Role of crystal deposits in the pathogenesis of osteoarthritis

Basic calcium phosphate (BCP) crystals are found in the synovial fluid of 30-60% of patients with osteoarthritis. According to A. Swan et al. (1994), calcium-containing crystals are found in the synovial fluid of a much larger number of patients with osteoarthritis; however, due to the extremely small size of the crystals or their small number, they are not identified using conventional techniques. The presence of BCP crystals in the synovial fluid correlates with radiographic signs of articular cartilage degeneration and is associated with a larger volume of effusion compared to effusion in knee joints without crystals. A study of factors influencing the radiographic progression of gonarthrosis showed that the deposition of calcium pyrophosphate dihydrate (CPPD) crystals is a predictor of an unfavorable clinical and radiographic outcome. In a study of elderly patients, osteoarthritis was found to be associated with chondrocalcinosis, particularly in the lateral tibiofemoral compartment of the knee and the first three metacarpophalangeal joints. It is not uncommon for both types of crystals, OFC and PFC, to be found in patients with osteoarthritis.

Clinically, articular cartilage degeneration caused by calcium crystal deposition differs from that seen in primary osteoarthrosis. If crystals were a simple epiphenomenon of cartilage degeneration, they would be found in the joints most often affected by primary osteoarthrosis, i.e., the knees, hips, and small joints of the hands. In contrast, crystal deposition diseases most often affect joints that are not typical for primary osteoarthrosis, such as the shoulder, wrist, and elbow. The presence of crystals in the joint (effusion) fluid is associated with more severe articular cartilage degeneration. The question of which is the cause and which is the effect, crystal deposition or cartilage degeneration, is debated. An intermediate position is occupied by the following assumption: a primary anomaly in cartilage metabolism leads to its degeneration, and secondary deposition of crystals accelerates its degradation (the so-called amplification loop theory).

The exact mechanism by which calcium crystals damage articular cartilage is unknown is summarized below. Theoretically, calcium crystals may directly damage chondrocytes. However, histological examination rarely reveals crystals near chondrocytes, and even more rarely are they ingested by them. The most likely mechanism is phagocytosis of crystals by synovial lining cells, followed by release of proteolytic enzymes or secretion of cytokines that stimulate chondrocyte release of enzymes. This concept is supported by a study of the role of PFKD-induced synovitis in the development of rapidly progressive osteoarthritis in pyrophosphate arthropathy. In this study, calcium pyrophosphate dihydrate crystals (1 or 10 mg) were injected weekly into the right knee of rabbits with osteoarthritis induced by partial lateral meniscectomy. It turned out that after 8 injections, the right knee joint showed significantly more serious changes compared to the left. The intensity of synovial inflammation correlated with intra-articular injections of calcium pyrophosphate dihydrate crystals and their dose. Despite the fact that the doses of CPPD crystals used in this study exceed those in vivo, the results indicate the role of CPPD-induced inflammation in the progression of osteoarthritis in pyrophosphate arthropathy.

Potential mechanisms of induction of articular cartilage damage by calcium-containing crystals are associated with their mitogenic properties, the ability to induce MMPs and stimulate prostaglandin synthesis.

Mitogenic effect of calcium-containing crystals. In crystal-associated arthropathies, proliferation of synovial lining cells is frequently observed, with the crystals themselves being only partially responsible for this process. The increase in the number of synovial cells is accompanied by increased secretion of cytokines, which promote chondrolysis and induce the secretion of proteolytic enzymes. OFC crystals at concentrations found in human joint pathology dose-dependently stimulate mitogenesis of resting skin fibroblast cultures and canine and mouse synovial fibroblasts. Crystals of calcium pyrophosphate dihydrate, urate, sulfate, carbonate, and calcium phosphate stimulate cell growth. The onset and peak of ( ^3H )-thymidine incorporation induced by these crystals are shifted by 3 h compared to stimulation of cells with blood serum. This period of time may be necessary for phagocytosis and dissolution of crystals. Addition of control crystals of the same size (e.g., diamond dust or latex particles) did not stimulate mitogenesis. Sodium urate monohydrate crystals had weak mitogenic properties and were significantly inferior to those of calcium urate, indicating the importance of the calcium content of the crystals in mitogenesis. Synthetic OFC crystals had the same mitogenic properties as crystals obtained from patients with chondrocalcinosis. The mitogenic effect of calcium-containing crystals was not the result of an increase in the calcium content of the surrounding nutrient medium in vitro, since dissolution of basic calcium phosphate crystals in the nutrient medium did not stimulate the incorporation of ( ^3H )-thymidine by fibroblasts.

