Human Knee Synovial Fluid Cytokines Correlated with Grade of Knee ...

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Bulletin of the NYU Hospital for Joint Diseases 2011;69(2):122-7

Human Knee Synovial Fluid Cytokines Correlated with Grade of Knee Osteoarthritis A Pilot Study C. Thomas Vangsness, Jr., M.D., Wendy S. Burke, P.T., D.P.T., M.S., O.C.S., Steven J. Narvy, M.D., Robert D. MacPhee, Ph.D., and Alexander N. Fedenko, M.D., Ph.D.

Abstract The purpose of this pilot study was to evaluate the cytokine profile of human knee synovial fluid and correlate this with the subject’s degree of articular cartilage degradation, radiographic score, and synovial histology. Materials and Methods: Synovial fluid was withdrawn before knee meniscectomy in 12 subjects with varying degrees of osteoarthritis and assayed for 21 cytokines, using a multiplex cytokine assay and flow cytometry instrumentation. Articular cartilage surfaces were scored by a single orthopaedic surgeon on the basis of the International Cartilage Repair Society (ICRS) classification during the arthroscopy, and posterior-anterior knee radiographs were graded using the Kellgren-Lawrence (KL) classification. Synovial biopsies were taken in four zones at the time of surgery for histological analysis. Results: Significant concentration differences in IL-2, IL-5, MCP-1, and MIP-1 were found between subjects with advanced arthritis and subjects with little or no arthritis on the ICRS scale (p < .05). No such differences could be appreciated using KL scores. There was no correlation between C. Thomas Vangsness, Jr, M.D., is from the Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles. Wendy S. Burke, P.T., D.P.T., M.S., O.C.S., is from the Department of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles. Steven J. Narvy, M.D., is from the Keck School of Medicine, University of Southern California, Los Angeles. Robert D. MacPhee, Ph.D., is from the Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles. Alexander N. Fedenko, M.D., Ph.D., is from the Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles. Correspondence: C. Thomas Vangsness, Jr., M.D., Suite 2000, Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, 1520 San Pablo Street, Los Angeles, CA 90033; [email protected].

histology samples and visualized surface osteoarthritis. Conclusion: This data suggests a molecular basis of disease progression, with higher levels of cytokines indicative of greater degrees of osteoarthritis. These results add pilot data that can assist investigators in conducting a comparative observational study of the levels of inflammatory cytokines with radiologic and arthroscopic assessments of osteoarthritis.

T

he study of the synovium is critical to understanding the pathogenesis of arthritis and other joint diseases. The synovial membrane is a thin, weak layer of fibrous connective tissue, only a few cells thick, that lines the joint cavity. The synovial lining lacks a basement membrane and merges with the underlying loose connective tissue; the absence of the basement membrane allows for quick exchange between blood and synovial fluid.1 The synovium controls the environment within the joint by acting as a selectively permeable membrane that screens the entry and exit of nutrients and other molecules in the joint.1,2 It consists of three types of synovial cells: A cells (synovial macrophage), B cells (synovial fibroblast), and D cells (dendritic cells), with each cell type having a different morphology and expressing different surface antigens.1,3 Synovial fluid functions as a transport medium for nutritional substances to the avascular articular cartilage and aids in the mechanical function of joints by lubricating the articulating surfaces.2,3 It enters the cartilage by diffusion and is further dispersed by compression and relaxation of the cartilage during motion. The fluid originates from ultrafiltration through the rich vascular network of the synovial tissue, and type B synovial cells secrete hyaluronic acid into the mix to form synovial fluid.1,3 Physical and chemical changes that occur in the synovial fluid during disease reflect basic pathological processes, such as osteoarthritis, occurring in the joint. The characteristics of

Vangsness CT, Burke WS, Narvy SJ, MacPhee RD, Fedenko AN. Human knee synovial fluid cytokines correlated with grade of knee osteoarthritis: a pilot study. Bull NYU Hosp Jt Dis. 2011;69(2):122-7.


