Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFNgamma production

Abstract

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN gamma production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFNgamma but was enhanced by prostaglandin E(2) (PGE(2)). IL-23-induced IL-17 production was increased by PGE(2) and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFNgamma-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFNgamma but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNFalpha, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFNgamma production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

Fig. 1.

The IL-23/IL-17 axis is essential for the neutrophil migration in antigen-induced arthritis. (A) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or its vehicle (saline, Sal) in immunized (mBSA Im, closed bars) or sham-immunized (Sham-Im, open bars) mice treated with a co-injection of IgG control (α-CTL), α-IL-23 (700 ng/cavity), or α-IL-17 (700 ng/cavity) antibodies. (B) The levels of IL-23 and IL-17 in joint homogenate were determined 3 h after challenge with saline or mBSA (10 μg/cavity) in immunized mice. (C and D) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-23 (1–10 ng/cavity, closed bars) or saline (Sal, open bar) in mice treated with a co-injection of IgG control (α-CTL), anti-IL-23 (5 μg/cavity), or anti-IL-17 (10 μg/cavity) antibodies. *P < 0.05 vs. saline control group; ^#P < 0.05 vs. IL-23 or mBSA (immunized) groups. Data are mean ± SEM, n = 5, representative of 3 experiments.

Fig. 2.

IL-23-induced neutrophil migration in AIA is prostanoid dependent. (A) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-23 (closed bars in the right side of the panel) in naïve mice or 24 h after intra-articular injection of mBSA (10 μg/cavity) or saline (Sal) in immunized (mBSA Im, closed bars) mice or mBSA in sham-immunized (Sham-Im, open bar) mice treated 30 min before with indomethacin (Indo, 0.5, 2.7, or 5 mg/kg, s.c.) or etoricoxib (Etori, 45 mg/kg, s.c.) or not treated. (B) Expression of COX-2 mRNA in peritoneal cells harvested 0.5, 1.5, and 3 h after IL-23 (10 ng/cavity) injection into mouse peritoneal cavity. (C) The levels of PGE₂ in joint homogenate were determined 3 and 12 h after challenge with saline (Sal) or mBSA (10 μg/cavity) in immunized mice. (D) The levels of PGE₂ in cultured lymph node cells stimulated with IL-23 (100 ng/ml) or saline (Sal, IL-23 diluent) for 36 h. *P < 0.05 vs. IL-23 or mBSA (immunized) groups. Data are mean ± SEM, n = 5, representative of 3 experiments.

Fig. 3.

PGE₂ enhances IL-23-induced neutrophil recruitment by increasing IL-17 synthesis via suppressing the IL-12/IFNγ axis. (A) Neutrophils harvested from the articular cavity 24 h after intra-articular injection of mBSA (1 or 10 μg/cavity) or saline (Sal) in immunized (mBSA-Im, closed bars) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar) mice treated with a co-injection of PGE₂ (30 pg/cavity), IL-12 (10 pg/cavity), IFNγ (100 pg/cavity), or α-IFNγ antibody (700 ng/cavity). Some mice were pretreated 30 min earlier with indomethacin (Indo, 5 mg/kg, s.c.), as indicated. (B and C) Neutrophils harvested from articular cavity 6 h after intra-articular injection (1 or 10 ng/cavity) or saline (open bar) in wild-type, IL-12p40^−/−, or IFNγ^−/− mice. Some mice were treated with a co-injection of PGE₂ (1 pg/cavity), IL-12 (0.1 ng/cavity), or IFNγ (1 ng/cavity) or were pretreated with indomethacin (Indo, 5 mg/kg, s.c. 30 min earlier). (D and E) Lymph node cells harvested from immunized mice were cultured with or without mBSA (100 μg/ml), indomethacin (Indo, 50 μg/ml), etoricoxib (Etori, 50 μg/ml), PGE₂ (1 μM), α-IFNγ (10 μg/ml), or α-IL-23 (100 ng/ml) for 36 h. IL-17 and IFNγ concentrations in culture supernatants were determined by ELISA. *P < 0.05 vs. medium (RPMI) controls; ^#P < 0.05 vs. mBSA (10 μg/cavity; immunized), mBSA (100 μg/ml), IL-23 (10 ng/cavity) or IL-23 (100 ng/ml); ⁺P < 0.05 vs. IL-23 (1 ng/cavity) or mBSA (1 μg/cavity; immunized). Data are mean ± SEM, n = 5, representative of at least 2 experiments.

