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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Feb 1, 2012.
Published in final edited form as:
PMCID: PMC3260472
NIHMSID: NIHMS348040

Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship with symptoms

Abstract

Objective

Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.

Methods

Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.

Results

Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.

Conclusion

In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.

Keywords: Meniscectomy, meniscal injury, inflammation, synovium, synovitis

Introduction

Joint injury predisposes individuals to develop OA (1, 2). Among the most common knee joint injuries associated with increased OA risk are meniscal injuries. Recent longitudinal data from the Multicenter Osteoarthritis Study indicate that meniscal damage is associated with a 6-fold increased risk (OR 5.7, 95% CI 3.4–9.4) of developing radiographically visible OA changes (3). Furthermore, in patients with established OA, meniscal damage is associated with risk of progression (4). Anatomic patterns of meniscal tear are often utilized to discriminate between traumatic and degenerative meniscal pathology; traumatic tears occurring in an otherwise normal meniscus are reported to present with longitudinal (sometimes “bucket-handle” type tears) or radial orientations, while horizontal, flap or complex tears and maceration are interpreted as degenerative tears, i.e. those occurring in a meniscus structurally weakened by degenerative change (5). Both patterns of meniscal alteration are associated with elevated risk of OA (68), but risk associated with degenerative-type tears appears to be higher (9). Biomechanical factors play a role in the structural changes in both patterns of meniscal pathology, but cellular and molecular processes that lead to increased risk of OA are not understood. Furthermore, these injuries are often asymptomatic (10), and factors contributing to symptoms such as pain have not been defined.

In patients with OA, inflammation is one factor associated with risk of both progression of cartilage loss (11, 12) and symptoms (1315). Inflammation in OA joints manifests as synovial membrane (SM) mononuclear cell infiltration observed in early and late stages of disease (1620). However, it is unclear whether inflammation pre-dates or is a consequence of OA development. Roemer and colleagues (21) recently noted an association between meniscal damage and synovial effusion on MRI, but the cellular and molecular nature of this inflammation was not defined. Pessler et al. (22) noted a mild synovitis with histologic features similar to OA in a group of patients with “orthopedic arthropathies,” including some with meniscal tears. However, prevalence of inflammation in patients with meniscal injuries in the absence of preexistent OA has not been well studied.

The present study was designed to define the prevalence and characteristics of synovial inflammation in patients undergoing arthroscopic partial meniscectomy for traumatic meniscal injury in the absence of antecedent evidence of OA. Furthermore, we sought to determine whether synovial inflammation is associated with pre-operative clinical symptoms. A histologic scoring system to grade inflammation was validated using independent evaluators and comparisons made with previously characterized synovial tissue from patients with OA.

Materials and Methods

Patients

The study was approved by the Institutional Review Board (IRB) of the New England Baptist Hospital (NEBH), and all patients gave written, informed consent. Patients aged 18 to 60 years who suffered a traumatic knee injury and were scheduled for arthroscopic partial meniscectomy for treatment of symptomatic meniscal tears were recruited from the NEBH Department of Orthopedic Surgery. The inclusion criteria were patient recall of a knee injury which initiated their symptoms and occurred within six months of presentation, and a meniscal tear identified on pre-operative MRI and considered the cause of symptoms. We excluded: (i) those with known inflammatory arthritis or symptoms to suggest systemic inflammatory arthritis (i.e. > 30 minutes of morning stiffness, multiple joint complaints, concurrent back pain) (ii) patients with radiographic evidence of OA (osteophytes or joint space narrowing), and (iii) patients with meniscal tears affecting the vascular portion of the meniscus thought to be amenable to surgical repair rather than resection. The latter was done to increase homogeneity of the patient population. For comparative evaluation of histopathology, meniscectomy patients were compared to a group of 20 knee OA patients whose synovium had been previously biopsied (20). These patients included 14 with advanced knee OA who met ACR (clinical + radiographic) criteria and had undergone total knee replacement surgery and 6 patients with earlier-stage knee OA who had undergone arthroscopic surgery with intra-operative evidence of cartilage loss or fibrillation, but without advanced radiographic changes (Kellgren-Lawrence x-ray scores ≤ 2).

