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Elevated serum granzyme B levels are associated with disease activity and joint damage in patients with rheumatoid arthritis

Objectives Little is known about the roles of granzyme B in rheumatoid arthritis (RA). We aimed to evaluate the serum level of granzyme B in patients with RA and determine relationships with clinical features and joint destruction of RA. Methods We enrolled 100 patients with RA, 50 patients with osteoarthritis (OA), and 50 healthy controls (HC). Granzyme B serum concentrations were measured by ELISA; we then analyzed associations between granzyme B levels, clinical features, and joint destruction by calculating Sharp scores and disease activity as measured by Disease Activity Score-28 based on erythrocyte sedimentation rate (DAS28-ESR) in patients with RA. Results Compared with HC and patients with OA, serum granzyme B levels in patients with RA were remarkably elevated. Serum granzyme B levels did not differ between patients with OA and HC. Granzyme B levels correlated with ESR, rheumatoid factor, swollen joint counts, joint erosion scores, total Sharp scores, and DAS28-ESR. Moreover, patients with RA with high disease activity had higher granzyme B levels. Conclusions Serum granzyme B levels were elevated significantly in patients with RA and correlated positively with disease activity and joint destruction. Serum granzyme B may have potential applications in laboratory evaluation of patients with RA.


Introduction
Rheumatoid arthritis (RA) is a chronic immune-mediated disease characterized by degradation of articular cartilage and bone; it affects 0.2% to 1.0% of the population worldwide. 1 With progressive destruction of bone, patients with RA can develop obvious joint deformities and may suffer severe work disability. These outcomes have a considerable impact on patient quality of life and economic burden, both of which have received attention. 2 Previous studies have demonstrated that cytotoxic T cells and natural killer (NK) cells are significantly increased in rheumatoid synovial tissue, 3,4 and these cells contain a family of homologous serine proteinases called granzymes. 5 Granzymes are important members of the serine protease family and have a wide range of functions. 6 In particular, granzyme B has been shown to serve a vital role in apoptosis of target cells through the gran-zymeÀperforin apoptotic pathway. 7 Several studies have indicated the presence of granzyme-positive cells in synovial tissue from patients with RA, and expression of granzyme B was shown to be higher in rheumatoid synovial tissue than in osteoarthritic synovial tissue. 8,9 Recent work revealed a potential role of granzymes in increasing degradation of extracellular matrix (ECM), which might accelerate cartilage and bone destruction in RA. 10 Colombo et al. 11 found that granzyme B serum levels of 18 patients with RA were correlated with disease activity measured by Disease Activity Score 28-joint count-C-reactive protein (DAS28-CRP), and levels of granzyme B were significantly decreased after treatment. However, the significance of granzyme B, and especially its role in RA joint damage, for patients with RA remains largely unclear. Therefore, in this study, we aimed to investigate the potential functions of granzyme B in RA. We compared serum levels of granzyme B in patients with RA and osteoarthritis (OA) and in healthy individuals. We also analyzed correlations between serum granzyme B levels and clinical characteristics, disease activity, and joint damage of patients with RA.

Ethical approval
All participants gave written informed consent, and the study was approved by the ethical committee of Xuanwu Hospital.

Patients
Patients with RA (n ¼ 100) fulfilling the 1987 American College of Rheumatology (ACR) revised criteria 12 and 2010 ACR/European League Against Rheumatism (EULAR) classification criteria 13 were recruited in our hospital. Patients with OA (n ¼ 50) fulfilling the 1995 ACR criteria 14 and matched by sex and age were enrolled as disease controls in our hospital. All the patients with OA were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), which includes 24 items of pain (0-20), stiffness (0-8), and physical function (0-68), with a total score of 96. 15 The average WOMAC score of patients with OA in this study was 67.1 AE 7.9. Healthy individuals (n ¼ 50) were recruited from the local community.

Clinical data
Clinical data included age, sex, duration of disease, and counts of tender and swollen joints. The following laboratory indices were recorded: white blood cell (WBC) count, red blood cells (RBC) count, hemoglobin (Hb), platelet (PLT) count, immunoglobulin (Ig)G, IgA, IgM, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibody (anti-CCP antibody). Disease activity of patients with RA was scored using the 28-joint Disease Activity Score (DAS28) calculated using the Nijmegen formula based on ESR. 16 A DAS28-ESR >5.1 is considered indicative of high disease activity in accordance with the EULAR recommendations. 17

Joint damage assessment
All patients with RA had X-rays of the hands and wrists, and joint damage was assessed by the van der Heijde modified Sharp score. 18 Total Sharp scores were calculated on the basis of 17 areas evaluated for joint erosions and 18 areas evaluated for joint space narrowing. The assessments were completed independently by two experienced radiologists who were blinded to the patients' data.

