Role of crystal deposition in the pathogenesis of osteoarthritis

In 30-60% of patients with osteoarthritis, crystals of basic calcium phosphate (OFC) in articular fluid are detected. According to A. Swan and co-authors (1994), calcium-containing crystals are found in the joint fluid in a much larger number of patients with osteoarthritis, but because of the excessively small crystal sizes or small amounts, they are not identified using conventional techniques. The presence of crystals of basic calcium phosphate in the joint fluid correlates with the radiographic signs of articular cartilage degeneration and is associated with a large volume of effusion compared with effusions in knee joints without crystals. The study of factors influencing the radiologic progression of gonarthrosis showed that the deposition of calcium pyrophosphate crystals of dihydrate (PFCD) is a predictor of unfavorable clinical and radiologic outcome. In the study of elderly patients, it was found that osteoarthritis is associated with chondrocalcinosis, especially in the lateral tibiofemoral part of the knee joint and in the first three metacarpophalangeal joints. Often, in patients with osteoarthritis, both types of crystals are detected - OFC and PFCD.

Clinically, the degeneration of articular cartilage, caused by the deposition of calcium-containing crystals, differs from that in primary osteoarthritis. If the crystals were a simple epiphenomenon of cartilage degeneration, they would be found in joints that are most often affected by primary osteoarthritis, i.e. In the knee, hip, small joints of the hands. On the contrary, diseases of deposition of crystals often affect atypical for primary osteoarthrosis joints - shoulder, wrist, ulnar. The presence of crystals in the joint (exudate) fluid is associated with a more severe degeneration of the articular cartilage. The question of what is the cause and what is the consequence is the deposition of crystals or the degeneration of the cartilage. Intermediate position is assumed by the following assumption: the primary anomaly of cartilage metabolism leads to its degeneration, and the secondary deposition of crystals accelerates its degradation (the so-called theory of amplification loop).

The exact mechanism of damage to the articular cartilage with calcium-containing crystals is not known, its individual elements are given below. Theoretically, calcium-containing crystals can directly damage chondrocytes. However, with histological examination, the crystals are rarely localized near the chondrocytes, they are even less often absorbed by them. The most probable is the phagocytosis of the crystals by the cells of the synovial lining, followed by the release of proteolytic enzymes or the secretion of cytokines that stimulate the release of enzymes by chondrocytes. This concept is confirmed by the study of the role of PFCD-induced synovitis in the development of rapidly progressing osteoarthritis in pyrophosphate arthropathy. In this study, rabbits with osteoarthritis, induced by partial lateral meniscetectomy, received calcium pyrophosphate dihydrate (1 or 10 mg) once a week in the right knee joint. It turned out that after 8 injections in the right knee joint there were significantly more serious changes compared to the left one. Intensity of synovial inflammation was correlated with intraarticular injection of calcium pyrophosphate crystals dihydrate and their dose. Despite the fact that the doses of PFCD crystals used in this study exceed those in vivo, the results indicate the role of PFCD-induced inflammation in the progression of osteoarthritis in pyrophosphate arthropathy.

Potential mechanisms of calcium-containing crystal inducing damage to articular cartilage are associated with their mitogenic properties, the ability to induce MMP, and stimulate the synthesis of prostaglandins.

Mitogenic effect of calcium-containing crystals. In crystallosociated arthropathies, proliferation of synovial lining cells is often observed, and the crystals themselves are only partially responsible for this process. An increase in the number of synovial cells is accompanied by an increase in the secretion of cytokines, which promote chondrolysis and cause the secretion of proteolytic enzymes. OCK crystals in concentrations found in the pathology of joints in humans dose-dependently stimulate mitogenesis of resting skin fibroblasts culture, synovial fibroblasts of dogs and mice. Crystals of calcium pyrophosphate dihydrate, urate, sulfate, carbonate and whether calcium phosphate stimulate cell growth. The onset and the inclusion peak ( ³ H) -thymidine, induced by these crystals, are shifted by 3 h compared to stimulation of serum cells. Perhaps, this period of time is necessary for phagocytosis and dissolution of the crystals. The addition of control crystals of the same size (eg, diamond dust or latex particles) did not stimulate mitogenesis. The urate crystals of sodium monohydrate had weak mitogenic properties and were significantly inferior to those of calcium urate, which indicates an important value in mitogenesis of the calcium content in crystals. Synthetic crystals OFC possessed 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 in the cell surrounding the nutrient medium in vitro, since the dissolution of the crystals of basic calcium phosphate in the nutrient medium was not stimulated by the incorporation of ( ³ H) -thymidine fibroblasts.

One of the proposed mechanisms of RPC-induced mitogenesis is the following: the anomalous proliferation of synovial cells can be associated (at least in part) with endocytosis and intracellular dissolution of the crystals, which leads to an increase in Ca ²⁺ concentration in the cytoplasm of cells and activation of the calcium-dependent pathway, leading to mitogenesis. In support of this concept, there is a need for direct cell-crystal contact to stimulate mitogenesis, since exposing the cell culture to crystals caused cell growth, and exposing cells that lacked such contact did not cause their growth. To study the necessity of phagocytosis of the crystals following the cell-crystal interaction, the cells were cultured with ⁴⁵ Ca-OFK and ( ³ H) -thymidine. It turned out that containing ⁴⁵ Ca-OFC cells included a much larger amount ( ³ H) -thymidine than cells without labeled with basic calcium phosphate. In macrophage culture, cytochalasin inhibition of endocytosis in crystals caused inhibition of crystal dissolution, which also emphasizes the need for phagocytosis.