Outcome scores

The Short form-12 (SF-12 ®) health surveys and Lysholm questionnaires were administered pre-operatively. In addition, patients were asked to assess their knee pain on a visual analog scale (VAS). The Lysholm questionnaire is a knee-specific instrument for measuring symptoms (pain, swelling, limp, locking, instability) and functional disability (stair-climbing, squatting, use of supports) on a single scale (0–100). Originally developed to assess responses to ligamentous repairs (23), this instrument has been validated in patients undergoing meniscal procedures (24, 25). In contrast, SF-12® captures information on general physical and emotional well-being.

Assessment of meniscus and cartilage integrity

Surgical reports were available for 28 patients, and were reviewed to determine anatomic pattern of meniscal pathology (degenerative vs. traumatic). Cartilage integrity was assessed intra-operatively by the operating surgeon and noted in operative reports using the Outerbridge scoring system (26): 0=normal articular cartilage, 1=superficial softening, 2=superficial fissuring or fibrillation involving <1.25 cm area, 3=fibrillation or fissuring >1.25 cm area, 4=full-thickness cartilage wear with exposed subchondral bone. When operative reports were not available (n= 5) or cartilage condition was not recorded clearly (n=6), videotapes taken during surgery were reviewed by the principal surgeon, Brian McKeon, MD, who determined Outerbridge scores. The worst score from all three compartments (medial, lateral, patello-femoral) was utilized in this analysis.

Synovial tissue collection and preparation

Tissue from patients undergoing meniscectomy was obtained from three locations: suprapatellar pouch, medial and lateral gutters. Biopsies were taken from areas of synovium that appeared inflamed or thickened. When no inflammation was apparent, biopsies were taken from standard locations: the femoral aspects of the medial and lateral gutters, and the central supratrochlear region in the suprapatellar pouch. Biopsies from knee OA patients had been obtained only from the suprapatellar pouch (20). Tissue biopsies were formalin-fixed and paraffin-embedded before sectioning and H&E staining.

Histologic assessment of synovial inflammation

Both meniscectomy and OA synovial specimens were subjected to the same assessment protocol. To standardize evaluations, we analyzed only sections containing a clearly recognizable lining layer with underlying vascularized subintima, and evaluated inflammation at low-power (5X objective). As there are no published reports on synovial infiltrates in patients with meniscal injury only, inflammation was graded based on perivascular mononuclear cell infiltration in synovium from OA patients (20, 27) as follows: grade 0=none, grade 1=mild (0–1 perivascular aggregates per low-power field); grade 2=moderate (> 1 perivascular aggregate per low power field with or without focal interstitial infiltration); grade 3=marked aggregates (both perivascular and interstitial). To evaluate inter- and intra-reader reliability, 18 synovial specimens were scored by two independent readers (E.D., C.R.S.) and 8 were re-scored by one blinded reader (E.D.).

Synovial gene expression microarray analysis

Eight synovial biopsies were chosen for microarray analysis, four each from meniscectomy patients with synovial inflammation (grade 1 or 2) or without synovial inflammation (grade 0). The biopsies were from different patients and anatomic locations varied. Total RNA was extracted from homogenized SM samples using PerfectPure® RNA Fibrous Tissue kits (5Prime Inc., Gaithersburg, MD). All RNA was DNAse-treated, oligo-dT primed, and cDNA synthesized with SuperScript III® Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA). RNA integrity was determined by electrophoresis on a microfluidics-based platform (Agilent Technologies, Santa Clara, CA). RNA was hybridized to Affymetrix human U133 plus 2.0 chips at the Weill Cornell Medical College Core Facility. Data were analyzed using Genespring 10.0 software (Agilent Technologies) as follows. Raw data were transformed using the RMA algorithm with baseline transformation to the median of all arrays. Probesets were filtered by expression (20–100%), with the requirement that probes be present in at least 4 of 8 arrays. An unpaired t test was done on the filtered data. We found 3030 probesets differentially expressed in synovial inflammation samples (p<0.05); 260 were differentially expressed with a >2-fold difference. Pathway over-representation analysis was done utilizing algorithms available via the Innate DB database (http://www.innatedb.ca/index.jsp) (28). Innate DB is a database of genes, proteins, interactions and signaling responses involved in mammalian innate immune responses. Targets were then chosen for validation by real-time qPCR.