One proposed mechanism for OFC-induced mitogenesis is that abnormal synovial cell proliferation may be due, at least in part, to endocytosis and intracellular dissolution of crystals, which increases cytoplasmic Ca ²⁺ concentrations and activates the calcium-dependent pathway leading to mitogenesis. This concept is supported by the requirement for direct cell-crystal contact to stimulate mitogenesis, since exposure of cell cultures to crystals induced cell growth, while exposure of cells deprived of such contact did not. To study the requirement for crystal phagocytosis following cell-crystal interaction, cells were cultured with ⁴⁵ Ca-OPC and ( ³ H)-thymidine. It was found that cells containing ⁴⁵ Ca-OPC incorporated significantly more ( ³ H)-thymidine than cells without basic calcium phosphate labeling. In macrophage cultures, inhibition of crystal endocytosis by cytochalasin resulted in inhibition of crystal dissolution, further highlighting the necessity of phagocytosis.

Calcium-containing crystals are soluble in acid. After phagocytosis, crystals dissolve in the acidic environment of macrophage phagolysosomes. Chloroquine, ammonium chloride, bafilomycin A1, and all lysosomotrophic agents that increase lysosomal pH dose-dependently inhibit intracellular crystal dissolution and (3H)-thymidine uptake ⁱⁿ fibroblasts cultured with basic calcium phosphate crystals.

Addition of OFC crystals to a monolayer fibroblast culture caused an immediate tenfold increase in intracellular calcium, which returned to baseline after 8 min. The source of calcium was predominantly extracellular ion, since the basic calcium phosphate crystals were added to a calcium-free culture medium. The next increase in intracellular calcium concentration was observed after 60 min and lasted for at least 3 h. Here, the source of calcium was phagocytosed crystals dissolved in phagolysosomes.

It was found that the mitogenic effect of OFC crystals is similar to that of PDGF as a growth factor; like the latter, OFC crystals exhibit synergism with IGF-1 and blood plasma. Blockade of IGF-1 reduces cell mitogenesis in response to OFC. PG Mitchell et al. (1989) showed that induction of mitogenesis in Balb/c- ₃ T3 fibroblasts _by OFC crystals requires the presence of serine/threonine protein kinase C (PKC), one of the main mediators of signals generated during external stimulation of cells with hormones, neurotransmitters and growth factors. A decrease in PKC activity in Balb/c-3 T3 cells _inhibitsOFC - mediated induction of the proto-oncogenes c-fos and c-myc, but does not affect the stimulation of these oncogenes mediated by PDGF.

The increase in intracellular calcium following dissolution of phagocytized crystals is not the only signaling pathway for mitogenesis. When growth factors such as PDGF bind to their membrane receptor, phospholipase C (a phosphodiesterase) is stimulated, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to form the intracellular messengers inositol-3-phosphate and diacylglycerol. The former releases calcium from the endoplasmic reticulum by modulating the activity of calcium-dependent and calcium/calmodulin-dependent enzymes such as protein kinases and proteases.

R. Rothenberg and H. Cheung (1988) reported increased degradation of phosphatidylinositol 4,5-bisphosphate by phospholipase C in rabbit synovial cells in response to stimulation with OFC crystals. The latter significantly increased the content of inositol-1-phosphate in cells with labeled ( ^3H )-inositol; the peak was reached within 1 min and persisted for about 1 h.