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

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

It has been established that the mitogenic effect of RPC crystals is similar to that of PDGF as a growth factor; like the latter, the OFC crystals show synergy with respect to IGF-1 and blood plasma. The blockade of IGF-1 reduces the mitogenesis of cells in response to the OF. PG Mitchell and co-authors (1989) showed that the induction of Balb / c- ₃ T ₃ fibroblast induction with OFC crystals requires the presence of serine / threonine protein kinase C (PKC), one of the main mediators of signals generated by external stimulation of cells by hormones, neurotransmitters and factors growth. Reduction of PKC activity in Balb / c- ₃ T ₃ cells inhibits the OFK-mediated induction of proto-oncogenes c-fos and c-tus, but does not affect stimulation of these oncogenes mediated by PDGF.

An increase in the content of intracellular calcium after 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 (phospho-diestease) is stimulated, which hydrolyses phosphatidylinositol 4,5-bisphosphate to form intracellular messengers-inositol-3-phosphate and diacylglycerol. The former releases calcium from the endoplasmic reticulum, modulating the activity of calcium-dependent and calcium / calmodulin-dependent enzymes, such as protein kinases and proteases.

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

Diacyl-glycerol is a potential activator of calcium pyrophosphate dihydrate. Since the CRC crystals increase the activity of phospholipase C, which leads to the accumulation of diacylglycerol, one can therefore expect an increase in activation of PKC. PG Mitchell and co-authors (1989) compared the effect of RPC crystals and PDGFs on the synthesis of DNA by Balb / c- ₃ T ₃ fibroblasts . In cell culture, PKC was inactivated by incubation of cells with a tumor-fixing phorbol diester (TFA), an analog of diacylglycerol. Prolonged stimulation with low doses of TFA reduces the activity of PKC, whereas a single stimulation with a high dose activates. The stimulation of DNA synthesis by RPC crystals was suppressed after inactivation of PKC, which indicates the importance of this enzyme in the OFC-induced mitogenesis. Earlier, GM McCarthy and co-authors (1987) demonstrated the connection of the mitogenic response of human fibroblasts to the OFC crystals with activation of PKC. However, the OFC crystals do not activate phosphatidylinositol-3-kinase or tyrosine kinases, confirming the fact that the mechanism of cell activation by OPC crystals is selective.

Cell proliferation is controlled by a group of genes called proto-oncogenes. Proteins foe and thu, products of proto-oncogenes c-fos and c-shus are localized in the cell nucleus and are associated with specific DNA sequences. Stimulation of ZT3-fibroblasts by OCP crystals results in the expression of c-fos for several minutes, which reaches a maximum after 30 min after stimulation. The induction of transcription with -mouse OFC crystals or PDGF occurs within 1 hour and reaches a maximum after 3 hours after stimulation. At least 5 h cells support an increased level of transcription of c-fos and c-myc. In cells with inactivated PKC, stimulation of c-fos and c-muss crystals of RPA or TPD is significantly inhibited, while induction of these PDGF genes does not change.

Representatives of the mitogen-activated protein kinase family (MAP K) are key regulators of various intracellular signaling cascades. One of the subclasses of this family, p42 / p44, regulates the proliferation of cells by a mechanism involving the activation of the proto-oncogenes c-fos and c-jun. The OFC and PFCD crystals activate the protein kinase signaling pathway, in which both p42 and p44 participate, which indicates the role of this pathway in mitogenesis induced by calcium-containing crystals.

Finally, in the OFC-induced mitogenesis, the transcription nuclear factor KB (NF-kB), which was first described as the light chain immunoglobulin to (IgK) gene, is involved. This is an induced transcription factor, important for many signaling pathways, since it regulates the expression of various genes. The induction of NF-kB is usually associated with the release of inhibitory proteins from the cytoplasm, called 1kB. Following the NF-kB inducing, translocation of the active transcription factor to the nucleus occurs. OFC crystals induce NF-kB in Balb / c- ₃ T ₃ fibroblasts and human skin fibroblasts.

Several pathways may be involved in signaling after NF-κB activation, but all of them involve protein kinases that phosphorylate (and thus degrade) 1 kB. Based on the results of in vitro studies, it was previously assumed that 1 kB serves as a substrate for kinases (eg, PKC and protein kinase A). However, a 1 kB kinase complex with a large molecular weight was recently identified. These kinases specifically phosphorylate serine residues of 1 kB. Activation of NF-kV TNF-a and IL-1 requires the effective action of NF-KB-inducing kinase (NIC) and 1KB kinase. The molecular mechanism of NIC activation is not currently known. Despite the fact that OFC crystals activate both PKC and NF-kV, it is not known to what extent these two processes can be connected. Since the modification of the GKB kinase is carried out by phosphorylation, the role of PKC in induction by NF-KB crystal therapy by phosphorylation and activation of the GKB kinase is not ruled out. In support of this concept, inhibition of PKC by staurosporin OFC-crystalline-induced mitogenesis and NF-KB expression may serve as a support for this concept. Similarly, staurosporin can inhibit the GkV kinase, and therefore, inhibits protein kinase A and other protein kinases.

