Elevated levels of TNF-α, IL-1β and IL-6 in the synovial tissue of patients with labral tear: a comparative study with hip osteoarthritis

This study was approved by our Institutional Review Board (approval number: B19–259) and conducted based on the ethical standards of the 1964 Declaration of Helsinki and its later amendments. Written consent to participate was obtained from all participants. A total of 130 subjects who had ST samples extracted during an operation to treat HOA from October 2013 to March 2019 were enrolled. All subjects received non-steroidal anti-inflammatory drugs (NSAIDs) preoperatively. We excluded subjects with inflammatory diseases, including neoplastic disease and rheumatoid arthritis, and those with a history of hip surgery. Further, subjects were also excluded if examination of their samples failed due to laboratory error. Finally, we included 106 hips from 100 patients, with 32 hips with LT receiving hip arthroscopic surgery and 74 hips with late-stage OA receiving THA. HOA grade was assessed radiographically according to the Kellgren-Lawrence (K-L) classification [14]. Pain at rest and on activity was scored on a 0 (no pain) to 10 (worst pain imaginable) cm visual analog scale (VAS). In patients who received hip arthroscopy, we also determined the presence of FAI according to the proposed diagnostic criteria of the Japan Hip Society [15].

ST samples were extracted based on the gross and arthroscopic appearance of sites showing redness during surgery. A single experienced clinician performed all synovial tissue extractions and radiological assessments throughout the study. ST samples were immediately transferred to liquid nitrogen before storage in a freezer at − 80 °C until use for RNA extraction. RNA was extracted from ST samples, and levels of TNFA, IL1B, IL6, and COX2 mRNA were compared among all patients using real-time polymerase chain reaction (PCR). ST samples extracted from 10 patients (n = 5 each) were immediately digested using collagenase and used for cell culture.

ST samples and cultured synovial cells were subjected to a total RNA extraction process using TRIzol (Invitrogen, Carlsbad CA, USA) according to the manufacturer’s instructions. The resulting RNA formed the template for first-strand cDNA synthesis of TNFA, IL1B, IL6, and COX2. Briefly, Moloney murine leukemia virus reverse transcriptase (SuperScript III RT, Invitrogen) was used in 25-μL reaction mixtures comprising 2 μL cDNA, 0.2 μM specific primer pair, 12.5 μL N′,N′-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine dye reagent (SYBR Premix Ex Taq, Takara, Kyoto, Japan), and nuclease-free water. Primers were designed in Primer Blast (http:// www.​ncbi.​nlm.​nih.​gov/​tools/​primer-blast/​) and synthesized by Hokkaido System Science Co., Ltd. (Sapporo, Japan). PCR primer pair sequences are shown in Table 1. Amplified sequences were investigated for specificity using melt curve analysis. Quantitative PCR conducted using a Real-Time PCR Detection System (CFX-96; Bio-Rad, CA, USA) was used to examine relative mRNA expression. PCR cycle parameters were as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. mRNA levels of test genes were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Correlations between gene expression and BMI and VAS were examined.

To determine whether TNF-α is involved in regulating inflammatory cytokines (IL1β, IL-6) and COX-2, synovial fibroblasts were extracted from hip ST removed from patients with LT and late-stage HOA (n = 5 each). Mononuclear cells were extracted from ST samples by digestion with 20 mL of 0.1% type I collagenase, and were subsequently cultured in α-minimal essential media (ɑ-MEM) supplemented with 10% fetal bovine serum at 1 × 10⁴ cells/cm² in six-well plates for 7 days. We confirmed that almost all cultured cells (> 90%) were positive for the synovial fibroblast marker, CD90, and negative for the pan hematopoietic marker, CD45, using flow cytometry (Additional file 1: Figure S1). After the 7 days in culture, passage 0 synovial cells were subjected to stimulation with control (ɑ-MEM) or 10 ng/mL human recombinant TNF-α for 1 day. To analyze the effects of TNF-α stimulation, mRNA was extracted from stimulated cells and TFNA, IL1B, IL6, and COX2 levels were determined using real-time PCR.

Patients with LT were significantly younger than those with late-stage HOA (p <  0.001). VAS score on walking was significantly higher in patients with late-stage HOA than LT (P = 0.024). There was no significant difference in VAS score at rest (P = 0.636). The proportion of patients with each K-L grade differed significantly between HOA and LT groups (P <  0.001). All patients with LT had K-L Grade 1 or 2 and underwent arthroscopy, with 27 cases receiving suture anchors and the others receiving arthroscopic debridement and synovium resection. FAI was identified in 12 (37.5%) patients with LT. All patients with late-stage OA had K-L Grade 3 or 4 and underwent THA (Table 2).

TNFA, IL1B, and COX2 mRNA levels in ST were significantly higher among patients with LT than those with late-stage HOA (TNFA, p <  0.001; IL1B, p < 0.001; COX2, p = 0.001; Fig. 1). In contrast, no significant differences were observed in IL6 mRNA levels between patients with LT and late-stage HOA (p = 0.078).

Previous studies have reported that inflammatory cytokine levels are elevated in the synovium of patients with FAI [13]. We therefore examined whether FAI was associated with elevated levels of TNFA, IL1B, IL6 and COX2. We found no difference in the expression of these genes between patients with LT with and without FAI (TNFA, p = 0.546; IL1B, p = 0.559; IL6, p = 0.599; COX2, p = 0.124; Fig. 2).

No correlation was found between TNFA, IL1B, IL6 and COX2 expression levels and VAS and BMI in either the LT or HOA group (Table 3). However, IL6 expression was positively correlated with BMI in the LT group (ρ = 0.403, P = 0.022; Table 3).

BMI was correlated with gait pain in the HOA group (ρ = 0.292, P = 0.012; Table 4) but not in the LT group (ρ = − 0.130, P = 0.479; Table 4). No correlation was observed between pain at rest and BMI in either the LT (ρ = − 0.050, P = 0.746; Table 4) or HOA group (ρ = 0.341, P = 0.112; Table 4).

Given that we found differences in the expression of inflammatory mediators between patients with LT and late-stage HOA, we next evaluated whether synovial cells were functionally different between these groups of patients. Age and the proportion of patients with each K-L grade differed significantly between HOA and LT patients (age, P < 0.001; K-L grade, P < 0.001; Table 5). Compared to vehicle control, TNF-α stimulation significantly increased inflammatory cytokine (IL1B, IL6) and COX2 expression in synovial fibroblasts collected from patients with LT and late-stage HOA (IL1B, p = 0.043 and p = 0.043; IL6, p = 0.043 and 0.043; COX2, p = 0.043 and p = 0.080, respectively; Fig. 3). However, no differences in mRNA levels were observed between LT- and HOA-derived cell cultures following TNF-α stimulation.