IL-32 and IL-17 interact and have the potential to aggravate osteoclastogenesis in rheumatoid arthritis

Abstract

Introduction: Interleukin (IL)-32 and IL-17 play critical roles in pro-inflammatory responses and are highly expressed in the synovium of patients with rheumatoid arthritis (RA). We investigated the relations between these two cytokines (IL-17 and IL-32) for their ability to induce each other and to stimulate osteoclasts in RA fibroblast-like synoviocytes (FLSs) and T cells.

Methods: FLSs were isolated through surgical synovectomy obtained from patients with RA or osteoarthritis (OA). Real-time PCR were performed to evaluate the expression of IL-32, IL-17 and osteoclast-related genes. Immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to determine the distribution of inflammatory cytokines and the presence of osteoclastogenesis.

Results: IL-17 induced the expression of IL-32 in the FLSs from RA patients, as assessed by microarray. IL-32 production was increased by IL-17. IL-32 in the FLSs from RA patients induced the production of IL-17 in CD4+ T cells. IL-32 and IL-17 were colocalized near TRAP-positive areas in joint specimens. IL-17 and IL-32 synergistically induced the differentiation of osteoclasts, as demonstrated by the expression of osteoclast-related genes. IL-32 and IL-17 also could induce resorption by osteoclasts in a RANKL-dependent manner.

Conclusions: IL-17 affected the expression of IL-32 in FLSs of RA patients and IL-32 induced the production of IL-17 in CD4+ T cells. Both IL-17 and IL-32 cytokines can reciprocally influence each other's production and amplify the function of osteoclastogenesis in the in RA synovium. Separately, IL-17 and IL-32 each stimulated osteoclastogenesis without RANKL. Together, the two cytokines synergistically amplified the differentiation of osteoclasts, independent of RANKL stimulation.

Figure 1

IL-17 induced expression of interleukin (IL)-32 via NF-κB and PI3 kinase in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). (A) FLSs from RA patients (RA FLSs) were cultured with increasing doses (1 and 5 ng/ml) of IL-17 for 12 h. IL-32 mRNA levels were measured by real-time PCR. ***P < 0.001 (in comparison with nil), ** P < 0.01 (in comparison with nil). (B) RA FLSs were pretreated with the signal inhibitors parthenolide (10 μM) or LY294002 (10 μM) for 2 h and then cultured with IL-17 (10 ng/ml) for 12 h. The IL-32 mRNA level was measured by real-time PCR. *P < 0.05 (in comparison with IL-17), **P < 0.01 (in comparison with IL-17). A and B show the means ± SD of more than three separate experiments. (C) Expression of IL-17, IL-32, phospho-IkB (p-IkB), NF-κB (p50), NF-κB (p65), phospho-Akt (p-Akt) and AKT in the synovium of patients with RA or osteoarthritis (OA). Immunostaining was performed using specific antibodies. Data are representative of three experiments with similar results.

Figure 2

Interleukin (IL)-17/T helper (Th)17-induced IL-32 expression from rheumatoid arthritis (RA) patients. (A) Production of IL-32 by RA fibroblast-like synoviocytes (FLSs) in contact with CD4⁺ T cells. RA FLSs and CD4⁺ T cells from healthy donors were co-cultured. FLSs (1 × 10⁵) were cultured with CD4⁺ T cells (1 × 10⁶) with or without anti-IL-17 blocking antibody (10 μg/ml). IL-17 production was then measured by sandwich ELISA, and the IL-32 mRNA levels of RA FLSs were determined by real-time PCR. ***P < 0.001 (compared with FLS+CD4⁺ T cells). (B) Induction of RA FLS IL-32 production by the supernatant of Th17-polarized cell cultures. RA FLSs and the culture supernatants of Th17-polarized cells from healthy donors were co-cultured. CD4⁺ T cells were incubated with membrane-bound anti-CD3 antibody (2 μg/ml), IL-6 (5 ng/ml), IL1β (5 ng/ml), IL-23 (10 ng/ml), TGF-β (5 ng/ml) with or without an anti-IL-17 blocking antibody incubated for 2 h before the next incubation) for 3 days to induce Th17.polarization. FLS (1 × 10⁵) were cultured with the culture supernatants of these Th17 polarized cells. IL-17 production was measured by sandwich ELISA and the IL-32 mRNA levels in RA FLSs were determined by real-time PCR. **P < 0.01 (compared with FLSs + culture supernatant of Th17 cells), *** P < 0.001 (compared with FLSs + culture supernatant of Th17 cells). The data are representative of three experiments with similar results.

