Role of enzymes and cytokines in the pathogenesis of osteoarthritis

In recent years, much attention of researchers has been focused on identifying proteases responsible for the degradation of the articular cartilage ECM in osteoarthrosis. According to modern concepts, matrix metalloproteases (MMPs) play an important role in the pathogenesis of osteoarthrosis. In patients with osteoarthrosis, an increased level of three MMPs is detected - collagenases, stromelysins and gelatinases. Collagenase is responsible for the degradation of native collagen, stromelysin - type IV collagen, proteoglycans and laminin, gelatinase - for the degradation of gelatin, collagens 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, is assumed.

Three types of collagenases have been identified in human articular cartilage, the levels of which are significantly elevated 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 articular cartilage suggests that each of them plays its own specific role. Indeed, collagenases-1 and -2 are localized mainly in the superficial and upper intermediate zone of articular cartilage, while collagenase-3 is found in the lower intermediate zone and in the deep zone. Moreover, the results of immunohistochemical study demonstrated that as osteoarthritis progresses, the level of collagenase-3 reaches a plateau and even decreases, while the level of collagenase-1 gradually increases. There is evidence that in osteoarthritis, collagenase-1 is mainly involved in the inflammatory process in articular cartilage, while collagenase-3 is involved in tissue remodeling. Collagenase-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, three have also been identified in human stromelysin: stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11). Today, it is known that only stromelysin-1 is involved in the pathological process in osteoarthrosis. Stromelysin-2 is not detected in the synovial membrane of patients with osteoarthrosis, but it is found in very small quantities in the synovial fibroblasts of patients with rheumatoid arthritis. Stromelysin-3 is also found in the synovial membrane of patients with rheumatoid arthritis near fibroblasts, especially in fibrosis zones.

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

Recently, another group of MMPs has been identified that are localized on the surface of cell membranes and are called membrane-type MMPs (MMP-MT). This group includes four enzymes - MMP-MT1 - MMP-MT-4. MMP-MT expression has been found in human articular cartilage. Although MMP-MT-1 has collagenase properties, both enzymes MMP-MT-1 and MMP-MT-2 are capable of activating gelatinase-72 kDa and collagenase-3. The role of this group of MMPs in the pathogenesis of OA requires clarification.

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

The biological activity of MMPs is controlled by specific TIMPs. To date, three types of TIMPs have been identified that are found in human articular tissues: TIMP-1–TIMP-3. A fourth type of TIMP has been identified and cloned, but it has not yet been detected in human articular tissues. These molecules specifically bind to the active site of MMPs, although some of them are able to bind the active site of 72 kD progelatinase (TIMP-2, -3, -4) and 92 kD progelatinase (TIMP-1 and -3). Evidence suggests that in OA, there is an imbalance between MMPs and TIMPs in articular cartilage, resulting in a relative deficiency of inhibitors, possibly due in part to an increase in the level of active MMPs in the tissue. TIMP-1 and -2 are found in articular cartilage and are synthesized by chondrocytes. In osteoarthrosis, only type I TIMP is detected in the synovial membrane and synovial fluid. TIMP-3 is found exclusively in the ECM. TIMP-4 shares almost 50% of its amino acid sequence with TIMP-2 and 38% with TIMP-1. In other target cells, TIMP-4 is responsible for modulating the activation of 72 kD progelatinase on the cell surface, indicating an important role as a tissue-specific regulator of ECM remodeling.

Another mechanism for controlling the biological activity of MMPs is their physiological activation. It is believed that enzymes from the serine and cysteine protease family, such as AP/plasmin and cathepsin B, respectively, are physiological activators of MMPs. Increased levels of urokinase (uAP) and plasmin have been found in the articular cartilage of patients with osteoarthritis.