ELISA detection of serum granzyme B levels
Serum samples were obtained from all patients with RA and OA, and HC; samples were coded and stored at À80 C until analysis. Serum granzyme B levels were detected by using DuoSet sandwich ELISA kits (catalog number DY2906-05) and DuoSet Ancillary Reagent Kit 2 (catalog number DY008) from R&D Systems (Minneapolis, MN, USA). In accordance with the manufacturer's instructions, detection was performed as follows. Briefly, plates were coated with capture antibody against human granzyme B overnight at room temperature (RT). Then, plates were washed three times in 0.05% Tween 20 in PBS and blocked with 1% bovine serum albumin in PBS for 1.5 hours at RT. The plates were washed as above. Standards and samples were added and plates incubated for 2 hours at RT. The plates were washed as above. Next, biotinylated antihuman granzyme B antibody and horseradish peroxidase (HRP)-conjugated streptavidin were used to detect the reaction. Tetramethylbenzidine was added as the substrate solution, and the reaction was stopped with 2 N H 2 SO 4 . The optical density was immediately measured using an automatic ELISA reader at 450 nm with a correction wavelength of 570 nm. The concentration was calculated from the standard curve.

Statistical analysis
Statistical analyses were performed using SPSS 20.0 for Windows (IBM Corp., Armonk, NY, USA). Data are presented as mean AE standard deviation or median [interquartile range (IQR)]. The group differences were assessed by one-way ANOVA test with Dunnett's T3 post hoc test (as appropriate), two-tailed independent samples Student's t-test, or MannÀWhitney rank-sum test. The correlations between two variables were analyzed by Spearman's correlation coefficient. The regression analysis was analyzed by logistic regression. A P-value < 0.05 was considered statistically significant.  (Figure 1a). However, there was no difference in granzyme B levels between female and male patients with RA (Z ¼ À0.072) (Figure 1b), and no correlation between granzyme B levels and duration of RA or age of patients with RA (duration: r ¼ 0.033; age: r ¼ À0.007) (Figure 1c, d).  anti-CCP antibody (r ¼ À0.059), or counts of tender joints (r ¼ 0.156) (data not shown). We then conducted multivariate logistic regression and the results showed that there was no independent risk factor for serum granzyme B level (odds ratio ¼ 1.665; P ¼ 0.006).

Granzyme B levels and joint damage in patients with RA
We found that granzyme B levels were associated with bone damage in patients with RA. Granzyme B levels were positively correlated with total Sharp scores (r ¼ 0.222, P ¼ 0.026) and joint erosion scores (r ¼ 0.202, P ¼ 0.044); no correlation was found between granzyme B levels and joint space narrowing scores (r ¼ 0.189) (Figure 3a-c).

Granzyme B levels and disease activity score in patients with RA
In accordance with the recommendations from EULAR, the DAS28 based on ESR was evaluated as described. We found that serum levels of granzyme B were positively correlated with DAS28-ESR (r ¼ 0.322, P ¼ 0.001) (Figure 4a). Furthermore, patients with RA were classified into two groups based on their DAS28-ESR: a DAS28-ESR score 5.1 defined the low/ moderate activity group (51 patients) and a DAS28-ESR score >5.1 defined the high activity group (49 patients). We then compared serum granzyme B levels among the two groups. Granzyme B levels were significantly higher in the high activity group (59.84 AE 23.12 pg/mL) than in the low/moderate activity group (51.02 AE 21.74 pg/mL) (Z ¼ À2.100, P ¼ 0.036) (Figure 4b).