Quantitative PCR analysis

After accounting for the histological analysis, there was sufficient tissue available from 37 biopsies for analysis of gene expression by real-time PCR. Twelve of the 37 specimens were suprapatellar, 14 from the medial and 11 from the lateral gutter, and represented biopsies from 18 patients. RNA was extracted and cDNA synthesized as described for the microarray analysis. mRNA levels of four chemokines and one chemokine receptor identified by pathway analysis of the microarray data (IL-8, CCR7, CCL19, CCL21 and CCL5) were measured by real-time PCR using specific primers (sequences available upon request) and iQ Sybr-Green Supermix (BioRad, Hercules, CA). Primers spanned introns and yielded a single product. After normalizing Ct values to GAPDH, expression levels were calculated relative to the mean of specimens without inflammation.

Statistical Analysis

Inter- and intra-reader reliability of inflammation scores is reported as a weighted kappa statistic. Given the small sample size and some irregularly distributed variables, nonparametric tests were used. Between-group differences were evaluated with Mann-Whitney t-tests, and Spearman’s correlation coefficients were calculated using Prism 5.0 software (GraphPad, Inc., San Diego, CA). Generalized Estimating Equations (GEE) with an unstructured correlation matrix were applied when multiple data points per patient were analyzed (relative expression levels), to adjust for lack of independence of the data. Multiple linear regression analysis was performed to examine the association between suprapatellar inflammatory score and baseline Lysholm scores. Age, gender, BMI and time between injury and surgery were included as independent covariates.

Results

Patient characteristics

Thirty-three patients who met criteria were recruited. Demographics of these patients and OA patients used for comparison are presented in Table 1. For meniscectomy patients, the median interval between knee injury and surgery was 14.8 weeks (range 1–42 weeks). Most (26, 82%) had medial meniscal tears; six had lateral tears; and one both medial and lateral tears. Surgical reports were available for 28 patients; twenty-five indicated the presence of complex tears (with horizontal cleavages and flap lesions, one described as macerated). Only two had radial tears, one with both a medial radial and lateral complex tear, and two were unrecorded. Despite excluding patients with radiographic OA, only seven patients (21%) exhibited Outerbridge grade zero (normal) cartilage in all compartments. The remainder had grade 1 (n=6), grade 2 (n=7), or grade 3 (n=7) lesions in one or more compartments, with 6 exhibiting focal grade 4 chondral lesions but no diffuse full-thickness cartilage loss. Median Body Mass Index (BMI) was similar in meniscectomy and OA patients, but OA patients were older (medians, 64 vs. 48 years, p<0.0001) and more likely to be female (Fisher’s exact test, p<0.05).

Table 1
Patient Characteristics

Histologic assessment of synovial inflammation

In five cases, biopsies were mishandled or mislabeled during specimen procurement and preparation, resulting in only 28 patients contributing biopsies sufficient for evaluation. Inflammation was graded 0–3 in both meniscectomy and OA patients. Inter-rater and intra-reader weighted kappas for the inflammation score were 0.87 and 1.0 respectively. Figure 1 shows photomicrographs of biopsy specimens from representative meniscectomy patients with typical grade 0, 1 and 2 inflammation. None exhibited grade 3 inflammation.

Figure 1
Histology of synovial membrane inflammation in meniscectomy patients. Synovial biopsies from meniscectomy patients taken at the time of surgery were fixed, embedded, and thin-sectioned before being stained with Haematoxylin and Eosin. Inflammation was ...

