The roles of IL-19 and IL-20 in the inflammation of degenerative lumbar spondylolisthesis

The antibodies of IL-19, IL-20, IL-20R1, and IL-20R2, and IL-1β monoclonal antibodies were purchased from R&D Systems, Minneapolis, MN. Monocyte chemotactic protein (MCP)-1and tumor necrosis factor (TNF)-α were purchased from Sigma-Aldrich Co., St Louis, MO.

Thirteen consecutive patients (4 men; 9 women; average age: 68 years old) who had been diagnosed with DLS of L4–5 and undergone posterior spinal surgery of L4–5 including posterior decompression, posterior instrumentation, and posterior lateral fusion with or without posterior lumbar interbody fusion in National Cheng Kung University Hospital were enrolled in this study. The written informed consents for participation in the study were obtained from participants, and no participants are children. All the procedures were approved by the Human Experiment and Ethics Committee of National Cheng Kung University Medical Center (IRB approval: ER-96-163.), and were done in accordance with the Guidelines of the Declaration of Helsinki. The DLS diagnosis was based on the clinical presentation of lower back pain with sciatica or intermittent claudication and a radiograph showing slippage of theL4–5 lumbar vertebrae; Meyerding grades 1 or 2, with the degenerative changes of disc space narrowing; facet joint hypertrophy; and endplate spurs [2, 14]. All patients received MRI examination, which revealed degenerated discs (one patient with Grade II, four patients with Grade III, six patients with Grade IV, and one patient with Grade V based on Pfirrmann’s criteria [28]), and hypertrophic facet joints (eight patients with bright signal on T2 weighted image), and buckling of ligamentum flavum as well as lateral recess or central stenosis over L4–5 level. Surgical indications included progressive motor deficit, cauda equina syndrome, intermittent claudication, mechanical back pain or persistent sciatica of L5 after failed conservative treatment of more than three months. We collected the surgical specimens of degenerated intervertebral disc, facet joint, and ligamentum flavum tissue from the thirteen elderly patients with DLS receiving posterior spinal surgery. A block of L4–5 facet joint (symptomatic side) along with the capsule and synovial tissue was harvested using a bone chisel, ligamentum flavum of L4–5 harvested using a curved curette, and intervertebral disc of L4–5 harvested using a disc rongeur. A half retrieved disc tissues were used for immunohistochemical staining, and another half disc tissues were collected for isolation of disc cells.

Immunohistochemically (IHC) staining with the monoclonal antibodies for IL-1β, TNF-α, MCP-1, IL-19, IL-20, IL-20R1, and IL-20R2 were investigated to analyze the expression of these cytokines and chemokines and the procedures was briefly described as below. Tissue samples were rinsed twice in phosphate-buffered saline (PBS), stored overnight in 3.7% formaldehyde, dehydrated and then embedded in paraffin. Paraffin-embedded sections were then deparaffinized, rehydrated and antigen-retrieval with Tris-EDTA buffer (10 mmol/L Tris Base, 1 mmol/L EDTA, 0.05% Tween-20, pH 9.0) at 95–100 °C for 30 min for immunohistochemistry staining. Sections were then incubated with 3% H₂O₂ to block intrinsic peroxidase, after washing, the sections were incubated with blocking reagent for 30 min and then with the monoclonal antibodies against IL-19, IL-20, IL-20R1, and IL-20R2 (as described previously [29]), TNF-α (1:50 diluted), IL-1β (1:100 diluted), and MCP-1(1:20 diluted) at 4 °C for overnight. Isotype mouse IgG₁ was used as the negative control. After washing thrice with PBST (PBS-0.05% Tween-20), sections were then incubated with HRP conjugated secondary antibody for 40 min at room temperature. After washing thrice with PBST, DAB chromogen was incubated with sections at room temperature for 2–10 min (ImmPACT™ DAB, Vector Laboratories, Burlingame, CA USA). And then counter stained with hematoxylin (Merck, Darmstadt, Germany). After washing with running tap water, the sections were then dehydrated and then cleared in xylene and mounted with mounting medium (Entellan®, Merck, Darmstadt, Germany). At least two sections from each patient’s specimen were analyzed for IHC staining. The whole section from each patient’s specimen was evaluated for each case. At least 100 cells for each cell type were counted. Specific cell types were deemed positive if > 1% of the cells were stained. The immunostained sections were evaluated by two senior pathologists in an observer-blinded fashion.

