1. Introduction

Synovial fluid is a clear, viscous, straw-colored fluid in the synovial cavity of a joint that has biomechanical, metabolic, and regulatory functions. The molecular and cellular components of SF endow it with unique properties and functions to maintain joint homeostasis and frictionless movement. SF is produced by the cells of the synovial membrane, is rich in hyaluronic acid, and is infiltrated from the superficial capillaries and venules along with low molecular weight proteins. In a healthy joint, the SF acts as a medium for transporting essential nutrients, enzymes, cytokines and growth factors responsible for cartilage remodeling. The volume of synovial fluid normally depends on the size of the joint. Its maximum volume in normal knee and hip joints reaches 3.5 ml. In inflammatory processes, the volume of SF often increases, but even with a normal amount of joint fluid can not exclude the pathology of the joint. Synovial fluid contains immune cells, including mast cells, monocytes, synovial membrane cells and inflammatory cytokines, which together reflect the degree of inflammation in the affected area [2,5,9]. In musculoskeletal disorders such as RA and OA, inflammatory markers and nerve signal transducing substances have been found in the CSF samples of patients with knee OA. Sohn D.H. et al. identified 108 proteins of the SL in OA, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage renewal, and proteins involved in inflammation and immunity [3,4-7]. Synovial fluid has been shown to be a useful substrate in the diagnosis of joint disease [1,6-9].

In the present study, using synovial fluid samples from patients with RA, we investigated the features of immunologic parameters and interleukin profile of the intra-articular process.

2. Methods

2.1. Study Population and Methods

This observational study was conducted to assess the physicochemical and immunological characteristics of synovial fluid in patients with rheumatoid arthritis (RA) compared to a control group with posttraumatic hemarthrosis.

The study protocol was approved by the Institutional Review Board of Samarkand State Medical University. All procedures were carried out in accordance with the ethical standards of the Helsinki Declaration. Written informed consent was obtained from each participant.

The RA group comprised patients diagnosed based on the American College of Rheumatology/European League Against Rheumatism classification criteria for RA. The control group included patients who had experienced joint trauma without any autoimmune disorder. Demographic data, including age, sex, and disease duration, were recorded.

Synovial fluid samples were aspirated from the knee joints using sterile techniques. Samples were centrifuged to remove cellular debris and either analyzed immediately or stored at -80°C for subsequent assays.

Visual Inspection: The color of the synovial fluid was noted immediately after aspiration.

Viscosity Measurement: A rotational viscometer was used, calibrated before each measurement session.

Biochemical Profiling: Total protein was measured using the Bradford assay, and glucose levels were determined by an enzymatic colorimetric method.

Leukocyte and erythrocyte counts were performed using a light microscope and a Neubauer counting chamber. Differential cell counts were conducted to identify and quantify rhegocytes.

Lymphocyte Subpopulations: Flow cytometry was used to identify CD3+, CD4+, CD8+, CD16+, CD20+, CD25+, and CD95+ lymphocytes. Fluorochrome-conjugated antibodies specific to these markers were used.

Autoantibodies and Acute Phase Reactants: RF and CRP levels were quantified using high-sensitivity ELISA kits.

Immunoglobulin Quantification

IgA, IgM, and IgG levels in synovial fluid were measured using sandwich ELISA techniques. Standards and samples were run in duplicate to ensure accuracy.

Radiographic Analysis

Joint radiographs of RA patients were taken and classified into stages according to established radiographic criteria. The presence and extent of joint erosions and other changes were recorded.

2.2. Statistical Analyses

In this prospective observational study we utilized descriptive statistics for an overview of the study population's demographic and clinical characteristics. We calculated means, medians, and proportions to highlight baseline features and disease duration.

For the longitudinal aspect, repeated measures analyses (mixed-effects models) assessed changes in disease activity and patient outcomes over time. Survival analysis, employing Kaplan-Meier curves and Cox proportional hazards models, provided insights into the duration until events like treatment response or disease flare.

Subgroup analyses explored the heterogeneity within the RA population, comparing outcomes based on factors such as seropositivity or treatment regimens. Correlation and regression analyses identified relationships between variables and predictors of outcomes, adjusting for confounders.

Handling missing data involved multiple imputation techniques, ensuring robustness with sensitivity analyses. Reputable statistical software (R-studio) validated the analyses for accuracy.

3. Results

In RA patients, the synovial fluid predominantly exhibited a slightly greenish and moderately turbid appearance, deviating from the clear, straw-colored fluid seen in the control group. This change in coloration and clarity may indicate the presence of inflammatory processes within the joint. Additionally, a significant reduction in the viscosity of the synovial fluid was observed in RA patients (3.51±0.21 mPa·s) compared to the control group (20.12±0.78 mPa·s), a change that reflects a fundamental alteration in the fluid's composition and is likely attributed to the inflammatory environment in RA-affected joints (table 1).

