp55. Soluble cytokine receptors are truncated forms of normal receptors, binding to cytokines, they interfere with their binding to membrane-associated receptors of target cells, acting by the mechanism of competitive antagonism.

The main precursor of soluble receptors are membrane-bound IL-1PP. The affinity of rIL-IP with respect to IL-1 and IL-1 PA is different. Thus, pIL-1PH has a greater affinity for IL-1p than for IL-1 PA, and pIL-1PI shows a greater affinity for IL-1RA than for IL-ip.

For TNF also there are two types of soluble receptors - pp55 and pp75, like soluble IL-1 receptors, they are formed by "sheeding" (dumping). In vivo, both receptors are found in the tissues of the affected joints. The role of soluble TNF receptors in the pathogenesis of osteoarthritis is debated. It is suggested that in low concentrations they stabilize the three-dimensional structure of TNF and increase the half-life of the bioactive cytokine, while high concentrations of pp55 and pp75 may reduce TNF activity through competitive antagonism. Apparently, pp75 can act as a carrier of TNF, facilitating its binding to the membrane-associated receptor.

The third class of natural inhibitors of cytokines is represented by a group of anti-inflammatory cytokines, which include TGF-beta, IL-4, IL-10 and IL-13. Anti-inflammatory cytokines reduce the production of pro-inflammatory, as well as some proteases, stimulate production of IL-1RA and TIMP.