Figure 5

Increased IL-17 mRNA expression in the joints of transgenic mice. (A) Cells from the lymph nodes and thymi of untreated B6 wild-type (Wt) or transgenic (Tg) mice were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (500 ng/ml) in the presence of BD GolgiPlug for 4 hours, followed by staining with TCRβ, CD4, CD8, CD44 and intracellular IL-17. One representative stain is shown (right). The statistical analysis of the percentage of IL-17⁺ cells in TCRβ⁺CD4⁺CD8^-CD44^hi cells (three mice in each group) is shown (left). (B) Naïve CD4⁺ T cells (CD4⁺CD44^lowCD62L^hi) from B6 Wt, IL-10-knockout (IL-10Ko) and Tg mice were sorted and stimulated with coated anti-CD3 and anti-CD28 antibodies under Th17 differentiation conditions in the presence or absence of IL-10 (100 μg/ml). Seven days later IL-17 production was measured by intracellular staining. The results of one of three representative experiments are shown. (C) The relative levels of IL-17 mRNA from the joints of DBA/1J Wt and Tg mice with collagen-induced arthritis (on day 50 postimmunization) were measured by real-time PCR. (D) Cells from draining lymph nodes of immunized C57BL/6 Wt or Tg mice (on day 7 postimmunization) were stimulated with the same procedure described above for IL-17 intracellular staining. One representative stain showing lymphocytes (left) or CD3⁺TCRδ⁺ T cells (middle) upon gating is shown. The statistical analysis for the percentage of IL-17⁺ cells in CD3⁺TCRδ⁺ cells (three mice in each group) is shown (right). (E) The same population of cells described in (D) was also stimulated for IL-17 intracellular staining upon gating on lymphocytes (left) or CD4⁺ T cells (middle). Statistics analysis for the percentage of IL-17⁺ cells in TCRβ⁺CD4⁺CD8^-CD44^hi cells (three mice in each group) is shown (right).