**5. Exoglycosidases activity in cell cultured synoviocytes**

The patterns of compartmental distribution of exoglycosidases activity in cultured synovial cells of RA and JIA patients are presented in Fig. 6.

The activity of HEX (total), HEX A, and Glu A in the intracellular compartment in cultured synoviocytes of RA and JIA patients is over 3-fold higher than in the extracellular compartment. In contrast, no activity of GAL, MAN and FUC in the extracellular compartments of these cultured synoviocytes was detectable. It is most likely that these exoglycosidases were released extracellularly in small quantities, i.e. below the limits of detection of our colorimetric procedure. Shikhman et al. (2000) have demonstrated similar results in cultured human articular chondrocytes. The high level of activity of exoglycosidases in the intracellular compartment of cultured synoviocytes indirectly confirms the suggestion that the degradation of glycosaminoglycans predominantly takes place in the intracellular compartment. However, Woynarowska et al. (1992) reported data indicating the contribution of extracellular HEX activity in glycosaminoglycan degradation.

The high activity of exoglycosidases in synovial tissue of RA and JIA patients (Popko et al.2006) suggest the value of synovectomy in treatment of rheumatoid diseases (Fig. 5).

We recommend the usefulness of synovectomy in patients with RA and JIA, as the removal of diseased synovial tissue (Fig. 5) could slow down the destructive process of the joint

The patterns of compartmental distribution of exoglycosidases activity in cultured synovial

The activity of HEX (total), HEX A, and Glu A in the intracellular compartment in cultured synoviocytes of RA and JIA patients is over 3-fold higher than in the extracellular compartment. In contrast, no activity of GAL, MAN and FUC in the extracellular compartments of these cultured synoviocytes was detectable. It is most likely that these exoglycosidases were released extracellularly in small quantities, i.e. below the limits of detection of our colorimetric procedure. Shikhman et al. (2000) have demonstrated similar results in cultured human articular chondrocytes. The high level of activity of exoglycosidases in the intracellular compartment of cultured synoviocytes indirectly confirms the suggestion that the degradation of glycosaminoglycans predominantly takes place in the intracellular compartment. However, Woynarowska et al. (1992) reported data indicating the contribution of extracellular HEX activity in glycosaminoglycan degradation.

Fig. 5. Arthroscopic synovectomy a knee of patient with JIA.

cells of RA and JIA patients are presented in Fig. 6.

cartilage, and allow regeneration of a normal synovial membrane.

**5. Exoglycosidases activity in cell cultured synoviocytes** 

Fig. 6. Exoglycosidases activity of RA/JIA synovial fibroblast in the intra (In) / extracellular (Ex) compartment, expressed as pKat/3-5x105 cells.

IL-1 and TNF-α (tumor necrosis factor-α) are key proinflammatory cytokines whose concentration significantly increases in rheumatoid synovial fluid and joint tissues. We have established a profile of exoglycosidases in cell cultures stimulated by IL-1β of inflamed (RA, JIA), and post-injury human synoviocytes (Popko et al. 2008) (Fig. 7). Stimulation by IL-1β cultured synoviocytes taken from patients with ACL, JIA and RA causes much higher increases in activity of HEX and HEX A than remaining exoglycosidases (Fig. 7). On Fig. 7 one can see that the increase in HEX activity after IL-1β stimulation is more pronounced in the intracellular compartment of synoviocytes derived from rheumatoid patients than in ACL-injury, amounting 189.44 % increase (in comparison to untreated cultures) in synoviocytes from JIA, and 127.97 % in synoviocytes of RA patients. We noted a 201.18 % increase in HEX-A activity (in comparison to untreated cultures) in stimulated synoviocytes from JIA, and a 128.03 % increase in synoviocytes of patients with RA. In extracellular compartment of cultured rheumatoidal synoviocytes, stimulation by IL-1β cause only 33.4-72.44 % increases in HEX and HEX A activities. In extracellular compartment of cultured synoviocytes derived from injured knees of ACL patients, after stimulation by IL-1β the highest increase (121.80 %) was observed in HEX A activity. The mechanism of selective stimulation of HEX by IL-1β is not known. Shikhman et al. (2000) suggested that cytokines are involved in secretion of HEX from chondrocytes and stated that IL-1β could selectively up-regulate HEX synthesis and facilitate intra-compartmental transport of HEX from lysosomes/endosomes into the extracellular space, by modifying the mannose-6-phosphate receptor system.

Lysosomal Glycosidases in Degradation of Human Articular Cartilage 105

activity of RA leukocytes. Patients with long-standing RA had higher activity of lysosomal

We suggest that of the exoglycosidases, only HEX activity measurement in synovial fluid and serum has practical value (Popko et. al. 2006). The specific activity of HEX and its isoenzyme A in serum and synovial fluid from patients with different arthropathies is

> Control ACL OA RA JIA

**\*\* \*\***

**\*** p<0,05 **\*\***p<0,001

Serum SF Serum SF

HEX (total) HEX A

Fig. 8. Specific activity of N-acetyl-β-hexosaminidase and its isoenzyme A in serum and

In the serum of RA patients, the specific activity of HEX was significantly increased in comparison to control (Fig. 8). This increase of HEX activity in the serum of RA patients may depend on the increase in HEX activity of RA leukocytes (Sohar et al. 2002). In the serum of patients with JIA and OA, we observed a moderate increase in HEX activity, i.e. 25.46% and 17.3 % respectively. In the case of patients with ACL injury, the specific activity of HEX and

In the synovial fluid of JIA and RA patients, we found a significant increase in the specific activity of HEX and its isoenzymes, in comparison to HEX activity in synovial fluid from OA and ACL injuries. This suggests that release of HEX to synovial fluid is greatly

The activity of HEX in synovial fluid of patients with JIA and RA was significantly higher than the activity in OA and ACL patients. Additionally, we have found that in JIA and RA patients, the specific activity of HEX in synovial fluid is 6-8 times higher than in serum

enhanced by the autoimmunological inflammatory process in knee joint cavity.

**\* \***

glycosidases in their leukocytes than those with disease of shorter duration.

**\*\***

**\*\***

presented in Fig. 8.

0

5

**\* \*\* \*\***

synovial fluid (µkat/kg of protein).

its isoenzymes in serum behaved similarly as in the control.

10

Specific

 activity

(µkat/kg of protein)

15

20

25

30

35

Fig. 7. Effect of interleukin 1β (IL-1β) on intra-(In) and extracellular (Ex) activity of lysosomal exoglycosidases in cultured human synovial cells from patients with anterior cruciate ligament (*ACL*) injuries, juvenile idiopathic arthritis (JIA), and rheumatoid arthritis (RA). Synoviocytes were stimulated with IL-1β for 24h at 37ºC. After IL-1β stimulation, cultured cells and exracellular fluid were analyzed for exoglycosidase activities. The effect of IL-1β was expressed as the enzymatic activity in stimulated versus unstimulated cultures.

Rheumatoid synoviocytes exhibit altered morphology and show certain similarities to tumor cells (Mor et al. 2005). Our data confirmed the observation that synoviocytes obtained from patients with JIA and RA are more active in the synthesis and secretion of exoglycosidases than those obtained from patients with injured knees or healthy joints (Popko et. al.2008). (Popko et. al.2008).