Prevalence and anatomic variation of inflammatory infiltrates

The three locations biopsied in meniscectomy patients were compared first. Inflammation was observed most often in suprapatellar biopsies (43%, or 12/28), compared with medial or lateral (26%, 7/27) gutters. When suprapatellar inflammation was observed, it was often found in at least one gutter as well (7/12). Five patients exhibited suprapatellar inflammation only; two had inflammation only in the gutters. When analyzed according to side (medial or lateral) of meniscal injury (ipsi- or contralateral) there was no predilection for inflammation in the gutter on the side of the meniscal pathology (data not shown). We next compared extent (grade) and prevalence of synovial inflammation in meniscectomy and OA patients. As biopsies from OA patients had been taken from the suprapatellar pouch (27), comparison was made only at this location. Inflammation was observed less often in meniscectomy than in OA patients (Table 1; 43% vs. 75%), and tended to be of lower grade.

Association of inflammation with patient characteristics and Lysholm scores

Meniscectomy patients were stratified according to presence (n=12, grade 1–2) or absence (n=16, grade 0) of suprapatellar inflammation. Lower Lysholm scores (indicating greater knee-related symptoms/disability) were observed in patients with suprapatellar inflammation than in patients without (difference between means = −19.9, 95% CI −9.20 to −30.7, p=0.0008). No significant differences in SF-12® (−0.85, 1.08 to −2.79) or VAS scores (0.44, 2.27 to −1.40) were observed. Patients with inflammation were significantly older (51.3±7.3 years vs. 40.2 ±11.6, p=0.007), and interval between injury and surgery was significantly shorter (10.2±8.8 weeks vs. 18.5 ± 11.5, p=0.047). Inflammatory infiltrates were observed in some patients presenting for surgery within a few weeks of their reported injury. Although difference in mean Outerbridge cartilage scores was not significant, patients with synovial inflammation tended to have higher Outerbridge scores (2.3±1.2 vs. 1.3±1.5, p=0.07). Only one of seven patients with normal (grade 0) cartilage demonstrated inflammation. Of the six patients with focal grade 4 lesions, five were female, but otherwise these six were not clearly distinguishable from the rest, and Lysholm scores varied widely (40–90). Synovial biopsies were available for four of six: two exhibited synovial infiltrates (grade 1); two did not. There was no correlation between Outerbridge scores and Lysholm scores (r=0.03, p=0.86).

Multivariate analysis

Multiple linear regression analysis was performed to determine whether the relationship between suprapatellar synovial inflammation and Lysholm scores was independent of known OA risk factors and degree of cartilage abnormality. Age, gender, Outerbridge score, BMI and time between injury and surgery were included as independent covariates. Both inflammatory score (p=0.001, effect estimate −15.3 ± 4.7 per point) and BMI (p=0.004, effect estimate −1.3 ± 0.4 per kg/m2) were significantly associated with Lysholm score after adjusting for the above variables. Outerbridge score (p=0.69) and age (p=0.30) were not.

Analysis of synovial gene expression in patients with or without synovial inflammation

Figure 1 shows H & E stained sections from one non-inflammatory biopsy (panel a) and one inflammatory biopsy (panel c) subjected to microarray analysis of gene expression patterns. Two-hundred sixty genes were differentially expressed ≥2 fold (p<0.05) between biopsies with and without inflammation. Inflammatory pathway over-representation analysis (28) of differentially expressed genes revealed twenty-two “pathways” (transcripts which cluster into functional categories or molecular pathways) significantly enriched with corrected p values < 0.05. We focused on the seven clusters which included more than three gene products (Table 2). Of these seven, a signature of chemokines and their receptors was the top up-regulated pathway in biopsies exhibiting inflammation. The six transcripts in this signature are shown in Table 3, with their respective fold-change and p-values. Of other pathways identified (Table 2), “Primary Immunodeficiency” and “Hematopoietic cell lineage” were composed of cell surface receptors and genes associated with infiltrating leukocyte populations (i.e. CD19, IL2RG, IL7R, CIITA, CD1D, CD2). Three additional pathways included overlapping lists of cytokine receptor chains (IL2RB, IL2RG), an intra-cellular signaling molecule (JAK3), and cytolytic enzymes (GZMA, GZMB) expressed by T and NK cell populations. This was expected since we had defined inflammation as perivascular mononuclear cell aggregates, which are largely composed of lymphocytes (27). The seventh pathway, “Cytokine-cytokine receptor interactions,” was comprised of the same six chemokine/receptor transcripts and cytokine receptor chains (IL2RB, IL2RG). For the purpose of the present analysis, we focused our attention on the chemokines because of their potential contribution to early events in lymphocyte accumulation in synovium.

