Rheumatoid Arthritis Morning Stiffness Is Associated With Synovial Fibrin and Neutrophils

Abstract

Objective: Morning stiffness is a hallmark symptom of rheumatoid arthritis (RA), but its etiology is poorly understood. This study was undertaken to determine whether any histologic features of synovium are associated with this symptom.

Methods: Data on patient-reported morning stiffness duration and severity, and Disease Activity Score in 28 joints (DAS28) were collected from 176 patients with RA undergoing arthroplasty. Synovium was scored for 10 histopathologic features: synovial lining hyperplasia, lymphocytes, plasma cells, Russell bodies, binucleate plasma cells, fibrin, synovial giant cells, detritus, neutrophils, and mucin. Fibrinolysis of clots seeded with various cell types was measured in turbidimetric lysis assays.

Results: Stiffness severity and morning stiffness duration were both significantly associated with DAS28 (P = 0.0001 and P = 0.001, respectively). None of the synovial features examined were associated with patient-reported stiffness severity. The presence of neutrophils and fibrin in RA synovial tissue were significantly associated (P < 0.0001) with patient-reported morning stiffness of ≥1 hour, such that 73% of patients with both synovial fibrin and neutrophils reported morning stiffness of ≥1 hour. Further, neutrophils and fibrin deposits colocalized along the synovial lining. In in vitro analyses, fibrin clots seeded with necrotic neutrophils were more resistant to fibrinolysis than those seeded with living neutrophils or no cells (P = 0.008). DNase I treatment of necrotic neutrophils abrogated the delay in fibrinolysis.

Conclusion: In RA, prolonged morning stiffness may be related to impaired fibrinolysis of neutrophil-enmeshed fibrin deposits along the synovial membrane. Our findings also suggest that morning stiffness severity and duration may reflect distinct pathophysiologic phenomena.

Figure 1:

Association of morning stiffness severity and duration with disease activity A. Frequency of patients reporting any morning stiffness stratified by DAS28 score severity. B. Duration of morning stiffness stratified by DAS28 score severity. C. Severity of stiffness stratified by DAS28 score severity. D. Distribution of responses to questions regarding duration of morning stiffness and severity of stiffness. Each dot represent one patient response. The black dots represent the 43% of patients with discordant responses (less than one hour of morning stiffness but stiffness severity ≥6/10). P values represent results of chi-squared (A) and Kruskal-Wallis (B and C) tests.

Figure 2:

Fibrin and neutrophils associate with duration of morning stiffness Percent of patients with more than one hour of morning stiffness according to presence of synovial fibrin (A) and neutrophils (B). P values represent results of Fisher’s exact tests, using the Bonferroni correction to account for multiple comparisons. C. Percentages of patients with more than one hour of morning stiffness according to having synovium with neither fibrin nor neutrophils, fibrin only, neutrophils only, or both fibrin and neutrophils. P values represent results of Cochran-Armitage trend test. D. Severity of stiffness according to presence of neutrophils and fibrin. P values represent results of Mann-Whitney tests.

Figure 3:

Neutrophils colocalize with fibrin deposits along the synovial lining A. Representative image of H&E stained RA synovial tissue, 20x magnification. Bracket highlights eosinophilic synovial fibrin deposition along the synovial membrane. The scale bar indicates 100μm. B. Representative image of H&E stained RA synovial tissue, 40x magnification. The arrows highlight neutrophils intermixed within fibrin, which appear as pink fibrillary material along the synovial lining. The scale bar indicates 50μm. C. Image of H&E stained rice body, 40x magnification. The arrows highlight neutrophils intermixed within fibrin. The scale bar indicates 50μm.

Figure 4:

Necrotic neutrophils impede fibrinolysis A. Clotting as measured by turbidity (OD=optical density) of EDTA plasma incubated with calcium while shaking or not shaking over time. Data presented are mean and standard deviation of the optical density (absorbance at 405nm) and represent one of two experiments. (m=minutes) B. Clotting as measured by turbidity of EDTA plasma alone or seeded with either live PMN, or necrotic PMN, before and after incubation with calcium for one hour. Data presented are mean and standard deviation of the optical density (absorbance at 405nm), normalized to the value obtained just prior to addition of calcium. For A and B, *** indicates P<0.0001 in unpaired t-tests. C. Left panel: Fibrinolysis as measured by normalized turbidity of clots alone or seeded with either live PMN or necrotic PMN and treated with plasmin. Right panel: Normalized turbidity of fibrin clots from left panel at 120 minutes. Data represent one of six independent experiments. D. Left panel: Fibrinolysis as measured by normalized turbidity of clots alone or seeded with either necrotic PMN or necrotic PMN pretreated with DNase1 and then lysed with plasmin. Right panel: Normalized turbidity of fibrin clots from left panel at 270 minutes. Data represent one of three independent experiments. For C and D, data presented are mean and standard deviation of optical density (absorbance at 405nm), normalized to the value obtained at time 0, just after addition of plasmin. P values in left panels represent results of mixed-effects model for clot conditions over time (*** indicates condition*time P<0.0001) and right panel represents ANOVA with Dunnett’s multiple comparisons test, using the no cells group as the reference. NS= not significant.