Diacylglycerol is a potential activator of calcium pyrophosphate dihydrate. Since OFC crystals increase phospholipase C activity, which leads to accumulation of diacylglycerol, consequently, one can expect an increase in PKC activation. PG Mitchell et al. (1989) compared the effects of OFC crystals and PDGF on DNA synthesis by Balb/c- _{3T3fibroblasts}. In cell culture, PKC was inactivated by incubation of cells with tumor-supporting phorbol diester (TPD), a diacylglycerol analog. Long-term stimulation with low doses of TPD decreased PKC activity, whereas a single stimulation with a high dose activated it. Stimulation of DNA synthesis by OFC crystals was suppressed after PKC inactivation, indicating the importance of this enzyme in OFC-induced mitogenesis. Previously, GM McCarthy et al. (1987) demonstrated a link between the mitogenic response of human fibroblasts to OFC crystals and PKC activation. However, OFC crystals do not activate phosphatidylinositol 3-kinase or tyrosine kinases, confirming that the mechanism of cell activation by OFC crystals is selective.

Cell proliferation is controlled by a group of genes called proto-oncogenes. The proteins foe and mye, products of the proto-oncogenes c-fos and c-myc, are localized in the cell nucleus and bound to specific DNA sequences. Stimulation of 3T3 fibroblasts with OFC crystals results in c-fos expression within a few minutes, which reaches a maximum 30 min after stimulation. Induction of c-myc transcription by OFC crystals or PDGF occurs within 1 h and reaches a maximum 3 h after stimulation. The cells maintain an elevated level of c-fos and c-myc transcription for at least 5 h. In cells with inactivated PCD, stimulation of c-fos and c-myc by OFC or TFD crystals is significantly suppressed, while the induction of these genes by PDGF does not change.

Members of the mitogen-activated protein kinase (MAP K) family are key regulators of various intracellular signaling cascades. One subclass of this family, p42/p44, regulates cell proliferation through a mechanism that involves activation of the proto-oncogenes c-fos and c-jun. OFC and PFKD crystals activate a protein kinase signaling pathway that involves both p42 and p44, suggesting a role for this pathway in calcium-containing crystal-induced mitogenesis.

Finally, OFC-induced mitogenesis involves the transcription factor nuclear factor κB (NF-κB), which was first described as the immunoglobulin κ light chain (IgK) gene. It is an inducible transcription factor important in many signaling pathways because it regulates the expression of various genes. NF-κB induction is usually coupled with the release of inhibitory proteins called IκB from the cytoplasm. NF-κB induction is followed by translocation of the active transcription factor to the nucleus. OFC crystals induce NF-κB in Balb/c- _3T3 fibroblasts and human skin fibroblasts.

Several pathways may be involved in signal transduction following NF-κB activation, but all involve protein kinases that phosphorylate (and thus degrade) IκB. Based on in vitro studies, IκB was previously thought to serve as a substrate for kinases (e.g., PKC and protein kinase A). However, a large molecular weight IκB kinase complex has recently been identified. These kinases specifically phosphorylate serine residues of IκB. NF-κB activation by TNF-α and IL-1 requires efficient action of NF-κB-inducing kinase (NIK) and IκB kinase. The molecular mechanism of NIK activation is currently unknown. Although OFC crystals activate both PKC and NF-κB, the extent to which these two processes may be linked is unknown. Since GκB kinase modification occurs via phosphorylation, a role for PKC in the induction of NF-κB by OFC crystals via phosphorylation and activation of GκB kinase cannot be ruled out. This concept is supported by the inhibition of OFC crystal-induced mitogenesis and NF-κB expression by the PKC inhibitor staurosporine. Similarly, staurosporine can inhibit GκB kinase, and thus inhibits protein kinase A and other protein kinases.

Thus, the mechanism of OFC-crystal-induced mitogenesis in fibroblasts includes at least two different processes:

• a rapid membrane-bound event that results in activation of PKC and MAP K, induction of NF-κB and proto-oncogenes,
• slower intracellular dissolution of crystals, which leads to an increase in the intracellular content of Ca ²⁺, and then to the activation of a number of calcium-dependent processes that stimulate mitogenesis.

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Induction by MMP-calcium-containing crystals

The mediators of tissue damage by calcium-containing crystals are MMPs - collagenase-1, stromelysin, 92 kD gelatinase and collagenase-3.