Despite the fact that several types of cathepsins are found in joint tissues, cathepsin-B is considered the most probable activator of MMPs in cartilage. Physiological inhibitors of serine and cysteine proteases have been found in human joint tissues. The activity of the AP-1 inhibitor (IAI-1), as well as cysteine proteases, is reduced in patients with osteoarthritis. Similar to MMP/TIMP, it is the imbalance between serine and cysteine proteases and their inhibitors that can explain the increased activity of MMPs 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 92 kD gelatinase; collagenase-3 activates 92 kD gelatinase; MMP-MT activates collagenase-3, and gelatinase-72 kDa potentiates this activation; MMP-MT also activates gelatinase 72 kDa. Cytokines can be divided into three groups - destructive (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 Leukemia inhibitory factor Interleukin-17
Regulatory	Interleukin-4 Interleukin-10 Interleukin-13 Enzyme inhibitors
Anabolic	Mnsulin-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 IL-1 production, increase the production of the IL-1 receptor antagonist (IL-1RA) and reduce the level of NO synthase in chondrocytes. Thus, IL-4 counteracts IL-1 in three directions: 1) reduces production, preventing its effects, 2) increases production of the main "scavenger" IL-1RA and 3) reduces production of the main secondary "messenger" NO. In addition, IL-4 reduces enzymatic degradation of tissue. In vivo, the optimal therapeutic effect is achieved with a combination of IL-4 and IL-10. Anabolic factors such as TGF-β and IGF-1 do not actually interfere with the production or action of IL-1, but exhibit the opposite activity, for example, stimulating the synthesis of proteoglycans and collagen, suppressing the activity of proteases, and TGF-β also inhibits the release of enzymes and stimulates their inhibitors.

Proinflammatory cytokines are responsible for increased synthesis and expression of MMPs in articular 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 proinflammatory cytokines. In joints affected by osteoarthrosis, the role of the effector of inflammation is played mainly by cells of the synovial membrane. It is the synovocytes of the macrophage type that secrete proteases and inflammatory mediators. Among them, IL-f, TNF-a, IL-6, leukemia inhibitory factor (LIF) and IL-17 are most “involved” in the pathogenesis of osteoarthrosis.

Biologically active substances that stimulate the degradation of articular cartilage in osteoarthritis

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

Literature data indicate that IL-1 and, possibly, TNF-a are the main mediators of joint tissue destruction in osteoarthrosis. However, it is still unknown whether they act independently of each other or whether there is a functional hierarchy between them. Animal models of osteoarthrosis have shown that IL-1 blockade effectively prevents articular cartilage destruction, while TNF-a blockade only leads to a decrease in inflammation in joint tissues. Increased concentrations of both cytokines were found in the synovial membrane, synovial fluid, and cartilage of patients. In chondrocytes, they are able to increase the synthesis of not only proteases (mainly MMP and AP), but also minor collagens, such as types I and III, and reduce the synthesis of collagens types II and IX and proteoglycans. These cytokines also stimulate reactive oxygen species and inflammatory mediators such as PGE ₂. The result of such macromolecular changes in articular cartilage in osteoarthritis is the ineffectiveness of reparative processes, which leads to further degradation of cartilage.

The above mentioned proinflammatory cytokines modulate the processes of MMP suppression/activation in osteoarthrosis. For example, the imbalance between TIMP-1 and MMP levels in cartilage in osteoarthrosis may be mediated by IL-1, since an in vitro study demonstrated that an increase in IL-1 beta concentrations leads to a decrease in TIMP-1 concentrations and an increase in MMP synthesis by chondrocytes. AP synthesis is also modulated by IL-1 beta. In vitro stimulation of articular cartilage chondrocytes with IL-1 causes a dose-dependent increase in AP synthesis and a sharp decrease in iAP-1 synthesis. The ability of IL-1 to decrease iAP-1 synthesis and stimulate AP synthesis is a potent mechanism for plasmin generation and MMP activation. In addition, plasmin is not only an enzyme that activates other enzymes, it also participates in the process of cartilage degradation by direct proteolysis.

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

TNF-a is also synthesized as a membrane-bound precursor with a mass of 26 kDa; by proteolytic cleavage it is released from the cell as an active soluble form with a mass of 17 kDa. Proteolytic cleavage is carried out by TNF-a-converting enzyme (TNF-AC), which belongs to the adamalizin family. A. R. Amin et al. (1997) found increased expression of TNF-AC mRNA in the articular cartilage of patients with osteoarthritis.

Biological activation of chondrocytes and synovocytes by IL-1 and TNF-a is mediated by binding to specific receptors on the cell surface - IL-R and TNF-R. Two types of receptors have been identified for each cytokine - IL-IP types I and II and TNF-R types I (p55) and II (p75). IL-1PI and p55 are responsible for signal transmission in joint tissue cells. IL-1R type I has a slightly higher affinity for IL-1beta than for IL-1a; IL-1R type II, on the contrary, has a higher affinity for IL-1a than for IL- ip. It remains unclear whether IL-IP type II can mediate IL-1 signals or it serves only for competitive inhibition of the association of IL-1 with IL-1R type I. Chondroitides and synovial fibroblasts of patients with osteoarthrosis contain large amounts of IL-1PI and p55, which in turn explains the high sensitivity of these cells to stimulation by the corresponding cytokines. This process leads to both increased secretion of proteolytic enzymes and destruction of articular cartilage.