Fig. 4.

IL-17 mediates neutrophil migration via TNFα, leukotrienes, and CXC chemokines (CXCL1 and CXCL-5). (A and C) Neutrophils harvested from articular cavity 6 h after intra-articular injection of IL-17 (1–30 ng/joint), IL-23 (10 ng/cavity), or saline (Sal, open bars) in mice treated with a co-injection of α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies. Some mice also were treated 30 min before with repertaxin (RPTX, 30 mg/kg, s.c.) or 1 h before with MK886 (1 mg/kg, by gavage). (B) Neutrophils harvested from articular cavity 24 h after intra-articular injection of mBSA (10 μg/cavity) or saline in immunized mice (mBSA Im, closed bars) or mBSA (10 μg/cavity) in sham-immunized (Sham-Im, open bar) mice treated with a co-injection of IgG control (α-CTL), α-TNFα serum (5 μl/cavity), α-CXCL1 (700 ng/cavity), or α-CXCL5 (700 ng/cavity) antibodies or 1 h before with MK886 (1 mg/kg, by gavage). *P < 0.05 vs. saline control; ^#P < 0.05 vs. IL-23, IL-17, or mBSA (immunized) groups. Data are mean ± SEM, n = 5, representative of 3 experiments.

Fig. 5.

IL-17-mediated neutrophil chemotaxis depends on CXC chemokines released by these cells. (A) Neutrophils were harvested from bone marrow of mice (IL-12p40^−/−, IFNγ^−/− and their control C57BL/6, or 5-LO^−/− and their control 129S1/SvImJ [129]), treated for 30 min with indomethacin (Indo, 50 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (1–100 ng/ml), MIP-2 (positive control, 20 ng/ml) or RPMI (vehicle). (B) Neutrophils were harvested from bone marrow of C57BL/6 mice and treated for 30 min with IgG control (α-Ctl, 30 μg/ml), anti-CXCR2 antibodies (30 μg/ml), or repertaxin (RPTX, 30 μg/ml). Chemotaxis was determined in a microwell chamber initiated by stimulation for 1 h with IL-17 (50 ng/ml) or RPMI (vehicle). (C) The levels of CXCL1 in culture supernatants of neutrophils harvested from bone marrow of C57BL/6 mice stimulated or not with IL-17 (50 ng/ml) for 1 h. *P < 0.05 vs. RPMI; ^#P < 0.05 vs. IL-17. Data are mean ± SEM, representative of at least 3 independent experiments. Data presented in the RPMI control group are representative for the basal migration of all mouse strains.

Fig. 6.

Schematic representation of IL-23-induced neutrophil migration in antigen (mBSA)-induced arthritis. Lines terminating in blunt ends indicate inhibition. During antigen-induced neutrophil migration to the joints, IL-23 is released early and, together with several cytokines, stimulates IL-17 production that induces the release of TNFα, the CXC chemokines (CXCL1 and CXCL5), and LTB₄, which together contribute to neutrophil recruitment by inducing locomotion and the expression of adhesion molecules. IL-23 elicited during antigen challenge also can induce COX2 expression leading to the production of PGE₂, which contributes to neutrophil recruitment by enhancing the IL-23-induced production of IL-17 through the impairment of the IL-12/IFNγ axis. PGE also can induce IL-23 production via dendritic cells (40), thus providing an IL-23-COX2-PGE-IL-23 amplification circuit, perpetuating neutrophil migration and inflammation.