Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis which leads to cartilage degradation, subchondral bone erosions, and eventual disability [
Histopathological features of RA synovitis include hyperplasia of lining layer, inflammatory infiltration (mostly lymphocytes and plasma cells), and increased density of resident cells (fibrocytes, fibroblasts, and endothelial cells). Several grading systems have been developed for scoring the histopathological features of synovitis in RA, which are useful in providing information combined with morphological and molecular data and giving basic and standardized diagnostic information concerning the inflammatory character of the diseases and the degree of inflammation [
Matrix metalloproteinases (MMPs) are a large group of zinc-dependent proteases with the ability of degrading components of extracellular matrix such as collagen, elastin, gelatin, and casein. MMP-3 is one of the MMP family members produced in joints. It can aggravate inflammation via activating various pro-MMPs such as pro-MMP-1, pro-MMP-7, pro-MMP-8, pro-MMP-9, and pro-MMP-13 and cleave extracellular components including collagen types III, IX, and X and telopeptides of collagen types I, II, and XI [
Sixty-two Chinese patients with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR) [
Clinical data of all patients with RA were collected at baseline, including the 28-joint tender and swollen joint count (28TJC and 28SJC), patient global assessment of disease activity (PtGA), provider global assessment of disease activity (PrGA), pain visual analogue scale (Pain VAS), Chinese language version of Stanford Health Assessment Questionnaire (HAQ) [
Serum samples were collected from all RA patients and 34 healthy controls after overnight fasting and stored at −80°C until analysis. Serum level of soluble MMP-3 was measured with a human MMP-3 detection kit (AESKU Diagnostics, Germany) according to the manufacturer’s instructions. This detects total MMP-3 (pro- and active MMP-3) in human serum. Measurements were done in duplicate. Serum samples were placed in designated microwells. In addition, calibrators, negative, and positive controls were added to the designated microwells to construct a standard curve. The plates were then incubated for 30 min at 26°C and washed with wash buffer 3 times. Then 100
Closed Parker-Pearson needle biopsy was performed on knee joints of all patients with RA [
Serial sections of synovial tissues were stained with hematoxylin and eosin (H and E) and a 3-step immunoperoxidase method. Sections were deparaffinized with xylol, ethanol, and demineralized water. Antigens were then retrieved by boiling in 1 mM EDTA (pH 8.0) for 10 to 15 min. After the sections had been washed in demineralized water and phosphate buffered saline (PBS), the primary antibody was added and incubated overnight at 4°C. After washing with PBS, the sections were incubated with EnVision Mouse or Rabbit conjugate (Dako Corporation, Carpinteria, CA, USA) for 30 min at 37°C. The color reaction was completed with the DAB-positive substrate. Sections were counterstained with hematoxylin. Nonspecific isotype IgG was used as a negative control. Absence of staining due to technical failure was excluded by including appropriate positive control tissues (breast carcinoma) in each staining run.
Serial sections were stained with rabbit anti-human MMP-3 (clone EP1186Y, Novus Biological, Littleton, CO, USA) and the following commercial mouse anti-human antibody preparations (Invitrogen, Carlsbad, CA, USA): anti-CD20 (clone L26, B cells), anti-CD38 (clone SPC32, plasma cells), anti-CD3 (clone PS1, T cells), anti-CD68 (clone KP1, macrophage-like synoviocytes and macrophages), and anti-CD15 (clone My1, neutrophils), according to standard staining protocols. All were monoclonal antibodies. Parallel sections were incubated with irrelevant, isotype, and concentration-matched monoclonal antibodies as negative control.
At least three qualified tissue pieces containing both synovial lining and sublining layers for each specimen were included in the analyses. Histologic changes of RA synovitis in the H and E-stained sections were graded with Krenn’s synovitis score [
The percentage of lining MMP-3 and CD68 positive-staining cells was determined by manual counting in the lining layer observing 5 different fields at magnification of ×400. In sublining area, the densities of CD20, CD38, CD3, CD15, or CD68 positive-staining cells were determined by manual counting combined with cellular morphology in consecutive sections stained with H and E. A selection of 9 hpf (400x) of the sublining area was examined for each specimen by two independent observers (MJD and CLF). As one hpf revealed a synovial area of 0.11740 mm2, the densities of CD38, CD3, CD20, CD15, or CD68 positive-staining cells were counted as cells per mm2 [
Statistical analyses were performed with SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Data of categorical variables were presented as frequencies and percentages. Data of continuous variables were presented as median and interquartile range (IQR). Nonparametric tests (Mann-Whitney ranksum test between two groups or Kruskal-Wallis one-way analysis of variance on ranks among three groups for continuous variables) were used to compare the differences of serum MMP-3, synovial MMP-3, or clinical indicators in different groups, as data of the mentioned variables were not distributed normally. Correlations between serum or synovial MMP-3 expression and clinical or histological indicators were analyzed by Spearman’s rank order correlation test. The abilities of serum and synovial MMP-3 to distinguish high grade from low grade synovitis were assessed with receiver operating characteristic curve (ROC) analysis. Multiple linear regression models were developed by stepwise variable selection method to describe the linear association between synovial MMP-3 and histological indicators, including components of synovitis score and densities of sublining inflammatory cells. All significance tests were two-tailed and were conducted at the 5% significance level.