The involvement of IL-6 in the pathological process in osteoarthritis cannot be ruled out. This assumption is based on the following observations:

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

However, IL-6 is capable of inducing the production of TIMPs, but does not affect the production of MMPs, so it is believed that this cytokine is involved in the process of inhibiting the proteolytic degradation of articular cartilage, which is carried out by a feedback mechanism.

Another member of the IL-6 family is LIF, a cytokine produced by chondrocytes obtained from patients with osteoarthrosis in response to stimulation by the proinflammatory cytokines IL-1p and TNF-a. LIF stimulates cartilage proteoglycan resorption, as well as MMP synthesis and NO production. The role of this cytokine in osteoarthrosis has not been fully elucidated.

IL-17 is a 20-30 kD homodimer with an IL-1-like effect, but much less pronounced. IL-17 stimulates the synthesis and release of a number of proinflammatory cytokines, including IL-1p, TNF-a, IL-6, and MMP in target cells, such as human macrophages. In addition, IL-17 stimulates NO production by chondrocytes. Like LIF, the role of IL-17 in the pathogenesis of OA has been poorly studied.

The inorganic free radical NO plays an important role in the degradation of articular cartilage in OA. Chondrocytes isolated from patients with osteoarthritis produce higher amounts of NO both spontaneously and after stimulation with proinflammatory cytokines compared to normal cells. High NO content has been found in synovial fluid and serum of patients with osteoarthritis - this is a result of increased expression and synthesis of induced NO synthase (hNOC), the enzyme responsible for NO production. Recently, the DNA of chondrocyte-specific hNOC was cloned, and the amino acid sequence of the enzyme was determined. The amino acid sequence indicates 50% identity and 70% similarity with hNOC specific for endothelium and nervous tissue.

NO inhibits the synthesis of macromolecules of the 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 IL-IP antagonist (IL-1RA) by chondrocytes. Thus, an increase in the level of IL-1 and a decrease in IL-1RA leads to hyperstimulation of NO in chondrocytes, which in turn leads to increased degradation of the cartilage matrix. There are reports on the therapeutic effect in vivo of a selective hNOC inhibitor on the progression of experimental osteoarthrosis.

Natural cytokine inhibitors are able to directly prevent cytokines from binding to cell membrane receptors, reducing their proinflammatory activity. Natural cytokine inhibitors can be divided into three classes based on their mode of action.

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

IL-1RA is found in different forms - one soluble (rIL-1RA) and two intercellular (μIL-lPAI and μIL-1RAP). The affinity of the soluble form of IL-1RA is 5 times higher than that of the intercellular forms. Despite intensive scientific research, the function of the latter remains unknown. In vitro experiments have shown that inhibition of IL-1beta activity requires a concentration of IL-1RA 10-100 times higher than normal, while in vivo conditions require a thousandfold increase in IL-1RA concentration. This fact may partially explain the relative deficiency of IL-1RA and excess of IL-1 in the synovium of patients with osteoarthrosis.

The second class of natural cytokine inhibitors are soluble cytokine receptors. Examples of such inhibitors in humans that are related to the pathogenesis of osteoarthritis are rIL-1R and pp55. Soluble cytokine receptors are shortened forms of normal receptors; when they bind to cytokines, they prevent their binding to membrane-associated receptors of target cells, acting by the mechanism of competitive antagonism.

The main precursor of soluble receptors is membrane-bound IL-1RP. The affinity of rIL-IP for IL-1 and IL-1RA is different. Thus, rIL-1RN has a higher affinity for IL-1β than for IL-1RA, and rIL-1PI exhibits a higher affinity for IL-1RA than for IL-ip.

There are also two types of soluble receptors for TNF - pp55 and pp75, like soluble IL-1 receptors, they are formed by "shedding". In vivo, both receptors are found in the tissues of affected joints. The role of soluble TNF receptors in the pathogenesis of osteoarthrosis is debated. It is assumed 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 can reduce TNF activity by competitive antagonism. Probably, pp75 can act as a TNF carrier, facilitating its binding to the membrane-associated receptor.

The third class of natural cytokine inhibitors 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 and some proteases, and stimulate the production of IL-1RA and TIMP.