Table 2
Pathways analysis of the 260 transcripts differentially expressed in inflammatory vs. noninflammatory synovial biopsies.
Table 3
Chemokines and chemokine receptor transcripts identified by pathway analysis of microarray data.

Validation of chemokine expression by real-time PCR

mRNA levels of four chemokines and one chemokine receptor identified by microarray pathway analysis (IL-8, CCR7, CCL19, CCL21 and CCL5) were measured by real-time PCR. All available biopsies yielding sufficient cDNA quantities were utilized (37 samples representing 18 patients). Samples were stratified by inflammation score (±) and relative analyte expression levels were compared. Figure 2 demonstrates that relative expression levels of IL-8 (panel a), CCL5 (panel b), CCR7 (panel c) and CCL19 (panel d) were all detected more frequently in biopsies exhibiting inflammation. Each patient contributed up to three biopsies in this analysis, so GEE models were run on log-transformed data to adjust for lack of independence. The GEE model revealed statistically significant relationships between inflammation, CCL19 (p= 0.0096) and CCL5 (p=0.0307), and trends with IL-8 (p=0.0649) and CCR7 (p=0.066). CCL21 was undetectable in most specimens (data not shown).

Figure 2
Real-time PCR validation of A. IL-8, B. CCL5, C. CCR7 and D. CCL19 measured in thirty-seven synovial biopsies from eighteen meniscectomy patients. RE = Relative Expression calculated relative to the mean in biopsies without inflammation, after normalizing ...

Association of chemokine expression with baseline Lysholm scores

Associations between chemokine expression in suprapatellar biopsies and clinical outcome scores were assessed by Spearman correlation. CCR7 (Figure 3a) and CCL19 (Figure 3b) expression showed strong negative associations with Lysholm scores (r=−0.790, p=0.002 and r=−0.783, p=0.002, respectively) in meniscectomy patients. Higher Lysholm scores indicate a less symptomatic knee. IL-8 (r=−0.54, p=0.07) and CCL5 (r=−0.38, p=0.2) were moderately but not significantly associated with Lysholm scores. No associations were observed between chemokine expression and VAS pain or SF-12 scores.

Figure 3
Association of chemokine levels and Lysholm scores. Chemokine expression was measured by real-time PCR in suprapatellar synovial biopsies (n=12 for CCR7, n=9 for CCL19) from meniscectomy patients. Associations between chemokine expression and Lysholm ...

Discussion

Emerging evidence indicates synovitis is related to OA symptoms and progression (1115). Synovial inflammation and effusions also occur with meniscal injuries (21), even in patients without radiographic OA. However, cellular and molecular characteristics of synovitis associated with meniscal damage have not been reported. We sought to determine the prevalence and molecular features of synovial inflammation in patients who were (i) without preexistent radiographic features of OA, and (ii) undergoing arthroscopic meniscectomy for clinically-documented traumatic knee injury with MRI evidence of meniscal tears. Specifically, we wanted to determine whether synovial inflammation correlated with symptoms.