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

• rapid membrane-bound event, which leads to activation of PKC and MAP K, induction of NF-KB and proto-oncogenes,
• slower intracellular dissolution of the 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.

[1], [2], [3]

The induction of MMP-calcium-containing crystals

Mediators of tissue damage with calcium-containing crystals are MMP-collagenase-1, stromelysin, 92 kD gelatinase and collagenase-3.

Taking into account the relationship between the content of OFK crystals and the destruction of joint tissues, a hypothesis has been advanced, according to which crystals of RPA and, possibly, some collagens are phagocytosed by synovial cells. Stimulated synovvits proliferate and secrete proteases. This hypothesis was tested in vitro by the addition of natural or synthetic crystals of RPA, PFCD, and others to the culture of human or dog synovitises. The level of activity of neutral proteases and collagenases increased dose-dependently and was approximately 5-8 times higher than in the control culture of cells that were cultured without crystals.

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

OFC crystals also caused the accumulation of collagenase-1 mRNA and collagenase-2 in mature porcine chondrocytes with subsequent secretion of enzymes into the medium.

GM McCarty and co-authors (1998) studied the role of intracellular dissolution of crystals in the crystallized production of MMP. The increase in lysosomal pH with bafilomycin A, inhibited the intracellular dissolution of the crystals, and also weakened the proliferative response of human fibroblasts to OFC crystals, but did not inhibit the synthesis and secretion of MMP.

Neither crystals of basic calcium phosphate nor PFCD did not induce production of IL-1 in vitro, in contrast to sodium urate crystals.

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

Symptoms of osteoarthritis testify to the significant role of MMP in the progression of the disease. The presence of calcium-containing crystals enhances the degeneration of the tissues of the affected joints.

Stimulation of prostaglandin synthesis

Along with stimulation of cell growth, secretion of enzymes, calcium-containing crystals cause the release of prostaglandins from mammalian cell cultures, especially PGE ₂. The release of PGE- ₂ in all cases occurs within the first hour after exposure of the cells to the crystals. R. Rothenberg (l 987) determined that the main sources of arachidonic acid for the synthesis of PGE ₂ are phosphatidylcholine and phosphatidylethanolamine, and also confirmed that phospholipase A ₂ and NOH are the dominant pathways of PGE ₂ production .

In response to the effect of crystals of CPC, PGE1 can also be released. GM McCarty and co-authors ( 1993, 1994 ) studied the effect of PGE ₂, PGE, and its analogue misoprostol on the mitogenic response of human fibroblasts to OFC crystals. All three agents inhibited the mitogenic response in a dose-dependent manner, with PGE and misoprostal showing more pronounced inhibitory activity. PGE, and misoprostol, but not PGE _2, inhibited the accumulation of collagenase mRNA in response to the effect of OFC crystals.

MG McCarty and N. Cheung (1994) investigated the mechanism of RPC-mediated activation of PGE cells. The authors showed that PGE, a more powerful inductor of intracellular cAMP than PGE- ₂ and PGE, inhibits the OFC-induced mitogenesis and MMP production through the cAMP-dependent signaling pathway. It is possible that an increase in PGE production induced by OFK crystals weakens their other biological effects (mitogenesis and MMP production) by the feedback mechanism.

Inflammation induced by crystals

Calcium-containing crystals are often found in the synovial fluid in patients with osteoarthritis, but episodes of acute inflammation with leukocytosis are rare in osteoarthritis and in crystal-associated arthropathies (for example, the "shoulder joint of Milwaukee" syndrome). The phlogistic potential of the crystals can be modified by a number of inhibitory factors. R. Terkeltaub et al (1988) demonstrated the ability of serum and blood plasma to significantly inhibit the response of neutrophilic granulocytes to crystals of basic calcium phosphate. The factors that cause such inhibition are crystal-binding proteins. The study of one such protein - a _2- HS glycoprotein (AHSr) - showed that ANSG is the most potent and specific inhibitor of neutrophil granulocyte response to OF crystals. AHSr - whey protein of hepatic origin; It is known that in comparison with other proteins of blood serum it is in relatively high concentrations contained in bone and mineralized tissue. In addition, AHSr is present in the "non-inflamed" synovial fluid, and is also found on the crystals of basic calcium phosphate in the native synovial fluid. Thus, the probability of AHSr modulation of the phlogogenic potential of basic calcium phosphate crystals under in vivo conditions is not excluded .

Summarizing all of the above, we present two schemes of the pathogenesis of osteoarthritis, proposed by WB van den Berg and co-authors (1999) and M. Sarrabba and co-authors (1996), which combine mechanical, genetic and biochemical factors.