Figure 1.
Th17 cells expand in the draining lymph nodes of immunized CCR2−/− mice.
A. 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. 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. C–D. 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. E. 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.
Figure 2.
Serum and joint cytokine profiles favor Th17 cell expansion in CCR2−/− mice.
A. 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. C. 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. D. 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.
Figure 3.
Antigen-specific antibodies increase with B cell expansion in CCR2−/− mice.
A. 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. 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. C. 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.
Figure 4.
Neutrophil infiltration increases in the spleen and the joints of CCR2−/− mice.
Cells were isolated from the spleen and the hind paws of mice at day 48 after immunization and were stained for flow cytometry analysis. Ly6G+CD11b+ (Ly6C+) cells were gated as neutrophils. Shown are representative dot plots (A) and quantification of the number of neutrophils (B) of three experiments, **p = 0.02 in the spleen and 0.002 in the joints. C. Joint tissues were isolated from the hind paws of mice at day 48 after immunization. The expression of CXCL1, CXCL2, and RANKL mRNA was measured by real time PCR in WT (n = 5) versus CCR2−/− (n = 4) mice, p = 0.059, 0.016, 0.026, respectively.
Figure 5.
Treatment with anti-IL-17A antibodies reduces CIA in CCR2−/− mice.
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).
Figure 6.
Monocytes decrease in the spleen, but are abundant in the bone marrow and arthritic joints in immunized CCR2−/− mice.
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 L6ChighCD11b+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.