Introduction

RA is traditionally considered a chronic, inflammatory joint autoimmune disorder affecting extra-articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles (removed part) [1]. Activated macrophages over express Major Histocompatibility (MHC) class II molecules and produce pro- Antigen activated CD₄⁺ T cells stimulate monocyte, macrophages and synovial fibroblasts to produce the cytokines. Activated CD₄⁺ T cells stimulate B cells through cell surface Contact and through aIb2 integrin, CD154 (CD40 Legend) and CD28 to produce immunoglobulins, including rheumatoid factor.

Activated CD4+T cells also express RANKL (receptor-activator of nuclear factor kappa B legend). The interaction of RANKL with RANK (receptor activator of nuclear factor Kappa B), was found on osteoclast precursor cells and mature osteoclasts, is essential for osteoclasto genesis and osteoclast activation. Pannus is considered the most destructive element affecting the joint it can attack the articular cartilage and destroy it [2]. Conventional radiography is the recommended method for monitoring progression of structural joint changes in the routine management of RA patients as well as in clinical trials. It has a lower sensitivity than MRI for bone erosions.

Grading System of Rheumatoid Arthritis

Larsen system for grading RA and related conditions by using standard reference film radiograph.

·         Grade 0: No radiographic changes (Abnormalities not related to arthritis may be present).

·         Grade 1: slight abnormality (Peri articular swelling, para articular demineralization, slight joint space narrowing).

·         Grade 2: definite early abnormality (Definite presence of erosions except in weight-bearing joints, joint space narrowing).

·         Grade 3: Medium destructive abnormality (Definite presence of erosion in all types of joints, joint space narrowing).

·         Grade 4: Severe destructive abnormality (Erosions, joint space narrowing, bone deformation in weight-bearing joints).

·         Grade 5: Mutilating abnormality (Disappearance of the original articular forces, gross bone deformities in weight-bearing joints).

·         Note-A set of standard reference film radiographs has to be available for comparative purpose. Dislocation and bony ankylosis should not be considered in the grading.

Magnetic Resonance Imaging (MRI) is the best imaging modality for RA, a multi planar approach is useful for the distinction between erosions and pre-erosive changes and for the assessment of pannus. Up to 47% of patients may develop erosions within 1 year after onset of RA. In advanced stages, the inflammatory process may lead to massive bone erosions. Use of scored radiographs as an outcome measure can help estimating the progression of rheumatoid arthritis. The most commonly used methods.

·         Larsen methods: produce an overall score for erosion and joint space narrowing [3].

·         Sharp methods: provide separate scores for erosion and joint space narrowing [4].

·         Simple Erosion Narrowing Score (SENS): Provide scores for erosion and joint space narrowing that are summed thereafter to give a figure comparable to the sharp total score [5].

Cathepsin is a protease, a type of protein that breaks apart other proteins, found in many types of cells. There are approximately a dozen members of this family, which are distinguished by their structure and which proteins they cleave. Most of the members become activated at the low pH found in lysosomes. Thus, the activity of this family lies almost entirely within those organelles. Cathepsin B, K, L, S, V, F, are classes of lysosomal enzymes which are implicated various disorders including inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, tumors, coronary disease atherosclerosis, autoimmune diseases and infectious diseases.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodeling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis [6]. Although the degradation of the extracellular matrix in joints is clearly mediated by proteolytic activities, the nature of the individual proteases remains unknown in most cases. To date, two protease families have been implicated in cartilage degradation: Matrix Metalloproteinase (MMP) and cysteine proteases [7].

Traditionally, metalloproteinase have been favored as potential culprit enzymes over cysteine proteases, but inhibitors of both protease classes have proved to be equally, effective in reducing inflammation and cartilage erosion in animal models of RA. Cathepsins are potential drug targets to treat tissue degenerative and inflammatory processes [8]. Cathepsin K has been identified as the predominant osteoclastic protease with a unique and potent collagenolytic activity [9]. Two types of cathepsin K-containing cells appear to be present in the synovial lining: CD68⁺ and CD68^- cells. However, the dense population of cathepsin K-positive cells in the lining of the inflamed synovium suggests a role of the enzyme in cartilage- and bone-invasive processes. This role is further supported by the observation that cathepsin K- positive SFs accumulate at sites of bone and cartilage erosion [10].