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Table 1 Kellgren-Lawrence (KL) Classification

knee synovial fluid have been defined for immunoglobins, glucose, fibrous cartilage, uric acid, and other proteins, yet little work has investigated cytokine levels in non-rheumatologic joint conditions. Cytokines are a family of non-antibody immunoregulatory proteins or other biological factors, including lymphokines, interleukins, tumor necrosis factors, and interferons. They are secreted by inflammatory leukocytes and some non-leukocytic cells, such as synovium and chondrocytes, as well as in response to injury or inflammation.4-7 Cytokines were originally identified and classified into families on the basis of their cellular origin and principal biological activities. Of interest here is the action of cytokines on the articular chondrocytes, which are active in maintaining the integrity of the articular matrix and play a role in overall joint homeostasis.6 Such cytokines can be classified as being anabolic, catabolic, anti-catabolic, and modulatory, based on their regulatory action on chondrocyte function at the articular surface:8-12 • Anabolic cytokines act as growth and differentiation factors on chondrocytes to increase synthetic activity. These include insulin-like growth factor 1 (IGF1) and transforming growth factor β (TGFβ). • Catabolic cytokines induce target cells to produce more matrix-degrading products. They include interleukin-1 alpha and beta (IL-1α and -1β), IL-6, IL-8, IL-17, IL-18, and tumor necrosis factor alpha (TNFα). • Anti-catabolic cytokines inhibit the activity of the catabolic cytokines. They include IL-4, IL-10, IL-13, and IL-1 receptor antagonist (IL-1ra). • Modulatory cytokines have the capacity to alter the activity of other cytokines in a variety of ways. They include IL-11 and leukemia inhibitory factor. The purpose of this study was to critically evaluate the status of knee articular cartilage and relate this to the concentration of 21 different cytokines in the synovial fluid. Additionally, these cytokine levels were correlated with synovial inflammation from four biopsy sites within the knee. Our hypothesis was that progressive arthritic changes confirmed by arthroscopy and radiographs would demonstrate increased levels of cytokines associated with osteoarthritis and increased evidence of synovial tissue inflammation.

and graded by this same orthopaedic surgeon, using the International Cartilage Repair Society (ICRS) criteria [Table 2].14 Tissue biopsies of the synovium were obtained under direct visualization from the superior patellar area, the medial and lateral gutters. and the anterior compartment. These tissue samples underwent H&E (hematoxylin and eosin) staining and analysis, and grading according to the College of American Pathologists criteria as mild, moderate, or severe. The synovial fluid from each patient was subsequently thawed and assayed for 21 cytokines, using a LINCOplex™ Immunoassay (Linco Research, Inc., St. Charles, Missouri), with Luminex® 100 flow cytometry instrumentation (Luminex® Corporation, Austin, Texas), according to the manufacturer’s instructions. Data was collected and analyzed using the Student’s t-test to compare each cytokine under both the ICRS and KL systems of classification.

Materials and Methods

Results

Twelve patients underwent an arthroscopy of the knee for a meniscal tear by a single orthopaedic surgeon, fellowshiptrained in sports medicine. Prior to arthroscopy, the degree of arthritis in the surgical knee was evaluated on a posterioranterior (PA) radiograph by the orthopaedic surgeon, using the Kellgren-Lawrence (KL) classification (Table 1).13 Under IRB (Internal Review Board) protocol, synovial fluid was withdrawn from consented patients following the induction of anesthesia and before the arthroscopy, and then frozen. During the arthroscopy, the tibial, femoral and patellar articular cartilage surfaces were visually examined, palpated,

Twelve patients were enrolled in this pilot study. Nine patients sustained medial meniscus tears (MMT), one patient sustained a lateral meniscus tear (LMT), and two patients sustained both an MMT and LMT. Using the ICRS classification, two patients were scored as class I, two were scored as class II, five were scored as class III, and three were scored as class IV. Using the Kellgren-Lawrence classification, three patients were scored as class I, four were scored as class II, two were scored as class II, and three were scored as class IV. These results are shown in Table 3. Immunoassay performance proved to be capable of yield-

0

Normal (absence of osteoarthritis)

1

Doubtful osteophyte

2

Minimal-definite osteophyte

3

Moderate joint space narrowing

4

Severe joint space narrowing

Table 2 International Cartilage Repair Society (ICRS) Classification 0

Normal cartilage

1a

Soft indentation

1b

Superficial fissures and cracks Lesions extending down to less than 50% of cartilage depth Defects extending down more than 50% of cartilage layer