Baseline demographic and clinical features of all patients with RA were shown in Table
Baseline demographic and clinical features of RA patients.
Characteristic | |
---|---|
Demographic | |
Age, yrs, median (IQR) | 56 (48~62) |
Female, |
51 (82) |
Disease status | |
Disease duration, mo, median (IQR) | 30 (12 to 96) |
ESR (mm/h), median (IQR) | 72 (47~107) |
CRP (mg/dL), median (IQR) | 3.9 (1.0~5.6) |
Rheumatoid factor-positive, |
54 (87) |
Anti-CCP-positive, |
50 (81) |
SDAI, median (IQR) | 33 (24~44) |
CDAI, median (IQR) | 29 (20~40) |
DAS28, median (IQR) | 5.5 (4.6~6.3) |
Synovitis score, median (IQR) | 4 (4~6) |
High grade synovitis, |
27 (44) |
Previous medications, |
|
Corticosteroids | 26 (42) |
Methotrexate | 20 (32) |
Leflunomide | 6 (10) |
Sulfasalazine | 5 (8) |
Hydroxychloroquine | 7 (11) |
Etanercept | 4 (6) |
In synovium, MMP-3 is expressed predominantly in the endochylema of lining cells (both macrophage-like synoviocytes and fibroblast-like synoviocytes), while it is absent in the sublining area. As shown in Figure
Representative immunohistochemical findings of synovial MMP-3 expression. (a) Mild synovial MMP-3 expression in lining cells in a discoid meniscus patient. (b) Moderate synovial MMP-3 expression in lining cells in an OA patient. (c) and (d) Intensive synovial MMP-3 expression in lining cells in a RA patient. (a, b, c) original magnification ×400; (d) original magnification ×1000. (e) Percentage of lining MMP3+ cells in OrthA, OA, and RA patients.
The percentage of lining MMP3+ cells was significantly higher in RA patients with high grade synovitis than that in RA patients with low grade synovitis (median 51%, IQR 47%~56% versus median 42%, IQR 36%~49%,
Areas under the curve (AUC) of synovial and serum MMP-3 as biomarkers for distinguishing high grade synovitis.
High synovitis score versus low synovitis score | AUC |
|
95% CI | Tradeoff value | Youden’s index | Sensitivity |
Specificity |
---|---|---|---|---|---|---|---|
Synovial MMP-3 | 0.772 | <0.001 | 0.655~0.890 | 44% | 0.517 | 88.9 | 62.9 |
Serum MMP-3 | 0.668 | 0.024 | 0.532~0.803 | 190.4 ng/mL | 0.412 | 92.6 | 48.6 |
Synovial (a) and serum (b) MMP-3 expression between high and low grade groups of synovitis score or subscore.
Correlation between synovial (a) and serum (b) MMP-3 with histological synovitis score.
(a~j) Synovial MMP-3 expression and inflammatory cells in representative synovium from 2 different patients with RA. High and low MMP-3 expression in the endochylema of lining cells in RA synovium (a and f). Case one showed aggregated CD3+ T cells (b) and CD38+ plasma cells (c), together with diffuse infiltration of CD68+ macrophages (d) and CD15+ neutrophils (e). Case two showed a small number of CD3+ T cells (g), CD38+ plasma cells (h), CD68+ macrophages (i), and CD15+ neutrophils (j). In panels (a) to (j), immunohistochemical stains with DAB as chromogen (brown); original magnification ×400. (k) ROC curve showed synovial MMP-3 with the ability to distinguish high grade from low grade synovitis. (l~o) Spearman’s rank correlation analysis between synovial MMP-3 and CD3+ T cells (l), CD38+ plasma cells (m), CD68+ macrophages (n), and CD15+ neutrophils (o).
According to the tradeoff value of the percentage of lining MMP3+ cells for distinguishing high grade and low grade synovitis, RA patients were divided into high synovial MMP-3 expression (>44%) and low synovial MMP-3 expression groups (≤44%). Densities of CD3+ T cells, CD20+ B cells, CD38+ plasma cells, and CD68+ macrophages in sublining area of synovium in patients with high synovial MMP-3 expression were significantly higher than those in patients with low synovial MMP-3 expression (all
Multiple linear regression models for synovial MMP-3 were developed using the percentage of lining MMP3+ cells as dependent variable, while components of synovitis score and densities of sublining inflammatory cells correlated with synovial MMP-3 as independent variables, including hyperplasia of lining layer, inflammatory infiltration, activation of synovial stroma, CD3+ T cells, CD38+ plasma cells, CD68+ macrophages, and CD15+ neutrophils. The final model, shown in Table
Multiple linear regression analysis of variables associated with synovial MMP-3.