Cathepsin K-positive SFs were highly enriched not only at sites of vascularization and angiogenesis but also around necrotic vessels. It can be hypothesized that the protease plays a role in the loosening of the surrounding matrix to facilitate angiogenic growth on one side and contributes to the degradation of expired vessels on the other side [10]. Synovial fibroblast derived cathepsin K have also been directly linked to the degradation of articular cartilage and bone. Bone and cartilage erosions are considered an irreversible degenerative process leading to the loss of joint function [11].

The inhibition of cathepsin K in RA-derived SFs results in a lysosomal accumulation of undigested type II collagen fibrils whereas the inhibition of cathepsins L, B, and S has no effect. The specific activation of the protease activity toward types I and II collagen by cathepsin K-digested aggrecan contribute to the potential role of cathepsin K in RA- associated joint destruction [12].

Osteoclastic bone resorption requires two processes: demineralization of the inorganic bone components and degradation of the organic bone matrix. These two processes occur in the osteoclast in a coordinated fashion by two separate mechanisms. The first phase involves acid secretion by the osteoclast into the resorption lacunae, and the second phase is the organic matrix degradation by cysteine proteases. An acidic microenvironment is required for bone resorption, both to dissolve the mineral component of bone and to aid protein matrix digestion. This unique metabolic milieu is achieved by lowering the pH in the resorption lacunae via acid secretion by the osteoclast. Cathepsin K inhibitors represent a novel target for developing agents to treat osteoporosis and other disorders characterized by increased bone resorption Taken as a whole, these findings identified cathepsin K as a potential target for antiresorptive drugs in RA [13,14]. The aim of the work is to measure serum level of cathepsin K in patients with rheumatoid arthritis, compare it to age matched control group and correlate the results with radiological progression.

Methods

The study carried out on 40 patients suffering from rheumatoid arthritis according to the criteria of the American Rheumatism Association. 10 samples are taken from healthy blood donners of matched age and sex. (control group). All the subjects signed an informed concent.

Exclusion Criteria

Other cause of increase cathepsin K was excluded such as osteoporosis and osteolytic bone metastasis.

All subjects in this study (rheumatoid arthritis patient and control group) were subjected to the following:

·         Full history taking, particularly for age, sex, duration of the disease articular and extra-articular manifestations.

·         Through clinical examination joint examination and clinical assessment with Diseases activity score-28 (DAS 28) [15].

Laboratory Investigation

10ml venous blood was withdrawn from the patients and controls in the morning after 8 h fasting, blood samples were withdrawn from the antecubital vein with minimal stasis. They were divided as follows.

·         2 ml delivered into EDTA tubes for Complete Blood Count (CBC).

·         1.6 ml delivered into tubes containing sodium citrate for Erythrocyte Sedimentation Rate (ESR).

·         Serum was used for routine chemical tests.

Serum was used also for determination of the following serological test [16]:

·         Rose waaler test.

·         Latex Rheumatoid Factor (RF).

·         C - Reactive Protein (CRP).

Determination of Cathepsin K Level Using ELISA

It is an enzyme-linked immunosorbent assay for the detection of cathepsin K. in serum of rheumatoid arthritis patients.

Sample Collection

The assay was performed with a serum samples; grossly haemolysed or turbid samples were not used. Thoroughly thawed samples were mixed before assay, repeat freeze, thawing has been avoided. Samples were stored at -20 ºC till assay.

Assay Protocol

·         All reagents and samples must be at room temperature (18-26 ^°C) before use in the assay.

·         We marked position for BLANK/STD/SAMPLE/CTRL (Blank/ Standard/ Sample/ Control) on the protocol sheet.

·         We Added 50 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective wells, except blank.

·         200 µl CONJ (anti Cathepsin K-HRPO) was added into each well, except blank, and swirled gently.

·         The plate was covered tightly and incubated overnight (20-24h) at room temperature (18-26 ^°C) in the dark.

·         Wells was aspirated and washed 5x with 300 µl diluted WASHBUF (Wash buffer), removal of remaining WASHBUF by hitting plate against paper towel after the latest wash.

·         200 µl SUB (Substrate) was added into each well.

·         The plate was incubated for 30 min at room temperature (18-26 ^°C) in the dark.

·         50 µl STOP (Stop solution) was added into each well.

·         Absorbance was measured immediately at 450 nm with reference 620 nm. (removed part)

Radiological Examination of the Affected Joint

Plain x-ray was done for each affected joint to estimate the progression of rheumatoid arthritis.