Table 1. Physicochemical parameters, cellular elements of synovial fluid in RA patients

The total protein concentration in the synovial fluid of RA patients was markedly higher (24.50±0.08 g/L) than in the control group (15.98±0.83 g/L), exceeding it by 60.7%. This increase in protein content is indicative of an enhanced inflammatory response. Furthermore, the glucose concentration was significantly higher in RA patients (5.18± 0.14 mmol/L) compared to the control (4.33± 0.14 mmol/L), suggesting altered metabolic processes within the joint environment of RA patients.

The leukocyte count in RA patients was substantially higher (11.68±0.71 10^10/ml) than in the control group (2.69±0.37 10^10/ml), indicating an active inflammatory process. The erythrocyte count was lower in RA patients (12.41±0,67 10^9/ml) compared to the control group (17.36±0,14 10^9/ml), which could be attributed to the trauma-related hemarthrosis in the control group. The detection of macrophage cells (rhogocytes) with coarse-grained inclusions containing immunoglobulins and rheumatoid factor in all RA patients underscores the autoimmune nature of RA. These cells are typically associated with the immune response characteristic of RA.

The findings from our study elucidate key changes in the synovial fluid of RA patients. The alterations in color, viscosity, protein, and glucose levels, coupled with the increased leukocyte count and the presence of rhogocytes, are significant markers that can aid in differentiating RA from other musculoskeletal diseases. Early identification of these changes can facilitate timely diagnosis and the initiation of appropriate treatment strategies, potentially improving patient outcomes.

In the RA patient group, the level of RF was markedly elevated (7.91±1.13 MU/ml) compared to the absence of detectable levels in the control group. RF, an autoantibody commonly found in RA, plays a significant role in the disease's pathogenesis. Its presence and elevated levels are often associated with more severe disease activity and can be a predictor of joint damage progression in RA. Similarly, CRP levels were significantly higher in RA patients (5.30±0.78 mg/L), while being absent in the control group's synovial fluid. CRP is an acute-phase protein produced by the liver in response to inflammation. Elevated CRP levels are indicative of active inflammation and are used to assess disease activity and response to treatment in RA patients (table 2).

Table 2. Indicators of inflammation and autoimmune activity in synovial fluid in rheumatoid arthritis

The study of synovial fluid of patients with RA revealed that the development and progression of intra-articular pathologic process is reflected in changes in its immunologic properties. Thus, cellular immunity (Table 3) of patients with RA was characterized by a decrease in the level of CD4+ cells in synovial fluid and an increase in CD3+, CD8+ CD16+ and CD20 cells. The number of lymphocytes with markers of the early (CD25+) stage of activation in the synovial fluid of patients with RA did not change significantly and tended to increase.

Table 3. Changes in immunologic parameters (lymphocyte subpopulation composition) in synovial fluid in rheumatoid arthritis

We observed a significant increase in CD3+ and CD8+ lymphocytes in RA patients compared to the control group, indicating an activated T-cell mediated immune response. The slight decrease in CD4+ lymphocytes, although not statistically significant, might indicate a shift in the T-helper cell balance within the joint environment. Notably, there was a substantial increase in CD16+ (Natural Killer cells) and CD20+ (B cells) lymphocyte counts in RA patients. This suggests a heightened innate immune response and B-cell activation, which are critical in the pathogenesis of RA. The CD25+ lymphocytes (representing activated T cells) showed a slight increase in RA patients. More significantly, CD95+ lymphocytes, carrying the apoptosis readiness receptor, were markedly elevated in RA patients (10.1±0.25), highlighting the ongoing apoptotic processes within the inflamed joints. The elevated levels of specific lymphocyte subpopulations reflect the complex immune response characteristic of RA. The increase in CD95+ lymphocytes in particular is indicative of enhanced apoptosis, which plays a role in the perpetuation of inflammation and joint damage in RA. Understanding the immunologic landscape of RA synovial fluid can aid in the development of targeted therapies. For instance, therapies modulating T-cell, B-cell, or Natural Killer cell activities could be beneficial in managing RA. The distinct lymphocyte profile in RA synovial fluid provides a comparative framework for differentiating RA from other arthritic conditions like psoriatic arthritis or Reiter's syndrome, which may show different immunologic patterns.