The human IVD cells at the L4–5 level were isolated from a half of degenerated disc tissues after the surgery of posterior lumbar interbody fusion in patients with DLS. The primary cultures of IVD cells from the degenerated disc were collected as described [22, 30] for further in vitro study. The disc tissues were digested with 0.2% collagenase type II (Worthington, Lakewood, NJ) in Dulbecco modified Eagle medium and Ham F-12 medium (DMEM/F12) (Gibco, Grand Island, NY) for 6 h. After enzymatic digestion, the suspension was centrifuged and washed with medium. The isolated IVD cells were cultured in serum-free DMEM/F12 medium, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/L l-glutamine (Gibco, Grand Island, NY).

To mimic the hypoxic condition for elucidating the in vitro effect of IL-19 or IL-20 on the disc cells, primarily isolated IVD cells (3 × 10⁵ cells/well) were cultured in serum-free DMEM/F12 medium and incubated with IL-19 or IL-20 under CoCl₂-mimicked hypoxic condition for 6 h. CoCl₂ only (hypoxic condition control), and PBS only (normoxic condition control) as the control groups. Total RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA) and underwent reverse transcription using SuperScript III (Invitrogen) according to the manufacturer’s instructions. The expression of mRNA was analyzed using quantitative PCR with gene-specific primers for IL-1β, IL-6, IL-8, TNF-α, VEGF, and MCP-1. Detection of amplified template was accomplished with SYBR Green I (Applied Biosystems) chemistry using a StepOnePlus detection system. Individual PCR products were analyzed using melting point analysis. Samples were heated from 50 to 95 °C, and the decline in fluorescent signals of each individual sample were assessed. The fluorescence/time-dependent generation of signals was assessed using the manufacturer’s software program. For calculation of the relative expression, we normalized to the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and compared it with the mean control values.

To explore whether IL-19 and IL-20 are involved in the pathogenesis of DLS, we investigated the expression profile of IL-19, IL-20, IL-20R1, and IL-20R2 in different degenerated tissues of disc, facet joint, and ligamentum flavum from patients with DLS using IHC staining with specific antibodies (Fig. 1). In this study, it is difficult for us to quantify the cytokine levels in IHC staining because there are different cells in the degenerated tissue of facet joint, including fibroblasts, fibrocytes, chondrocytes, and synoviocytes; therefore, if there were positively stained cells in tissue specimen of the patients with DLS, the result of the IHC staining was categorized to have positive staining. To identify the specific sources of the cytokines, we mainly analyzed the immune reactivity of those cytokines and receptors in chondrocytes and fibrocytes/fibroblast in facet joint tissue, and both disc and ligamentum flavum tissues. We found that IL-19, IL-20, and receptors of IL-19 and IL-20 (IL-20R1 and IL-20R2) were positively stained in three different tissues, including disc, facet joint, and ligamentum flavum tissues from patients with DLS (Fig. 1 and Table1). To further clarify whether IL-19/IL-20 expression pattern is associated the other proinflammatory cytokines in DLS, we also analyze the expression pattern of three critical proinflammatory cytokines (IL-1β, TNF-α, and MCP-1) using IHC staining with specific antibodies. The ratios of positive immune reactivity of IL-19, IL-20, IL-1β, TNF-α, MCP-1, IL-20R1, and IL-20R2 in the discs, facet joints, and ligamentum flavum of patients with DLS was analyzed and summarized in Table 1.

In the discs of DLS patients, IL-19 was detected in chondrocytes in 3 of 6 samples (50%) and fibrocytes/fibroblasts in 1 of 6 samples (16.7%). IL-20R1 and IL-20R2 were stained in the chondrocytes in 6 of 6 disc tissues (100%), whereas IL-20 was only stained in chondrocytes in one disc tissue (1/6, 16.7%). TNF-α and MCP-1 were detected in chondrocytes in 3 of 6 samples (50%) and in fibrocytes/fibroblasts (2/6, and 1/6, respectively). IL-1β was only detected in 1 of 6 disc samples in chondrocytes (16.7%, Table 1).