Discussion
Rheumatoid arthritis is a chronic and progressive inflammatory disease affecting the synovial tissue in multiple joints, and it is characterized by a number of clinical symptoms. 19 Granzymes are a family of serine proteases derived from the granules of cytotoxic lymphocytes, and granzyme B is thought to be responsible for the apoptosis of target cells. 20 It has been demonstrated that the synovial tissue of RA is infiltrated by cytotoxic cells, which could then lead to a specific increase in granzyme B. 21  The first findings of our study showed a significant increase in serum granzyme B concentrations in patients with established RA compared with patients with OA and healthy controls. The marked elevation of serum granzyme B associated with RA indicated that granzyme B expression is somehow related to the inflammatory activation of RA, consistent with previous studies. 22,23 Patients with autoimmune diseases have a high tendency to develop autoantibodies against nuclear components. 24 Defective clearance of apoptotic cells is believed to be one of several causes of RA, and apoptotic cells are present in synovial tissues and serum. 25 However, the origin of the increase in serum granzyme is not fully known. Previous studies have reported that the level of granzyme B was higher in synovial tissue than in corresponding serum samples, which indicated that the increase of granzyme B in serum may originate from the synovial tissues of the inflamed joint. 26 Following release from cytotoxic cells in synovial tissue, granzymes migrate into the circulatory system and reach a high concentration in serum. Soluble granzyme B is found extracellularly in normal plasma and is elevated in a number of diseases, including viral and bacterial infections and autoimmune diseases; therefore, granzyme B might be produced and secreted by nonimmune cells. 5,27 Although the finding that patients with RA have higher granzyme B concentrations than patients with OA has been demonstrated in some studies, 9,26 correlations between serum levels of granzyme B and clinical features in RA have not been fully elucidated. Goldbach-Mansky et al. 28 revealed a weak correlation between granzyme B and RF in patients with RA, whereas no obvious correlations were found between granzyme B concentrations and other clinical features. However, Tak et al. 26 reported highly significant correlations between serum granzyme B levels and serum levels of RF. Additionally, weakly positive correlations were found between serum granzyme B level and CRP level and swollen joint counts. 26 Xu et al. 10 reported that the frequency of granzyme B-producing B cells was negatively correlated with disease activity in patients with RA. Our study also found that elevated granzyme B levels in the sera of patients with RA were correlated positively with RF and counts of swollen joints. Moreover, in the current study, we showed positive correlations between serum granzyme B levels and ESR and DAS28-ESR disease activity score. These discrepancies in reported results may be because Xu et al. 10 focused on granzyme B produced by B cells, whereas our study detected total serum granzyme B, which is produced by multiple cell types, including T cells, NK cells, B cells, and others. Although no evidence has yet shown a causal role for granzyme B in disease activity of RA, our results suggested that granzyme B may be an indicator by which to monitor disease activity.
The extracellular activity of granzyme B is an emerging focus of autoimmune disease research. 29 A study by Colombo et al. revealed that granzyme-positive cells were present at the cartilage-pannus junction, where damaged synovium invades cartilage and bone. The authors hypothesized that secreted granzymes may act as mediators of extracellular proteolysis and have potent ECM remodeling activity. 11 Therefore, in this study, we assessed the correlation between granzyme B levels and indices of bone destruction. As expected, levels of granzyme B were strongly and positively correlated with joint destruction scores in patients with RA, which indicated that granzyme B level might be a surrogate marker to predict the severity of joint damage.
There are several possible explanations for the relationship between granzyme B and joint destruction. First, granzymes are important mediators of apoptosis and might mediate cartilage destruction by inducing apoptosis in chondrocytes. 30 Second, granzyme B has enzymatic activity to cleave ECM proteins in the cartilage matrix, 31,32 and granzyme-mediated ECM degradation may facilitate the movement of cytotoxic lymphocytes and other leukocytes in vivo or the cleavage products may attract various immune cells, contributing to local inflammation. 33,34 Finally, extracellular granzyme B can cleave and activate several important proinflammatory cytokines, including interleukin (IL-6), IL-8, and tumor necrosis factor-a. 35 However, in vivo evidence for the contribution of granzyme B to the destructive process in RA is lacking and should be explored in further studies.
In the present study, we systematically measured serum levels of granzyme B in patients with RA and correlated these measures with clinical features and joint destruction. Our findings suggest that measurement of granzyme B may be of use in laboratory evaluations of patients with RA. Further functional studies are needed to fully elucidate the role of granzyme B in development of RA.

Declaration of conflicting interest
The authors declare that there is no conflict of interest.

Funding
The present study was supported by the National Natural Science Foundation of China