2 3a 3b 3c 3d 4

Defects down to calcified layer Defects down to but not through the subchondral bone layer Delamination Severely abnormal, with penetration through subchondral plate

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Table 3 Patient Demographics Patient No. 1 2 3 4 5 6 7 8 9 10 11 12

Gender

Age at Surgery (Years)

KL

ICRS

Diagnosis

M M F M F F F M M M M M

18 73 74 79 63 49 59 54 58 16 50 40

2 2 4 4 1 2 3 4 3 1 2 1

1 3 4 4 3 3 2 4 3 3 2 1

MMT MMT, LMT MMT MMT, LMT MMT MMT MMT MMT MMT MMT LMT MMT

KL, Kellgren-Lawrence; ICRS, International Cartilage Repair Society. MMT, medial meniscus tear; LMT, lateral meniscus tear.

ing reproducible patterns when the cytokine panels were used to analyze replicate aliquots of a single synovial sample. When grouped by severity of arthritis under the ICRS criteria (mild, I-II vs advanced, III-IV), a statistically significant difference in levels of IL-2, IL-5, MCP-1, and MIP-1 could be appreciated in patients with advanced arthritis (Figs. 1 and 2, p < .05). Borderline significance (p < .10) could be appreciated for IL-1β, IL-6, IL-8, IL-12p70, and IFNγ. When patients were grouped by the KL criteria, none of the cytokines tested were elevated to a statistically significant level, even setting the decision criteria to a significance level of 0.10 (Figs. 3 and 4). Histological analysis demonstrated no qualitative difference in cellular inflammation for either the ICRS or KL cartilage surface grading. Figure 5A demonstrates normal synovial histology, and can be compared with a representative portrayal

of the synovial biopsies with varying degrees of inflammation, as shown in Figure 5B, C, and D.

Discussion To our knowledge, this is the first study to extensively evaluate and profile cytokines in the synovial fluid of the osteoarthritic knee in humans, using high-throughput flow cytometry. Prior studies have investigated this linkage using antibody-based radioimmunoassays in rheumatologic joint injury. Of particular interest is the study by Raza and colleagues,15 in which a test for 23 cytokines in rheumatoid synovium revealed a distinct but transient cytokine profile in patients with synovitis, destined to develop rheumatoid arthritis that was significantly different from that of patients with established disease.

Figure 1 Overall knee cytokine profile by the International Cartilage Repair Society (ICRS) classification.


125

Figure 2 Cytokine profiles for grouped ICRS classification. 300

250

200

150

Group I (mean); n = 3 Group II (mean); n = 4 Group III (mean); n = 2 Group IV (mean); n = 3

100

50

0

Figure 3 Overall knee cytokine profile by KL Classification.

300

250

200

150

Grade I-II (mean); n = 7 Grade III-IV (mean); n = 5

100

50

0

Figure 4 Cytokine profiles for grouped Kellgren-Lawrence (KL) classification.

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Figure 5 A, Normal synovial tissue. B, Mild synovial inflammation. C, Moderate synovial inflammation. D,Severe synovial inflammation.

The optimal manner in which to correlate a specific disease process with changes in cytokine levels requires the analysis of each sample for multiple cytokines. The most common method for the quantitation of secreted cytokines is the enzyme-linked immunosorbent assay (ELISA). Though ELISA shows good sensitivity and specificity, it has many drawbacks in assaying a panel of multiple cytokines in one sample. As detailed by Martins and coworkers,16 each ELISA can measure only one cytokine per well and requires up to 200 µL of sample per test. When attempting to assay six or seven cytokines, sample volume limitations become an issue, especially when working with tissue culture supernatant. Even with assays from the same manufacturer, sample dilutions, labeled antibodies, and incubation times vary greatly between tests, making it difficult to run several assays at once. Several research groups have validated the use of flow cytometry as an alternative to ELISA for measuring various biomarkers, including cytokines.17-19 Other studies have demonstrated linearity in results for multiplex cytokine assays used in conjunction with flow cytometry.16,20-23 However, these studies tested a limited number of cytokines and did not deal explicitly with synovial fluid. Synovial fluid, being a viscous, complex aggregate of hyaluronic acid and other proteins, brings forth unique procedural and technical challenges not found in culture matrices or in serum where osmolarity is generally a constant. Therefore, finding an appropriate dilution that best yields linearity for all cytokines tested is crucial in delivering accurate results. We tested several dilution parameters and confirmed linear, reproducible measures for cytokines in human knee synovial fluid. As noted in Figures 1 to 4, a pattern in the cytokine profile between arthritic and healthy knees emerged when patients were grouped under either the ICRS or KL classification system; statistical significance in this pilot study, however, could only be appreciated with grouping by ICRS criteria. Because