A previous study (22) reported similar synovial pathology in OA patients and patients with joint injury. We therefore compared histologic features of synovial inflammation in meniscectomy patients to those in patients with established knee OA (27). Appearance of cellular infiltrates was similar, but inflammation was less prevalent and extensive in meniscectomy patients. Contrary to what we anticipated, we did not see preferential localization of inflammation in the gutter on the side of the meniscal tear. One possible explanation is we did not analyze the perimeniscal SM directly adjacent to the tear. However, of the three locations biopsied, inflammation was identified most often in the suprapatellar location (in 43% of patients) suggesting synovial inflammation occurs within the joint at sites distant from the injury and is not only localized adjacent to the injury. Our findings are consistent with a recent report of the anatomic distribution of synovitis in knee OA defined by contrast-enhanced MRI techniques (34). In that study, the suprapatellar region was the second most common area (59.5% of patients) in which synovitis was detected. The explanation for involvement of the suprapatellar region remains unclear. We speculate certain sites within the joint may be uniquely sensitive to effects of proinflammatory factors produced in response to meniscal injury.

We investigated whether inflammation was associated with preoperative joint symptoms and dysfunction. When stratified according to presence or absence of suprapatellar inflammation, Lysholm scores were significantly lower (p<0.05) in patients with inflammation. Lower Lysholm scores indicate greater knee-related symptoms. No differences in SF-12® or VAS pain scores were observed. The Lysholm score is a knee-specific metric of symptoms and functional disability (25). It is a weighted score, with pain and instability-related symptoms having most weight (25 points each of 100 total). In contrast, the VAS scale only reflects knee pain, and the SF-12® health survey is not specific for knee-related issues. The unique association of inflammation with Lysholm scores and not VAS pain suggests symptoms other than pain (e.g., instability, swelling) captured by the Lysholm scale might account for this difference. The weighting of the scale may also contribute to our observation. In the future, other knee-specific instruments such as the KOOS (29) – in which pain, other symptoms, and function can be independently evaluated – may be helpful in addressing this question. Whether inflammation is a cause or consequence of knee-specific symptoms in these patients (such as mechanical instability introduced by meniscal damage) needs to be evaluated.

We next looked at patient characteristics (age, BMI, degree of cartilage abnormality, and time elapsed between injury and surgery) in the stratified data. Age and BMI (30) are known risk factors for OA. In this cohort, older patients were more likely to demonstrate synovial inflammation, but BMI did not differ with inflammation. We anticipated that infiltration of cells would increase with time elapsed between injury and surgery, but this did not appear to be true. Patients with inflammation tended to have shorter time intervals between injury and surgery. A possible explanation is that increased inflammatory symptoms prompt earlier intervention; however the present analysis did not address this issue. Multivariate analysis indicated the association between inflammation and Lysholm scores is independent of age, BMI, and interval between injury and surgery.

Our results cannot be generalized to all patients with meniscal tears. We studied a population in which an identifiable injury precipitated symptoms, and whose injuries did not involve the vascular portion of the meniscus. Also, despite a clear history of trauma most patients exhibited complex meniscal lesions. Although we excluded patients with radiographic OA, most patients demonstrated grade 1–4 Outerbridge cartilage lesions suggesting this population is enriched for patients with pre-radiographic disease. These observations show the presence of early joint degeneration in the majority of these patients (reviewed in (31)). Synovial inflammation is associated with progression of cartilage loss in patients with established OA (11, 12), thus we determined whether inflammation was related to the degree of cartilage abnormality. There was a trend toward greater inflammation in patients with cartilage abnormalities, but our multivariate model demonstrated that the association between inflammation and Lysholm scores was independent of degree of cartilage abnormality. Our finding of synovial inflammation in one of seven patients with normal cartilage suggests in some cases of meniscal injury, synovitis may pre-date cartilage changes. This finding is consistent with an earlier study noting synovial immune complex deposits in patients with normal cartilage undergoing meniscal surgery (32). It is possible synovitis contributes to alterations in structural and mechanical properties of meniscal tissues, resulting in susceptibility to meniscal injury and increased risk for development or progression of OA.

To obtain insight into molecular mediators that contribute to synovial inflammation, we did microarray analysis of synovial RNA. Four biopsies from patients with inflammation (grade 1 or 2) and four without (grade 0) were compared. Two hundred and sixty genes were differentially expressed between these two patient groups (>2 fold change). Pathway analysis, with a focus on genes involved in innate immune responses (28), revealed a set of chemokine and chemokine receptors among the most highly upregulated transcripts in biopsies with inflammation. Expression of these genes (Table 3) within synovium may promote recruitment of inflammatory cellular infiltrates, so we focused on this gene set for validation by real-time PCR.