Figure 3

Interleukin (IL)-32 induces the production of IL-17 in human CD4⁺ T cells. (A) CD4⁺ T cells from healthy donor human peripheral blood mononuclear cells (PBMCs) were cultured with membrane-bound anti-CD3 antibody and with/without recombinant human IL-32 (5 ng/ml) for 3 days. IL-17 production was measured by sandwich ELISA. **P < 0.01 (compared with anti-CD3). (B) Flow cytometry analysis of the expression of IL-17A by CD4⁺ T cells treated with membrane-bound anti-CD3 antibody (0.5 μg/ml) and with/without IL-32 (5 ng/ml) for 3 days. These cells were stained with anti-CD4-Percp cy7.7 and anti-IL-17-FITC, to determine the percentage of IL-17⁺ cells in the CD4⁺ gated population. *** P < 0.001 (compared with anti-CD3). (C) IL-17 and RORγt mRNA levels were measured by real-time PCR in stimulated CD4⁺ T cells with a membrane-bound anti-CD3 antibody with/without IL-32 (5 ng/ml). * P < 0.05 (compared with anti-CD3), *** P < 0.001 (compared with anti-CD3). (D) CD4⁺ T cells from healthy donors were cultured with membrane-bound anti-CD3 antibody (0.5 μg/ml), anti-CD28 (1 μg/ml), anti-IL-4 (2 μg/ml), anti-IFN-γ (2 μg/ml), IL-1β (20 μg/ml) and IL-6 (20 ng/ml) to induce Th17 polarization, and with/without recombinant human IL-32 (5 ng/ml) for 3 days. *** P < 0.001 (compared with Th17) (A), (B, left panel) and (D) are representative of three experiments with similar results. (B, right panel) and (C) represent means ± SD of three separate experiments.

Figure 4

Interleukin (IL)-32 induces IL-17 production in an autoimmune arthritis mouse model. (A) CD4⁺ T cells were isolated from the spleens of collagen-induced arthritis (CIA) mice, an autoimmune arthritis model. The cells were cultured with membrane-bound anti-CD3 (0.5 μg/ml) and with/without IL-32 (5 ng/ml) for 3 days. IL-17 production was measured by sandwich ELISA. ** P < 0.01 (compared with anti-CD3). (B-D) Expression of IL-17A by CD4⁺ T cells cultured under Th17-polarizing conditions for 3 days: anti-CD3 (0.5 μg/ml), anti-CD28 (1 μg/ml), anti-IFN-gamma (2 μg/ml), anti-IL-4 (2 μg/ml), anti-IL-2 (2 μg/ml), IL-6 (20 ng/ml), TGF-beta1 (2 ng/ml) with/without IL-32 (5 ng/ml) and/or type ll collagen with irradiated antigen-presenting cells, and then stimulated for 4 h with PMA and ionomycin, followed by intracellular cytokine staining. The percentage of IL-17⁺ cells in the CD4⁺ gated population (B), *** P < 0.001 (compared with Th17) and cytokine level (C) were measured by sandwich ELISA, *** P < 0.001 (compared with Th17) and mRNA levels (D) were measured by real-time PCR, * P < 0.05 (compared with Th17). B-D are representative of three experiment with similar results. (E) Expressions of IL-17, IL-32 and tartrate-resistant acid phosphatase (TRAP) in the synovium of the CIA and IL-1Ra-knock-out (KO) mice, two autoimmune arthritis mouse models. The results shown are representative of five experiments with similar results.

Figure 5

Interleukin (IL)-17 and IL-32 synergistically induce osteoclastogenesis. (A) Tartrate-resistant acid phosphatase (TRAP) staining for identification of osteoclasts. Osteoclast precursors were cultured in the presence of IL-17 (0.1 ng/ml) or/and IL-32 (5 ng/ml) with macrophage colony-stimulating factor (M-CSF; 25 ng/ml). The receptor activator of the nuclear factor kappa-B ligand (RANKL; 30 ng/ml)-treated group was the positive control. The medium and stimulus were changed every 3 days. After 15 days, the cells were stained for TRAP activity. (B) Numbers of multinucleated TRAP-positive cells per well. TRAP-positive cells containing two or more nuclei were scored as osteoclasts. TRAP-positive cells were counted three times by blind scoring. *P < 0.05 (compared with M-CSF+IL-32+IL-17), **P < 0.01 (compared with M-CSF+IL-32+IL-17). (C) The mRNAs of TRAP, Cathepsin K, calcitonin receptor (CTR) and matrix metallopeptidase 9 (MMP9), as osteoclast markers were quantified by real-time PCR. *P < 0.05 (compared with M-CSF+IL-32+IL-17), **P < 0.01 (compared with M-CSF+IL-32)+IL-17), ***P < 0.001 (compared with M-CSF+IL-32+IL-17). (D) Formation (left) and percent area (right) of resorption pits by osteoclasts on dentine discs. Cell culture was performed as described with dentine discs in 96-well plates. The osteoclast precursors were cultured in the presence of IL-17 (1 ng/ml) or IL-32 (5 ng/ml) with M-CSF (25 ng/ml) and RANKL (30 ng/ml). At day 21, the cells were removed from dentine. Resorption area was evaluated by light microscopy and measured using the image analysis software. (A), (B) and (D) are representative of two or three experiments with similar results. (C) Means ± SD of more than three separate experiments.