the ICRS categorization is based on direct visualization of the joint surface during arthroscopy, it can be assumed that the ICRS scale provides more accurate representation of the true degree of arthritis than may be evident on radiographs. Accordingly, the lack of apparent statistical significance in the KL groupings is attributed to miscategorization of the true joint condition on the basis of radiographic findings. The finding that IL-1 and TNFα, both noted in the literature as major contributors to cartilage catabolism in osteoarthritis, were not elevated in the advanced arthritis group as compared to the mild arthritis group in this study, is unexpected. Within the ICRS classification, based on the statistically elevated levels of MIP-1α (macrophage inflammatory protein-1α, which is known to induce the synthesis and release of IL-1 and TNF), borderline significance of IL-8 (a stimulator of TNF production) and borderline significance detected for IL-1β itself, we believe that sample size limitations are the likely cause. The findings of a statistically elevated level of MCP-1 (monocyte chemoattractant protein-1, linked to the recruitment of osteoclast precursors), and borderline elevation of the proinflammatory cytokines IFNγ, IL-12 (which stimulates production of IFNγ), and IL-6 are consistent with the clinical manifestations of osteoarthritis. Interestingly, both ICRS and KL data show overall higher levels of cytokines for grade III than for grade IV. As noted above, developing rheumatoid conditions have demonstrated a transient elevation in cytokine levels that was not present in patients with established disease. It is possible that a similar phenomenon occurs during the pathogenesis of osteoarthritis, that is, once extensive damage to the articular surface has occurred (grade IV on both scales), cytokine levels decline to a lower, albeit still elevated, level. Also worth noting is the finding of elevated cytokine levels even among the nonarthritic patients, compared to the normal level in uninjured knees described by Cameron and associates.24 Our results are compatible with the pattern of elevated cytokine levels detected by antibody staining among arthritic patients found by Smith and colleagues.25 No correction could be made in this study with respect to tissue inflammation on biopsies that were graded using the criteria of the College of American Pathologists. Limitations of this pilot study are primarily statistical. Given a small sample size of 12 patients, it is possible that the noted cytokine changes could be caused by another factor, aside from advancing severity of osteoarthritis, and, thus, would constitute a type I error. Such a problem could be overcome with a larger sample size, which would also increase statistical power and could demonstrate additional significant differences between study groups, irrespective of whether the cartilage surfaces were graded using the KL or the ICRS criteria. Comparable numbers of grade I-II subjects and grade III-IV subjects are also needed in order to make a meaningful comparison between the study groups. In the present study, this was not achieved under the ICRS classification: four subjects were grade I-II and eight


subjects were grade III-IV (as compared to seven subjects and five subjects, respectively, using the KL classification). In addition, the use of a healthy control group (grade 0 on both the KL and ICRS scales) could better characterize the biochemical manifestations of osteoarthritis at all stages of disease.

10.

11.

Conclusion To date, over 40 human cytokines have been identified as mediators in immune, autoimmune, inflammatory, and other responses in a variety of tissues and organs in the human body. This pilot study found that the profile of 21 of these cytokines from synovial fluid was technologically feasible, and increased knee osteoarthritis on the ICRS scale was associated with higher levels of IL-2, IL-5, and MCP-1 in knee synovial fluid. These results are not only of scientific interest, but also add pilot data to assist investigators in conducting a comparative observational study of the levels of inflammatory cytokines with radiologic and arthroscopic assessments of osteoarthritis. Further investigation using a larger number of study subjects will improve statistical power and may demonstrate a conclusive relationship. Disclosure Statement None of the authors have a financial or proprietary interest in the subject matter or materials discussed, including, but not limited to, employment, consultancies, stock ownership, honoraria, and paid expert testimony.

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