Rau and Herbon modification of Larsen Scoring Method for Rheumatoid Arthritis (SENS) [5]

·         32 Joints are evaluated

Eight PIP, Two IP of the thumb, Ten MCP, Two wrists and Ten MTP of the foot.

·         6 Stages are defined or follow.

0 = Normal, 1 = Soft tissue swelling and/or joint space narrowing/subchondral osteoporosis.

2 = Erosion with Destruction of the Joint Surface (DJS) £ 25%,3 =DJS26 - 50%.4 =DJS51 - 75%.

5 = DJS ³ 75%.

The score ranges from 0 to 160.

Method of Statistical Analysis

The collected data were coded, tabulated and statistically analyzed using a Microsoft, statistical package of social science version 15 (SPSS-15). Mean-median and standard deviation with used to express quantitative data.

a)                   Comparison between means of both groups was done by application of student t-test to compare between cases and controls.

b)                  Comparison between qualitative data of each group was assessed by Fisher Exact test.

c)                   Correlation between quantitative data of each group was assessed by calculation of Pearson’s correlation coefficient.

d)                  P value less than 0.05 was adopted as a level of significance.

Results

The present study was carried on 50 subjects admitted to “Alexandria Main University Hospital” divided into 2 groups:

·         Group I: 40 rheumatoid arthritis patients fulfilling ACR criteria for diagnosis of RA.

·         Group II: 10. healthy blood donners of matched age and sex.

Age and Sex

In the present study. Group I included 31 female patients (77.5%) and 9 male patients (22.5%), their age ranged from 19-50 years with mean age value 37.30 ± 7.58. Group II included 6 females (60%) and 4 males (40%), their age ranged from 34-42 years with mean age value 38 ± 2.94 Table 1.

Renal Function Test (Blood Urea Nitrogen Creatinine Level) Serum Uric Acid

These were no significant differences between 2 groups Table 1.

ESR (1st hour)

It ranged from 15-100 mm/h with mean value of (40.9 ± 22.19) mm/h in group I, while ranged from (8-20) mm/h with mean value of (11.0 ± 3.92) mm/h in group II with significant difference between the 2 groups (p<0.001) Table 1.

Cathepsin K

It ranged from (5.10-90.00) pmol/l group I with mean value of (26.87 ± 22.05) pmol/l, while it ranged from (4.00-12.00) pmol/l with mean value (7.94 ± 2.39) in group II with significant difference between the two groups (p<0.001) Table 2. There was statistically significant correlation between cathepsin K readings and morning stiffness, DAS 28, Larsen score (p 0.040^O.037, 0.007) respectively (Figures 1,2). There was significant correlation between erythrocyte sedimentation rate in the first hour with the result of cathepsin K (p equal 0.007). 23 patients had no limitation of movement; cathepsin K mean value was (20.22 ± 16.85), while 17 patients were complaining of limitation of movement, the mean value of cathepsin K was (35.88 ± 25.39), that show statistically significant difference. (P = 0.024).

Discussion

Our aim of the present work was to study cathepsin K levels and its association with radiological destruction in patients with rheumatoid arthritis (31 females and 9 males) their age ranged from 19-50 years, with mean value 37.3 ± 7.58. In addition, 10 healthy blood donners with matched age and sex were included as control group.

Serum Cathepsin K levels in patient group was significantly higher than control group (P < 0.001). Toren BR (2004) found that, bone resorption and formation is a well-balanced system and is mediated by osteoclast. Cathepsin k is essential for bone resorption, which depends on the production of cathepsin K by osteoclast and its secretion into the extra cellular department. This leads to degradation of the organic matrix between the osteoclasts and the bone surface [17].

Dodds RA and his colleagues (2001) found that, in vivo the activation of cathepsin K occurs intracellular before secretion into the resorbing lacunae and the onset of resorption, where by local factors may regulate the processing of pro-cathepsin K to mature cathepsin K [18]. Hou and his colleagues (2003) found that, cathepsin K has a potent aggreacan-degrading activity; where by the aggrecan cleavage products increase the collagenolytic effects of this protease on collagen type I. and type II. There were able to show that cathepsin K is also a critical protease in cartilage degradation by synovial fibroblast [19].

Increased expression of cathepsin K around lymphocytic infiltrates in synovial tissue seems to facilitate the movement of mononuclear cells through the perivascular matrix [20]. Pro inflammatory cytokines such as IL-IB and tumor necrosis factor alpha facilitate the expression of cathepsin K, its over expression in rheumatoid synovium, induced by IL-IB and tumor necrosis factor alpha due to increase of cathepsin K expressing cells, proves this protease to be available tool for bone research, and cathepsin K also may become a new and highly specific biomarker for RA [21].