We chose five genes for validation by real-time PCR: IL-8, CCL5, CCR7, CCL19, and CCL21. With the exception of IL-8, these belong to the “C-C” chemokine family which influences recruitment of monocytes, lymphocytes and eosinophils. IL-8, a “C-X-C” chemokine, promotes neutrophil chemotaxis to sites of inflammation. Although first described as a T-lymphocyte recruitment factor, CCL5 (or RANTES) has pleiotropic effects on multiple leukocyte subsets. CCR7 is the cognate receptor for both CCL19 and CCL21, which are involved in T-lymphocyte and dendritic cell migration. Interaction between these chemokines and their receptor mediates homing to secondary lymphoid tissues and appropriate migration of cells within lymphoid follicles (reviewed in (33)). Our analysis revealed that IL-8, CCR7 and CCL19 transcripts were often undetectable in specimens without inflammation (Fig 3), and GEE analysis demonstrated that relative expression levels of CCL5 and CCL19 were associated with inflammation, consistent with our microarray results. Levels of CCR7 and CCL19 transcripts, which represent a ligand/receptor pair, were strongly associated with Lysholm scores (Fig 4).

There are limitations to analysis of large datasets, such as those obtained by microarray, as they are prone to false positive results and not easily replicated (35). In the present study, our application of pathway analysis to this expression dataset lends face validity to our findings, as genes are clustered functionally as well as statistically. Furthermore, chemokines identified by this high-throughput technique were validated using the more accurate method of quantitative PCR applied to a larger set of patients. Still, the biomarker potential of the chemokine signature needs to be validated in larger, prospective studies to determine if these gene expression profiles have any diagnostic or prognostic biomarker potential. Patients enrolled in this study are being followed for 2 years, and their clinical course assessed to determine whether the inflammatory response and/or chemokine expression is associated with short- and long-term outcomes after arthroscopy. Further limitations of our present study include the small sample size and the cross-sectional design. However, given the role of these chemokines in recruitment of inflammatory cells, we speculate they may contribute to development of synovial inflammation in response to meniscal injury. Determining whether they directly affect development of pain or progression of cartilage damage will require further investigation.

Identification of cellular and molecular mechanisms associated with synovial inflammation is of considerable interest not only for development of potential diagnostic or prognostic markers in early symptomatic OA and meniscal injury, but also for development of therapeutic approaches to control clinical symptoms and potentially reduce risk of joint degeneration in patients with knee injuries. Our study provides insight into mechanisms driving inflammatory infiltration and demonstrates an association between synovial inflammation and clinical symptoms in patients with meniscal injury, irrespective of the presence of underlying cartilage degeneration.

Acknowledgments

Supported by funding from:

The New England Baptist Hospital Bone and Joint Institute, Boston, MA The Hospital for Special Surgery, New York, NY

American College of Rheumatology Research and Education Fund, Within Our Reach (SRG)

The Atlantic Philanthropies, American College of Rheumatology Research and Education Fund, John A. Hartford Foundation, and the Association of Specialty Professors (Career Development Award to CRS)

The authors would like to acknowledge the following individuals: Fae Williams for clinical coordination for portions of the project, Kumar Bharat Rajan, PhD for biostatistical support, Tibor Glant MD PhD and Anna Laszlo for guidance in microarray data analysis, and David Hunter MD for his input into the histologic score validation. In addition, we thank the Weill Cornell Medical College Microarray Core Facility for their technical expertise in performing microarray data collection.

Footnotes

Disclosures:

Rush University Medical Center and the Hospital for Special Surgery have filed a provisional patent application based upon results presented in this manuscript. BM owns stock in Parcus, Inc. and Conformis, Inc.

DML has equity in Synostics, Inc and is currently employed by Novartis Pharma AG. SRG has received consultant fees and honoraria from Merck Serono, Bone Therapeutics, Roche and Novartis.

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