In the facet joints of DLS patients, IL-19 and IL-20 were detected in chondrocytes in the facet joint tissues (6/12 and 5/12, respectively). IL-20R1 and IL-20R2 were generally detected in chondrocytes (12/12 and 12/12, respectively) and fibrocytes/fibroblasts (8/12 and 12/12, respectively) in facet joint tissues. TNF-α, MCP-1, and IL-1β were also detected in chondrocytes and fibrocytes/fibroblasts of the facet joint tissue. IL-1β was expressed in a higher ratio in facet joint tissue than in disc tissue (66.7% versus 16.7%, respectively). IL-1β was mainly detected in the chondrocytes (8/12, 66.7%) in comparison with fibrocytes/fibroblasts (2/12, 16.7%). TNF-α was detected in chondrocytes (10/12, 83.3%) and in fibrocytes/fibroblasts (10/12, 83.3%). MCP-1 was detected in the chondrocytes (9/12, 75%) and fibrocytes/fibroblasts (5/12, 41.7%) in the facet joint tissues (Table 1 and Fig. 1b).

In the ligamentum flavum of DLS patients, IL-19 and IL-20 were positively stained in chondrocytes (6/13 and 3/13, respectively) and fibrocytes/fibroblasts (3/13 and 3/13, respectively) in the ligamentum flavum tissues. IL-20R1 and IL-20R2 were generally detected in chondrocyte (12/13 and 12/13, respectively) and fibrocytes/fibroblasts (10/13 and 13/13, respectively). TNF-α and MCP-1were stained in chondrocytes (6/13 and 6/13, respectively) and fibrocytes/fibroblasts (7/13 and 2/13, respectively). IL-1β was only detected in chondrocytes (5/13) but not in fibrocytes/fibroblasts (0/13) (Table 1 and Fig. 1c).

According to the results of IHC staining of degenerated tissues of DLS, IL-19 and IL-20 were increased staining intensity and accompanied by abundant expression of TNF-α, IL-1β, and MCP-1 in facet joints of DLS patients except the equal expression of IL-19 in chondrocytes of disc tissues. Interestingly, IL-19 and IL-20’s receptors (IL-20R1 and IL-20R2) were expressed on chondrocytes and fibrocytes/fibroblasts in the disc, facet joint, and ligamentum flavum tissues from patients with DLS.

To further clarify the association of IL-19/IL-20 expression pattern with other proinflammatory cytokines TNF-α, IL-1β, and MCP-1 in degenerated tissues of DLS, we used correlative analyses and found that the expression of IL-20 and IL-1β in facet joint was significantly correlated (P = 0.018, Table 2), whereas none of the cytokine expression was significantly correlated in disc and ligamentum flavum.

We previously showed that IL-20 and its receptors were expressed in human herniated intervertebral disc (HIVD) tissues [22]. In this study, we further compared the expression of IL-19, IL-20, and their receptors (IL-20R1 and IL-20R2) in the discs between elderly patients with DLS and adult patients with HIVD. It was of interest that the percentages of positive staining of IL-19 and IL-20 were higher in the discs from adult patients with HIVD (83% and 65%, respectively) than in those from elderly patients with DLS (50% and 17%, respectively). Besides, IL-20R1 and IL-20R2 were all expressed in disc tissues from DLS patients (100% and 100%, respectively) and from HIVD patients (90% and 85%, respectively, Fig. 2). These data indicated that disc cells might be the possible target cells for IL-19 and IL-20 involved in the pathogenesis of DLS and HIVD.

Cartilage and disc tissues are hypoxic and avascular tissues. Previous studies indicated that hypoxia-inducible factors (HIF-1α and HIF-2α) are expressed in this tissue and play a role during intervertebral disc degeneration [10‐12]. To mimic the pathological environment of degenerative lumbar spondylolisthesis, we used CoCl₂-mimicked hypoxic conditions to increase HIF-α for in vitro culture system. To further clarify the role of IL-19 and IL-20 in DLS, we investigated whether IL-19 or IL-20 altered the in vitro expression levels of other proinflammatory cytokines and chemokines in the disc cells under CoCl₂-mimicked hypoxic conditions. Real-time PCR showed that both IL-19 and IL-20 induced the expression of IL-1β, IL-6, IL-8, TNF-α, VEGF, and MCP-1 in isolated disc cells (Fig. 3). In addition, we observed that IL-19 had a stronger effect than IL-20 for inducing cytokine and chemokine expression in disc cells derived from DLS patients.