Votta and his colleagues demonstrated high levels of cathepsin K expression in osteoclast at sites of extensive bone loss. According to this, they developed a peptide aldehyde inhibitor of cathepsin K that inhibits osteoclast. Mediated bone resorption in fetal rat long bone organ cultures and even in a human osteoclast. This inhibitor leads to significantly reduced bone loss [22]. In the synovium of RA, the cathepsin K protein was localized in synovial fibroblast, CD68⁺ macrophage like synoviocytes, stromal multinucleated giant cells. Highly interesting is the expression of cathepsin K by fibroblast and giant cells at sites of cartilage erosion. This was two to five times higher compared with osteoarthritic synovium. In normal synovium, cathepsin K expression was restricted to fibroblast like cells [23-25].

The Larsen score ranged from (0 -110) in our study with mean value (56.60 ± 31.71) with statistically significant correlations between cathepsin K and Larsen score (P = 0.007) the highest levels of cathepsin K were observed in patient with highest Larsen scores. The radiological destruction correlates significantly with cathepsin K [26]. DAS-28 ranged from (2.25-8.96) with mean value (5.94 ± 1.43) in patient group which show statistically significant correlation between it and cathepsin K. Skoumal M and his colleagues (2005) studied prospectively serum cathepsin K in rheumatoid arthritis patient and found that cathepsin K seems to be independent or weakly correlated with laboratory inflammation parameters. It was not associated with CRP (P = 0.27), but weak correlations were found with erythrocyte sedimentation rate (P = 0.03) and the disease activity score (P = 0.04). There was significant correlation with Larsen score, Cathepsin K levels showed an increase with the augmentation of radiological destruction (P = 0.035) [27].

Hou WS and his colleagues (2001), Found that there was significant correlation between cathepsin K and disease severity which determined by the selective and the critical role of cathepsin K in articular cartilage and articular bone erosion. It was further corroborated by the finding that cathepsin K has a potent aggrecan degrading activity and cathepsin K-generated aggrecan cleavage products specifically potentiate the collagenolytic activity of cathepsin K towards type I and type II collagens. But, no correlation was found between cathepsin K, erythrocyte sedimentation rate and CRP. Hou WS and his colleagues (2001), Found that there was significant correlation between cathepsin K level and joint incapacitation which determined by the Health Assessment Questionnaire disability index (HAQ), where bone and cartilage erosion derived by cathepsin K is irreversible degenerative process leading to loss of joint function [28].

Conclusion

This study demonstrates increased cathepsin K level in serum of patients with rheumatoid arthritis. The elevated serum levels of this protease are significantly correlated with the joint destruction, which in this study was assessed by Larsen score. Cathepsin K is a valuable biomarker for the assessment of bone metabolism in patients with established rheumatoid arthritis, its measurement will probably contribute to developing targeted therapies for the prevention of further bone destruction. So, we could recommend more studies to be performed to verify the presence of cathepsin K in patients with early RA and its value as a prognostic factor for bone destruction in RA.

Figure 1: Correlation between cathespin K and modified Larsen Score(SENS) in patient group.

Figure 2: Correlation between cathespin K and DAS 28 in patient group.

1.       Fox DA (2005) Rheumatoid arthritis. In: Arthritis and Allied Conditions. A textbook of Rheumatology: 1-51.

2.       Engelke K, Nagase S, Fuerst T, Small M, Kuwayama T, et al. (2014) The effect of the cathepsin K inhibitor ONO-5334 on trabecular and cortical bone in postmenopausal osteoporosis: the OCEAN study Bone Miner Res 29: 629-638.

3.       Larsen A (1995) How to apply Larsen score in evaluating radiographs of rheumatoid arthritis J Rheumatol 22: 1974-1975.

4.       Van der Heijde D, Dankert T, Nieman F, Rau R, Boers M (1999) Reliability and sensitivity to change of a simplification of the sharp/ van der Hejde radiological assessment in rheumatoid arthritis. Rheumatology (Oxford) 38: 941-947          .

5.       Boini S, Guillemin F (2001) Radiographic scoring methods as outcome measures in rheumatoid arthritis: properties and advantages. Ann Rheum Dis 60: 817-827.

