Adrenomedullin protects from experimental arthritis by down-regulating inflammation and Th1 response and inducing regulatory T cells

Abstract

Rheumatoid arthritis is a chronic autoimmune disease of unknown etiology characterized by chronic inflammation in the joints and subsequent destruction of the cartilage and bone. The present study proposes a new strategy for the treatment of arthritis: the administration of the immunomodulatory neuropeptide adrenomedullin. Treatment with adrenomedullin significantly reduced incidence and severity of collagen-induced arthritis, an experimental model of rheumatoid arthritis, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of adrenomedullin was associated with a striking reduction of the two deleterious components of the disease, ie, the Th1-driven autoimmune and inflammatory responses. Adrenomedullin also induced the generation and/or activation of efficient CD4+ CD25+ regulatory T cells in arthritis with capacity to suppress autoreactive response and restore immune tolerance, which could play a pivotal role in the therapeutic effect of adrenomedullin on experimental arthritis contributing to the restoration of immune tolerance.

Figure 1

AM decreases CIA incidence and severity. A: Severity of arthritis, assessed by clinical scoring or paw thickness measurement, in mice with established CIA injected intraperitoneally (arrow) either with PBS (control) or with AM (2 nmol/mouse) daily for 5 days or on a unique pulse. Numbers in parentheses represent incidence of arthritis (percentage of mice with arthritis score >2 at day 50). Images show representative examples of the paw swelling in mice of the different experimental groups. n = 8 to 16 mice per group. P < 0.001 versus control after day 32. B: Clinical scoring and disease incidence (parentheses) of CIA mice treated intraperitoneally daily (arrow) for 5 days with either with PBS or with different amounts of AM. n = 8 mice per group. P < 0.001 versus control for 1 to 10 nmol treatments after day 32. C: CIA mice were treated with CGRP (5 nmol/mouse) daily for 5 days from the onset of disease, and arthritis score was determined at day 45. Disease incidence for CGRP-treated mice was 100%. n = 5 mice per group. D: Histological analysis of trichromic-stained (top) or H&E-stained (bottom) sections of joints from CIA mice treated with PBS (control) or AM (2 nmol/mice, daily for 5 days). Arrows point to osteoclasts destroying bone. Scoring of inflammation, cartilage damage, and bone erosion of paws from untreated and AM-treated CIA mice. Myeloperoxidase activity measuring neutrophil infiltration in the joints. *P < 0.001 versus control.

Figure 2

AM inhibits inflammatory response in CIA. Systemic and local expression of inflammatory mediators in untreated (control) or AM-treated CIA mice assayed at day 35 after immunization. A: Cytokine/chemokine contents in joints. A paw from a nonimmunized mouse was analyzed simultaneously for assessment of the basal response. B: Serum TNF-α and IL-1β levels. n = 6 to 8 mice per group. *P < 0.001 versus controls. C: Cytokine/chemokine production by synovial membrane cells from CIA mice activated with CII in the absence or presence of different concentrations of AM or CGRP. n = 3 experiments performed in duplicate. *P < 0.001 versus control.

Figure 3

AM down-regulates Th1-mediated response in CIA. A: Proliferative response and cytokine production of DLN cells isolated at day 30 from untreated (control) or AM-treated CIA mice and stimulated in vitro with different concentrations of CII. Stimulation of DLN cells with anti-CD3 antibodies (▾, for untreated CIA mice; ▿, for AM-treated CIA mice) was used for assessment of nonspecific stimulation. A pool of three nonimmunized DBA/1 DLN cells was used for assessment of the basal response. No proliferation or cytokine production by T cells was detectable in the presence of an unrelated antigen (OVA). n = 5 mice per group. B: Number of CII-specific cytokine-producing T cells. DLN cells from untreated (control) or AM-treated CIA mice were restimulated in vitro with CII (10 μg/ml) and analyzed for CD4 and intracellular cytokine expression by flow cytometry. Dot plots show representative double staining for IFN-γ/TNF-α or IL-4/IL-10 expression in gated CD4 T cells. The number of IFN-γ-, IL-4-, and IL-10-expressing T cells relative to 10⁴ CD4 T cells is shown in the bottom panel. Data shown represent pooled values from two independent experiments. C: CII-specific proliferative response and number of cytokine-producing CD4 T cells in synovial membrane cells isolated from untreated (control) or AM-treated CIA mice and stimulated in vitro with CII (10 μg/ml) for 48 hours. Data show the results of pooled synovial cells from three animals per group. D: CII-specific IgG, IgG1, and IgG2a levels in serum collected at day 35 from untreated (control) or AM-treated CIA mice (8 to 12 mice per group). *P < 0.001 versus controls.

Figure 4

AM induces the emergence of regulatory CD4⁺CD25⁺ T cells in CIA. A: Flow cytometry analysis of DLN and synovial (joint) CD4⁺CD25⁺ cells isolated at day 30 from untreated (control) or AM-treated CIA mice. Numbers represent percentages of CD4⁺CD25⁻ and CD4⁺CD25⁺ cells. Histograms show the expression of CD45RB and Foxp3 in gated CD4⁺CD25⁻ and CD4⁺CD25⁺ cells in DLN. Similar profiles were observed for synovial cells. Dashed lines in histograms are isotype antibody controls. Data are representative of five mice per group. B: Number of CD4⁺CD25⁺Foxp3⁺ cells (black bars) and CD4⁺CD25⁻Foxp3⁻ cells (gray bars) per DLN and synovial membrane (joint) isolated from untreated (control) and AM-treated CIA mice (six mice per group). C: Proliferative response and cytokine production of autoreactive T cells isolated from CIA mice stimulated with spleen APCs and CII (10 μg/ml) in the absence (none) or presence of DLN T cells isolated from untreated (control) or AM-treated CIA mice. n = 3 experiments performed in duplicate. D: Proliferative response of autoreactive T cells (4 × 10⁵ cells) isolated from CIA mice co-cultured with increasing numbers of DLN T cells (regulatory T cells) from untreated (control) or AM-treated CIA mice (ratios from 1:64 to 1:1), and stimulated with CII (10 μg/ml) and splenic APCs. n = 3 experiments performed in duplicate. *P < 0.01 versus controls.

Figure 5

Involvement of Treg in the therapeutic effect of AM in CIA. A: CIA mice were treated with medium (control) or with AM (2 nmol/day) for 5 days and were treated with control Ig, anti-IL-10 (αIL10), anti-TGF-β (αTGFβ), or a combination of both antibodies on alternate days starting with the first AM administration (five to seven mice per group). Severity of arthritis was assessed by clinical scoring. P < 0.001 versus Ig control treatment for αIL10 and/or αTGFβ treatments after day 37. B: CD4⁺ cells were isolated from DLN of CIA mice treated with medium (CD4_control) or AM (CD4_AM) at the peak of disease and stimulated in vitro with CII. Some samples were depleted of CD25⁺ cells. Cells (5 × 10⁶) were injected intravenously into arthritic mice 2 days after disease onset (four to six mice per group). CIA mice without any treatment (untreated) were used as arthritic controls. P < 0.001 versus untreated control for CD4_AM treatment after day 32. P < 0.001 versus CD4_AM treatment for CD25-depleted CD4_AM cells after day 35.