Our research has explored the levels of major immunoglobulin classes in the synovial fluid of patients with rheumatoid arthritis (RA), offering insights into the role of humoral immunity in the disease. IgA, IgM, and IgG Levels: We observed a significant increase in all three major immunoglobulin classes (IgA, IgM, IgG) in the synovial fluid of RA patients compared to the control group. IgG showed the most pronounced increase (13.36±0.47 g/L in RA patients versus 3.58±1.54 g/L in the control group), followed by IgM (3.60±0.27 g/L in RA patients, compared to 0.62±0.05 g/L in the control group) and IgA (2.83±0.35 g/L in RA patients, versus 0.93±0.10 g/L in the control group). The elevated levels of IgG, IgM, and IgA suggest active B-lymphocyte involvement in RA. Since these immunoglobulins are the end products of CD20+ B cells, their increased levels indicate heightened B-cell activation and differentiation within the joint microenvironment of RA patients. The predominance of IgG, particularly in higher concentrations, is indicative of ongoing immune responses typically associated with chronic inflammation, as seen in RA. Elevated IgM and IgA levels also contribute to the understanding of the complex humoral immune response in RA, potentially reflecting the disease's severity and activity (Table 4).

Table 4. Changes in major classes of immunoglobulins in synovial fluid in rheumatoid arthritis

Our study delves into the complex relationship between the immunological profile of synovial fluid and the radiologic stages of rheumatoid arthritis (RA), offering insights into the disease's progression.

Early Stages (Stage II): In the second radiologic stage of RA, there was a notable increase in all indicators of cellular and humoral immunity. Specifically, CD16+ (Natural Killer cells) and CD20+ (B cells) showed significant elevations. This suggests an intensified immune response at this stage of RA (table 5).

Table 5. Immunologic parameters of synovial fluid in patients with different radiologic stages of rheumatoid arthritis

Advanced Stages (Stages III and IV): As RA progresses to more pronounced radiologic stages, we observed a decrease in overall cellular and humoral immune cells. However, there was a notable increase in CD25+ lymphocytes (markers of early-stage activation) and CD95+ lymphocytes (apoptosis readiness receptor). The elevated CD95+ cells suggest an increased apoptotic activity in these stages.

Across different radiologic stages, there was a marked increase in the levels of IgG, IgM, and IgA, with the most significant changes observed at the second stage. This increase aligns with the heightened immune response and B-cell activation. The distinct immunologic profiles observed at different radiologic stages of RA indicate that immune mechanisms play a crucial role in the disease's progression. In the early stages, the immune response is more pronounced, particularly in terms of B-cell and Natural Killer cell activity. As the disease progresses, the focus shifts towards apoptotic processes and T-cell activation. Understanding these stage-specific immunologic changes can inform targeted therapeutic approaches. For instance, treatments aimed at modulating B-cell activity may be more effective in the earlier stages, while therapies focusing on T-cell modulation and apoptosis regulation could be more relevant in advanced stages. These findings underline the importance of assessing immunologic parameters in synovial fluid as part of the diagnostic process and for prognostic evaluation in RA. The stage-specific immunologic changes could serve as valuable biomarkers for disease progression and treatment response.

4. Conclusions

Our comprehensive study of synovial fluid in patients with rheumatoid arthritis (RA) has yielded significant insights into the disease's pathophysiology. RA patients exhibited notable changes in the synovial fluid, including a slightly greenish and moderately turbid appearance, and a significant reduction in viscosity. These changes, coupled with increased total protein and glucose concentrations, underscore the presence of an active inflammatory process and altered metabolic activity within RA-affected joints. A marked increase in leukocytes, along with the presence of rhogocytes, indicates a robust immune response. The substantial elevation in lymphocyte subpopulations such as CD3+, CD8+, CD16+, and CD20+ cells reflects a heightened T-cell mediated and innate immune response. The increased levels of CD95+ lymphocytes highlight enhanced apoptotic activity, a critical aspect of RA pathogenesis.

The significant rise in immunoglobulin levels (IgG, IgM, IgA) in RA patients points to active B-lymphocyte involvement. This finding suggests that B-cell activation and differentiation play a crucial role in RA, contributing to the chronic inflammatory state.

The immune response in RA varies with the radiologic stages. In early stages, there is an intensified immune response, whereas in more advanced stages, a shift towards increased apoptosis readiness and T-cell activation is observed. This progression highlights the dynamic nature of immune involvement in RA.

These findings emphasize the importance of synovial fluid analysis in diagnosing RA, monitoring its progression, and guiding treatment strategies. The distinct immunological patterns across different radiologic stages of RA can serve as valuable biomarkers for disease progression and response to therapy. This understanding can aid in the development of targeted treatments, potentially slowing the progression of joint deformities and improving patient outcomes.