Dkk1 is released from activated platelets and binds to AECs during acute pulmonary inflammation. PRP, isolated from LM-challenged mice, was incubated with 100 μM SFLLRN for 20 minutes (A) or 5 to 20 minutes (B) and then centrifuged. The protein levels of Dkk1 and CD41 in the pellet were determined by western blotting (A) and the Dkk1 level in the supernatant was determined by ELISA (B). (C) Primary mouse AEC II was cultured for 4 days and then stimulated with TNFα and IL-1β (20 ng/mL of each) for 24 hours. Recombinant mouse Dkk1 was added to the cells at the indicated concentrations for 2 hours. Cell-based ELISA was performed to quantify the binding of Dkk1 to the stimulated AECs. (D) The mRNA levels of the Dkk1 receptors (KRM1, KRM2, LRP5, and LRP6) were analyzed by real-time PCR in the TNFα/IL-1β–stimulated AECs and normalized to control. Data are expressed as the mean ± SEM from 3 independent preparations.