CCL22 is constitutively expressed in vivo and in vitro. (A) CCL22 protein was quantified in tissue homogenates and in the serum of BALB/c mice by ELISA (n = 5 mice) and detected by immunohistochemistry in the lymph node (LN). PP, Peyer's patches. Bar, 100 µm. (B) 10⁶ freshly isolated lymph node cells and splenocytes from C57BL/6 mice were cultured, and CCL22 levels in the supernatants were determined at different time points by ELISA. (C) CCL22 mRNA expression from unsorted, CD11c-depleted (CD11c⁻), or CD11c-enriched (CD11c⁺) murine lymph node cells or splenocytes was determined by quantitative real-time PCR. (D) CD11b⁺CD8^neg, CD11b^negCD8⁺, B220⁺, CD103⁺, and CD103^neg cells were sorted from CD11c-enriched splenocytes, and RNA was isolated followed by quantitative real-time PCR. (E) CCL22 protein level in intestinal or skin-draining lymph node tissue homogenates of C57BL/6 mice measured by ELISA (n = 3 mice). (F) 10⁵ freshly isolated CD11c⁺ splenic DCs or 7-d differentiated BMDCs from C57BL/6 mice were cultured with or without 3 µg/ml CpG for 4 h, and CCL22 levels in the supernatants were determined by ELISA. Data are presented as mean ± SEM and are representative of two to four independent experiments (n = 3–5 mice per group). ***, P < 0.001 (two-sided Student’s t test).