Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells

Abstract

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55(-/-) but not p75(-/-) mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55(-/-) mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

Figure 1.

Increased IL-17 and IFNγ production in CIA after blockade of TNFα. DBA/1 mice with CIA were treated with TNFR-Fc or isotype control mAb (100 μg/mouse on alternate days) from the time of disease onset. (A and B) LN cells were taken 10 d after disease onset and levels of IL-17 (A) and IFNγ (B) were determined by ELISA in the supernatants without further stimulation (Nil) or after stimulation with type II collagen (CII) or anti-CD3 mAb (CD3). Data show individual mice (n = 8; *, P < 0.05). (C) Clinical scores were assessed over the 10-d period in TNFR-Fc–treated and control mice. The data are representative of at least three experiments. Error bars show SEM.

Figure 2.

Amplification of Th17 and Th1 cell activity in p55 TNFR^−/− mice. LN cells from WT, p55 TNFR^−/−, and p75 TNFR^−/− mice were taken 14 d after immunization with type II collagen in CFA. (A) LN cells were either unstimulated or stimulated with collagen or with anti-CD3 mAb, and the level of proliferation was determined by [³H]thymidine incorporation. The percentage of CD4⁺ T cells in the LN was determined by flow cytometry on day 14 after immunization. (B) Levels of IL-17 and IFNγ were determined by ELISA. (C) The proportion of CD4⁺ cells in the LN producing IL-17 and IFNγ were determined by flow cytometry. Histograms show mean ± SEM (n = 8). *, P < 0.05; **, P < 0.01. Data are representative of two experiments.

Figure 3.

TNFα inhibits expression of IL-12/IL-23 p40. Thioglycolate-elicited macrophages were cultured in the presence or absence of 30 or 100 ng/ml TNFα for 8 h and then stimulated for a further 18 h with 1 ng/ml LPS. (A–C) Levels of p40, IL-1β, and IL-6 protein were determined in the culture supernatants by ELISA. (D) Relative levels of p40 mRNA from WT, p55 TNFR^−/−, and p75 TNFR^−/− LN cells 14 d after immunization were determined by real time PCR. Histograms show mean ± SEM (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

Blockade of IL-12/IL-23 blocks the expansion of Th1/Th17 cells. (A–C) LN cells from immunized WT (white bars) or p55 TNFR^−/− (gray bars) mice treated with control Ig (Ig) or rat anti–mouse p40 Ab (p40) were either unstimulated or stimulated with collagen or anti-CD3 mAb. Levels of IL-17 (A) and IFNγ (B) were determined by ELISA, and the proportion of CD4⁺ cells in the LN producing IL-17 and IFNγ were determined by flow cytometry (C). Histograms show mean ± SEM (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of two experiments.

Figure 5.

Anti-TNFα therapy prevents the accumulation of Th1/Th17 cells in the joint. Arthritic DBA/1 mice (n = 6) were treated once every 3 d for a total of 14 d with anti-TNFα mAb (TN3-19.12; 300 μg/mouse) or control Ab. CD4⁺ cells from the inguinal LN and joints (obtained by enzymatic digestion of synovial tissue) were analyzed for intracellular cytokine expression by flow cytometry after stimulation with PMA/ionomycin. A, CD4⁺IL-17⁺ cells in LN; B, CD4⁺IL-17⁺ cells in joints; C, CD4⁺INFγ⁺ cells in LN; D, CD4⁺INFγ⁺ cells in joints. **, P < 0.01. Data are representative of two experiments. Error bars show SEM.