Follistatin-like protein 1 promotes arthritis by up-regulating IFN-gamma

Abstract

Follistatin-like protein-1 (FSTL-1) is a poorly characterized protein that is up-regulated in the early stage of collagen-induced arthritis and that exacerbates arthritis when delivered by gene transfer. The current study was designed to determine the mechanism by which FSTL-1 promotes arthritis. FSTL-1 was injected into mouse paws, resulting in severe paw swelling associated with up-regulation of IFN-gamma transcript and the IFN-gamma-induced chemokine, CXCL10. Mice depleted of T cells were protected. A central role for IFN-gamma was confirmed by the finding that mice deficient in IFN-gamma failed to exhibit paw swelling in response to injection of FSTL-1. Furthermore, IFN-gamma secretion from mouse spleen cells exposed to a weak TCR signal was increased 5-fold in the presence of FSTL-1. FSTL-1 could be induced by innate immune signals, including TLR4 agonists and the arthritogenic cytokine, IL-1beta, via an NFkappaB pathway. Finally, FSTL-1 was found to be overexpressed in human arthritis and its neutralization inhibited murine collagen-induced arthritis and suppressed IFN-gamma and CXCL10 production in arthritic joints. These findings demonstrate that FSTL-1 plays a critical role in arthritis by enhancing IFN-gamma signaling pathways and suggest a mechanism by which FSTL-1 bridges innate and adaptive immune responses.

FIGURE 1

FSTL-1 enhances T cell IFN-γ production. a, DBA/1 male mice were injected i.p. with 0.4 mg of the anti-TCR mAb, H57 (black symbols). One week later (day 0), hind paws were injected with 1 × 10⁹ particles of Ad(FSTL-1) or Ad(BglII). Paw swelling above pretreatment baseline was measured. The data represent the mean ± SEM of eight paws/group for each condition. T cell depletion of H57-treated mice was confirmed at the end of the study by flow cytometry on spleen cells. p values are shown for Ad(FSTL1) vs Ad(FSTL1)+ H57. A similar group of mice were sacrificed on days 1, 3, 6, and 8 and mRNA from hind paws (n = 2/group) was extracted and amplified by real time PCR for IFN-γ (b), CXCL10 (c), or IL-1β (d). e, IFN-γ knockout mice and wild type BALB/c controls were treated as in a. The data represent four hind paws/group. f, DBA/1 spleen cells were cultured in medium containing 10% FCS for 3 days with or without FSTL-1 (5 μg/ml) in the presence of the indicated titers of the anti-CD3 mAb, 2C11. Supernatants were assayed for IFN- γ by ELISA. Each bar represents the mean ± SEM of triplicate wells.*, p < 0.05; **, p < 0.01 by paired t test. Experiments were performed three times to ensure reproducibility.

FIGURE 2

FSTL-1 is induced in response to mediators of innate immunity. a, Control and super IκB-expressing MC3T3 cells were cultured for 3 days in triplicate in medium, TGF-β (2 ng/ml), LPS (100 ng/ml), or IL-1β (10 ng/ml) and supernatants were assayed by ELISA for FSTL-1. Each bar represents the mean and SEM and p values are comparing each group’s medium control. DBA/1 mice were injected intradermally in the paws with 50 μg of LPS (b), 50 μl of CFA (c), or 5 × 10⁸ particles of Ad(IL-1β) (d). Mice were sacrificed at the indicated times and the paws were subjected to real-time PCR analysis. Each bar represents the mean ± SEM of four hind paws from two mice. No increase was seen in response to injection of buffer (data not shown).*, p < 0.05; **, p < 0.01 by paired t test. Experiments were performed twice to ensure reproducibility.

FIGURE 3

FSTL-1 is over-expressed in RA and CIA synovium. a, Human synovial tissues from RA patients (n = 5) or control patients undergoing knee arthroscopic anterior cruciate ligament repair (n = 12), and (b) paws from CIA mice on day 35 or untreated controls (5 paws/group) were subjected to real-time PCR for FSTL-1 mRNA. Each bar represents the mean ± SEM.*, p = 0.03; **, p = 0.0001 by paired t test.

FIGURE 4

Neutralization of endogenous FSTL-1 suppresses CIA. Affinity purified polyclonal rabbit anti-mouse FSTL-1 was shown to bind specifically to mouse FSTL1 by Western blot (a) where lane 1 is a m.w. marker standard, lane 2 is 0.15 μg of purified mouse FSTL1 protein generated in Sf9 cells, lane 3 is 6 μl of day 2 serum from a DBA/1 mouse treated with 1 × 10⁹ particles of Ad(FSTL-1), and lane 4 is serum from a DBA/1 mouse treated with the control Ad(BglII). Mice were immunized with CII on days 0 and 21 and injected i.p. on the days indicated by the arrows with 200 μg of rabbit anti-FSTL-1 IgG or polyclonal rabbit IgG as a control (nine mice per group). A third group received no Ab. The hind paw arthritic index (b), the percentage of mice with a maximum arthritic index of 16 (c), and paw swelling above preimmunization baseline (d), were scored by a blinded observer. p values are shown for anti-FSTL-1 vs rabbit IgG and were performed by paired t test except for b which was by Exact Wilcoxon test. e, Hind paws from unimmunized mice (control) or from day 35 CII-immunized mice treated with anti-FSTL-1 or rabbit IgG (n = 6 mice/group) were subjected to real-time PCR for IFN-γ, CXCL10, and IL-1β mRNA. Each bar represents the mean ± SEM. Severe synovitis with destruction of cartilage and bone can be seen in representative day 35 paw sections from rabbit IgG-treated mice (f), while joints of anti-FSTL-1-treated mice (g) are well preserved. J = joint, S = synovium (magnification ×100). *, p < 0.05; **, p < 0.01.