Figure 6. Circulating Dkk-1 is from platelets upon allergen challenge or parasitic infection.

(A) Female C57BL/6 mice were challenged with PBS (n=3) or HDM extract (10 μg/challenge/mouse, n=5) as in Figure 1B. (B) Eight-week old female C57BL/6 mice were challenged with HDM extract (30 μg/challenge, n=4) or PBS (n=5) for 4 and 24 hours. Plasma samples were analyzed by ELISA. (C) Dkk1^d/d mice (n=7) and their wildtype littermate controls (n=7) were challenged with HDM extract as in Figure 1B. Harvested lungs were analyzed by immunohistochemistry. Original magnification is 10×. Scale bar in lower right panel represents 10 μm. (D) Peripheral blood was collected by cardiac puncture with or without EDTA. (E) Eight-week-old female C57BL/6 mice were injected with platelet depletion antibody (n= 5) or isotype control antibody (n=4). Twenty-four hours later, plasma samples were measured. (F) HDM allergen was challenged as in Figure 1B, and platelets were depleted for 12 hours. (G) Bacterial ds DNA from E.coli (B-ds DNA)(30 μg/mouse,n=4), LPS (3 μg/mouse, n=5), CpG-ODN 1585 (25 μg/mouse, n=4), and House Dust Mite (HDM) allergen extract (50 μg/mouse, n=4) were challenged intranasally in 6 week-old C57BL/6 mice. Twenty-four hours later, plasma samples were collected and Dkk1 concentrations were determined by ELISA. Control (n= 7) mice were given 20 μl of 0.9% NaCl saline. (H) At each time point plasma from female BALB/c mice (6-week old) that were infected with L. major was measured. (I) Platelets were depleted 4 hours prior to parasite infection. Plasma samples from 5-week old female BALB/c mice were analyzed 72 hours after infection. (J) Ten weeks after infection, platelets were depleted for 12 hours in BALB/c mice. (K) Human platelets from four healthy volunteers were activated with sLMAG for 1 hr. S1-S4 designates each healthy donor. X-axis shows dilution of sLMAG in the culture. 1:50 is equivalent to 1×10⁶ parasites. (L) Human platelets (1×10⁸/ml, n=5) were activated with sLMAG (1:50) in the presence of PKCα inhibitor, or PKCβ inhibitor for 1 hour. A representative of two independent experiments is shown. Small horizontal lines indicate the mean (± s.e.m.). Student's t-test (A, D, E), One-way ANOVA with Dunnett's post-hoc test (B, I) and One-way ANOVA with Bonferroni's post-hoc test (F,G,H,J,L) was performed. ***, p< 0.0005, **, p<0.005, *, p<0.05. See also Supplementary Figure 4.