Given the relationship between OFC crystal content and joint tissue destruction, a hypothesis was advanced that OFC crystals and possibly some collagens are phagocytosed by synovial cells. Stimulated synovocytes proliferate and secrete proteases. This hypothesis was tested in vitro by adding natural or synthetic OFC, PFCD, and other crystals to cultured human or canine synovocytes. The activity of neutral proteases and collagenases increased dose-dependently and was approximately 5-8 times higher than that of the control cell culture grown without crystals.

In cells cultured in a crystal-containing medium, co-induction of collagenase-1, stromelysin, and gelatinase-92 kDa mRNA was detected, followed by secretion of enzymes into the medium.

OFC crystals also induced the accumulation of collagenase-1 and collagenase-2 mRNA in mature porcine chondrocytes, followed by secretion of the enzymes into the medium.

GM McCarty et al. (1998) studied the role of intracellular crystal dissolution in crystal-induced MMP production. Elevation of lysosomal pH with bafilomycin A inhibited intracellular crystal dissolution and also attenuated the proliferative response of human fibroblasts to OFC crystals, but did not inhibit MMP synthesis and secretion.

Neither basic calcium phosphate nor PFCD crystals induced IL-1 production in vitro, but sodium urate crystals did.

Current data clearly indicate direct stimulation of MMP production by fibroblasts and chondrocytes upon contact with calcium-containing crystals.

Symptoms of osteoarthritis indicate a significant role of MMP in the progression of the disease. The presence of calcium-containing crystals increases the degeneration of tissues of the affected joints.

Stimulation of prostaglandin synthesis

Along with stimulation of cell growth and secretion of enzymes, calcium-containing crystals cause the release of prostaglandins from mammalian cell cultures, especially PGE2 _. The release of PGE2 _in all cases occurs within the first hour after exposure of cells to crystals. R. Rothenberg (1987) determined that the main sources of arachidonic acid for the synthesis of PGE2 _are phosphatidylcholine and phosphatidylethanolamine, and also confirmed that phospholipase A2 _and NOX are the dominant pathways for PGE2 _production.

PGE1 can also be released in response to OFA crystals. GM McCarty et al. (1993, 1994) studied the effects of PGE2 _, PGE, and its analog misoprostol on the mitogenic response of human fibroblasts to OFA crystals. All three agents inhibited the mitogenic response in a dose-dependent manner, with PGE and misoprostol exhibiting more pronounced inhibitory activity. PGE2 and misoprostol, but not PGE2 _, inhibited the accumulation of collagenase mRNA in response to OFA crystals.

M. G. McCarty and H. Cheung (1994) investigated the mechanism of OFC-mediated activation of cells by PGE. The authors showed that PGE, a more powerful inducer of intracellular cAMP than PGE2 _, and PGE, inhibits OFC-induced mitogenesis and MMP production via a cAMP-dependent signal transduction pathway. It is possible that the increase in PGE production induced by OFC crystals weakens their other biological effects (mitogenesis and MMP production) via a feedback mechanism.

Crystal induced inflammation

Calcium-containing crystals are often found in the synovial fluid of patients with osteoarthrosis, however, episodes of acute inflammation with leukocytosis are rare both in osteoarthrosis and in crystal-associated arthropathies (for example, Milwaukee shoulder syndrome). The phlogistic potential of crystals can be modified by a number of inhibitory factors. R. Terkeltaub et al. (1988) demonstrated the ability of blood serum and plasma to significantly inhibit the response of neutrophilic granulocytes to basic calcium phosphate crystals. The factors that cause such inhibition are crystal-binding proteins. A study of one of these proteins, a ₂ -HS glycoprotein (AHSr), showed that AHSР is the most potent and specific inhibitor of the response of neutrophilic granulocytes to OFC crystals. AHSr is a serum protein of liver origin; It is known that, compared with other serum proteins, it is found in relatively high concentrations in bone and mineralizing tissue. In addition, AHSr is present in "non-inflamed" synovial fluid and has also been detected on basic calcium phosphate crystals in native synovial fluid. Thus, the possibility of AHSr modulating the phlogogenic potential of basic calcium phosphate crystals in vivo cannot be ruled out.

To summarize all of the above, we present two schemes of osteoarthritis pathogenesis proposed by WB van den Berg et al. (1999) and M. Carrabba et al. (1996), which combine mechanical, genetic and biochemical factors.