6.       Goto T, Yamaza T, Tanaka T (2003) Cathepsins in the osteoclast. J Elctron Microsc (Tokyo) 52: 551-558.

7.       Bresnihan B (1999) Pathogenesis of joint damage in rheumatoid arthritis. J Rheumatol 26: 707-719.

8.       Littlewood-Evans A, Kokubo T, Ishibashi O (1997) Localization of cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistry. Bone 20: 81-86.

9.       Kafienah W, Bromme D, Buttle DJ, Croucher LJ, Hollander AP (1998) Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J 331: 727-732.

10.    Walsh DA (1999) Angiogenesis and arthritis. Rheumatology 38: 103-112.

11.    McQueney MS, Amegadzie BY, D’Alessio K, Hanning CR, McLaughlin MM, et al. (1997) Autocatalytic activation of cathepsin K. J Biol Chem 272: 13955-13960.

12.    Jackson JR, Minton JA, Ho ML, Wei N, Winkler JD (1997) Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin -1 beta. J Rheumatol 24: 1253-1259.

13.    Stoch SA, Wagner JA (2008) Cathepsin K Inhibitors: A Novel Target for Osteoporosis Therapy. Clinical Pharmacology & Therapeutics 83: 172-176.

14.    Wu-Shiun Hou, Zhenqiang Li, Ronald E, Chan GK, Klein MJ (2001) The potential role of cathepsin K in RA-associated joint destruction. American Journal of Pathology 159: 2167-2177.

15.    Prevoo MLL, Van't Hof MA, Kuper HH (1995) Modified disease activity scores that include twenty-eight-joint counts. Arthritis Rheum 38: 44-48.

16.    Lewis SM (200) Miscellaneous tests. In: Lewis SM, Bain BJ, Bates I, (eds). Dacie and Lewis practical hematology. 9^th edition. 527-590

17.    Troen BR (2004) The role of cathepsin K in normal bone resorption. Drug News Prospect 17: 19-28.

18.    Dodds RA, Tames IE, Reimon D, Ahern R, Hwang SM, et al. (2001) Human osteoclost cathepsin K is processed intracellular prior to attachment and bone resorptions. J Bone Miner Res 16: 478-486.

19.    Hou WS, Li Z, Buttner FH, Barthnik E, Bramme D (2003) Cleavage site specifity of cathepsin K cartilage proteoglycans and protease complex formation. Boil Chem 284: 891-897.

20.    Skoumal M, Haberhauer G, Kolarz G, Hawa G, Woloszczuk W, et al. (2005) Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction Arthritis Res Ther 7: R65-R70.

21.    Hou WS, Li Z, Keyszer G, Weber E, Levy R (2002) Comparison of cathepsin K and S expression within the rheumatoid arthritis and osteoarthritis synovium. Arthritis Rheum 46: 663-674.

22.    Svelander L, Erlandsson-Harris H, Astner L, Grabowska U, Klareskog L, et al. (2009) Inhibition of cathepsin K reduces bone erosion, cartilage degradation and inflammation evoked by collagen-induced arthritis in mice Eur J Pharmacol 613: 155-162.

23.    Kaneko M, Tomita T, Nakase T, Obsawa Y, Seki H, et al. (2001) Expression of proteinases and inflammatory cytokines in subchondral bone regions in the destructive joint of rheumatoid arthritis. Rheumatology Oxford 40: 247-255.

24.    Li Z, Hou WS, Bromme D (2000) Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates. Biochemistry 39: 529-536.

25.    Li Z, Yasuda Y, Li W, Bogyo M, KIatz N, et al. (2004) Regulation of collagenase activities of human cathespins by glysosaminoglycans. J Boil Chem 279: 5470-5479.

26.    Skoumal M, Haberhauer G, Kolarz G, Hawa G, Woloszczuk W, et al. (2008) The imbalance between osteoprotegerin and cathepsin K in the serum of patients with longstanding rheumatoid arthritis. Rheumatol Int 28: 637-641.

27.    Skoumal M, Hebarhauer G, Kolors G, Hawa G, Klingler A (2005) Serum cathepsin K in patient with long standing rheumatoid arthritis. Arthritis Res Ther: 65-70.

28.    Hou WS, Li Z, Ronald E, Gordon E, Chan K, et al. (2001) Cathepsin K is a critical protease in synovial fibroblast mediated collagen degradation. American Journal of Pathology 159: 2167-2177.