Variable | Unstandardized coefficients | Standardized coefficients |
|
|
95% CI | |
---|---|---|---|---|---|---|
|
Std. error | |||||
Components of synovitis score | ||||||
Hyperplasia of lining layer | — | — | — | 1.767 | 0.083 | — |
Inflammatory infiltration | — | — | — | 0.361 | 0.719 | — |
Activation of synovial stroma | 6.226 | 1.568 | 0.374 | 3.970 | <0.001 | 3.086~9.366 |
Sublining inflammatory cells | ||||||
CD3+ T cells | — | — | — | 0.948 | 0.347 | — |
CD38+ plasma cells | — | — | — | 0.528 | 0.599 | — |
CD68+ macrophages | 0.006 | 0.002 | 0.284 | 3.016 | 0.004 | 0.002~0.009 |
CD15+ neutrophils | 0.022 | 0.006 | 0.368 | 3.897 | <0.001 | 0.011~0.033 |
Constant | 26.071 | 2.645 | — | 9.857 | <0.001 | 20.775~31.368 |
The level of serum MMP-3 was elevated in RA patients compared with healthy controls (median 257 ng/mL, IQR 174~481 ng/mL versus median 32 ng/mL, IQR 18~53 ng/mL,
Serum MMP-3 in RA patients with high grade synovitis was significantly higher than that in patients with low grade synovitis score (median 325 ng/mL, IQR 233~528 ng/mL versus median 207 ng/mL, IQR 141~392 ng/mL,
This study provided relatively large scale of synovial tissues for histological analysis of MMP-3 from RA patients. Our results showed that synovial MMP-3 was elevated in RA synovium and positively correlated with synovitis assessed by comprehensive histological analysis including Krenn’s synovitis score and inflammatory cells by immunohistochemistry. As a result of correlation test and multiple linear regression analysis, synovial MMP-3 showed stronger association with activation of synovial stroma and sublining infiltration of macrophages and neutrophils than other histological indicators. Serum MMP-3 was significantly correlated with synovial MMP-3 and Krenn’s synovitis score and had the ability to distinguish high grade from low grade synovitis in RA.
RA results in a chronic and systemic inflammatory disorder that may affect many tissues and organs especially joints. Synovial tissues in RA can produce cytokines which aggravate inflammatory response, and uncontrolled synovitis leads to joint damage. Previous studies did not support traditional inflammatory biomarkers and cytokines as biomarkers of synovitis. CRP is a widely used inflammatory biomarker produced by hepatocytes in response to proinflammatory cytokines such as TNF and interleukins during inflammation, but it has poor value in the assessment of cumulative synovitis [
Inflammatory infiltration is one of the main histopathological features in RA synovium. Kobayashi et al. has reported positive correlation between synovial MMP-3 and semiquantitative score of synovial inflammatory cell infiltration [
Cellular proliferation is another histopathological feature of synovitis in RA, which includes hyperplasia of lining layer and activation of synovial stroma. Hyperplasia of lining layer always combines with promoting fibroblast-like synoviocytes adhesion, invasion, and synthesis of MMPs [
Serum MMP-3 was elevated in RA patients and was reported as a predictor of joint destruction in clinical studies [
As mentioned in method, serum or synovial total MMP-3 including pro- and active MMP-3 was detected in this paper. However, only active MMP-3 has the ability to cleave extracellular matrix proteins and to aggravate inflammation. Recently Sun et al. successfully developed an assay measuring active MMP-3 detection in human serum and found that serum active MMP-3 only correlated with inflammatory markers CRP and ESR, but not to other measures of disease burden in RA (DAS, HAQ) which implies that active MMP-3 reflects other aspects of disease than total MMP-3 [
Our results indicated that MMP-3 was involved in pathogenesis of synovitis especially the activation of synovial stroma and sublining infiltration of macrophages and neutrophils, and serum MMP-3 may be an alternative noninvasive biomarker of histological synovitis which is helpful for diagnosis of RA. Further studies are required to investigate whether serum active MMP-3 is more sensitive and reliable serological biomarker of synovitis than total MMP-3.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Jian-Da Ma and Jing-Jing Zhou contributed equally to this work.
The authors thank all the patients and medical staff who generously contributed to this study. This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20130171110075) and Guangdong Natural Science Foundation, China (Grant no. S2013010014396).