Enhanced Th17-Cell Responses Render CCR2-Deficient Mice More Susceptible for Autoimmune Arthritis

CCR2 is considered a proinflammatory mediator in many inflammatory diseases such as rheumatoid arthritis. However, mice lacking CCR2 develop exacerbated collagen-induced arthritis. To explore the underlying mechanism, we investigated whether autoimmune-associated Th17 cells were involved in the pathogenesis of the severe phenotype of autoimmune arthritis. We found that Th17 cells were expanded approximately 3-fold in the draining lymph nodes of immunized CCR2−/− mice compared to WT controls (p = 0.017), whereas the number of Th1 cells and regulatory T cells are similar between these two groups of mice. Consistently, levels of the Th17 cell cytokine IL-17A and Th17 cell-associated cytokines, IL-6 and IL-1β were approximately 2–6-fold elevated in the serum and 22–28-fold increased in the arthritic joints in CCR2−/− mice compared to WT mice (p = 0.04, 0.0004, and 0.01 for IL-17, IL-6, and IL-1β, respectively, in the serum and p = 0.009, 0.02, and 0.02 in the joints). Furthermore, type II collagen-specific antibodies were significantly increased, which was accompanied by B cell and neutrophil expansion in CCR2−/− mice. Finally, treatment with an anti-IL-17A antibody modestly reduced the disease severity in CCR2−/− mice. Therefore, we conclude that while we detect markedly enhanced Th17-cell responses in collagen-induced arthritis in CCR2-deficient mice and IL-17A blockade does have an ameliorating effect, factors additional to Th17 cells and IL-17A also contribute to the severe autoimmune arthritis seen in CCR2 deficiency. CCR2 may have a protective role in the pathogenesis of autoimmune arthritis. Our data that monocytes were missing from the spleen while remained abundant in the bone marrow and joints of immunized CCR2−/− mice suggest that there is a potential link between CCR2-expressing monocytes and Th17 cells during autoimmunity

Treatment with anti-IL-17A antibodies reduces CIA in CCR2^−/− mice.

<p>CCR2^−/− mice were immunized with bovine type II collagen at day 0 and were treated 5 times in 2.5 weeks with either an anti-IL-17A antibody (Treated, n = 5) or an isotype control IgG1 (Control, n = 7) starting from day 14 after immunization. Arthritis score (A) and paw swelling (B) were recorded over time (Days). Data represent one of the two independent experiments performed. Differences of both arthritis scores and paw swelling during the course of disease between treated and control groups are significant (p<0.01).</p

Th17 cells expand in the draining lymph nodes of immunized CCR2^−/− mice.

<p><b>A.</b> CCR2^−/− and WT mice were immunized with bovine type II collagen in CFA at day 0. Arthritis was measured and recorded in arthritis scores and paw swelling over time. These results are representative of one of three experiments with a total of 25 mice. <b>B.</b> Representative photos of hind paws (day 26) and their histological sections with the H&E stain (day 48) from WT and CCR2^−/− mice are shown. Arrows indicate the extensive inflammation and bone erosion in the joint section of CCR2^−/− mice with CIA. <b>C–D.</b> Single cell suspension of the draining lymph nodes of immunized mouse was cultured in medium with or without anti-CD3 and anti-CD28 antibody stimulation for 6 hours. They were then harvested and stained with anti-CD4 antibody on the surface and anti-IL-17A and anti-IFN-γantibodies intracellularly. C shows the dot plots of cells gated on CD4⁺ cells in flow cytometry, and D demonstrates the average numbers of Th17 cells versus Th1 cells in the draining lymph nodes of CCR2^−/− and WT mice 14 days after immunization, each group contains 5 animals, **p = 0.017. <b>E.</b> Treg cells were identified from the spleen and lymph nodes of immunized mice as CD4⁺CD25⁺FoxP3⁺ cells. These data are results of at least three separate experiments.</p

Monocytes decrease in the spleen, but are abundant in the bone marrow and arthritic joints in immunized CCR2^−/− mice.

<p>Single cell suspensions of the spleen, joints, and bone marrow from collagen-immunized mice were labeled with corresponding antibodies. The monocyte populations were recognized by flow cytometry as L6C^highCD11b⁺Ly6G⁻ cells (A). The abundance of monocytes in each compartment was quantified as percentage (B) and number of monocytes per 10⁴ of total gated live cells (C) in immunized CCR2^−/− and WT mice. These data are results of at least three separated experiments, **p<0.05.</p

Antigen-specific antibodies increase with B cell expansion in CCR2^−/− mice.

<p><b>A.</b> Single cell suspensions were isolated from the draining lymph nodes of mice at day 14 after immunization. B cells were identified as CD45R/B220⁺ cells by flow cytometry. Data shown are average numbers of B cells from WT (n = 6) and CCR2^−/− mice (n = 4), **p = 0.0004. <b>B.</b> Blood sera were collected from WT (n = 7) and CCR2^−/− mice (n = 8) at day 26 after immunization, and levels of anti-type II collagen antibodies (anti-CII) were determined by ELISA, **p = 0.005. <b>C.</b> ELISA results of serum levels of BAFF of WT and CCR2^−/− mice at day 14 (n = 4 vs. 5), day 21 (n = 7 vs. 8), and day 26 (n = 7 vs. n = 5) after immunization are shown.</p

Serum and joint cytokine profiles favor Th17 cell expansion in CCR2^−/− mice.

<p><b>A.</b> Blood sera were collected at day 26 after immunization from WT and CCR2^−/− mice. Levels of cytokines were measured by Luminex assays. Significant differences indicated as asterisks (**) were found in IL-1β (p = 0.01), IL-6 (p = 0.0004), and IL-17 (p = 0.04). Eight animals in each group were used. B. Sera levels of cytokines were measured at day 48 after immunization by Luminex. Four animals in each group were used. <b>C.</b> Joint tissues were isolated from the hind paws of mice at day 48 after immunization. The expression of IL-17 and IFN-γ mRNA was measured by quantitative real time PCR in WT (n = 5) versus CCR2^−/− (n = 4) mice, **p = 0.009. <b>D.</b> Joint tissues were isolated from the hind paws of mice at day 48 after immunization. The expression of IL-1β and IL-6 mRNA was measured by real time PCR in WT (n = 5) versus CCR2^−/− (n = 4) mice, **p≤0.02.</p