Effector Pathways in Peanut-Induced Anaphylaxis Figure 7. Platelet Activating Factor (PAF) and platelets in PE-induced systemic anaphylaxis. C3H/HeOuJ (C3H) and C57BL/6 mice were exposed to PBS+CT (cont) or PE+CT (PE) during a 4 week period, as described in material and methods. A, Temperature after i.p. challenge. Prior to challenge, indicated groups received the PAF R inhibitor WEB2086. B, Temperature after i.p. challenge. Prior to challenge, in indicated groups platelets were depleted using a rabbit anti-platelet serum. *: p,0.05 compared to control. # p,0.05 compared to PE groups. doi:10.1371/journal.pone.0028917.g007 negative control, mice were challenged with PBS in the left ear. Before challenge and after 3 hours, ear thickness of both ears was measured in duplicate using a digital micrometer (Mitutoyo, the Netherlands). Ear swelling was calculated by correcting the thickness of the right ear with the left control ear minus the basal ear thickness before challenge. Measurement of systemic anaphylaxis In separate experiments, mice received a challenge of 1 mg PE intraperitoneally (i.p.) on day 35. As an objective parameter of anaphylactic shock, body temperature was measured by means of rectal thermometry every 10–20 minutes for 90 minutes after challenge. In addition, clinical symptoms were scored using a scoring system, as used before [19]. Cell and cell mediator antagonists PAF function was inhibited using the PAF-receptor antagonists WEB 2086 or CV 3988 (Biomol, USA), which were injected i.p. one hour before challenge at 5 mg/kg BW. Mouse blood platelets were depleted using a rabbit anti mouse platelet serum (Accurate Chemical & Scientific corp, USA) at 50 ml/mouse i.p. three hours before challenge [30]. Depletion of platelets was confirmed by whole blood analysis (Abbott Cell-Dyn CD-1800 Hematology Analyzer, USA) (figure S1). Basophils were depleted with one i.p. injection of 50 mg BA103, one day before oral sensitization or challenge as described previously [31] (figure S1). Monocytes/ macrophages were depleted 3 days before challenge using liposomes containing dicloromethylene-biphosphonate (clodro- nate), as described previously [32] at 5 mg/mouse i.p. Macro- phage depletion was confirmed by differential cell counts and flowcytometric analysis of F4/80 + cells in peritoneal fluid (figure S1). In addition, clodronate liposome treatment did not affect FceRI + cells (mast cells/basophils) in peritoneal fluid. Neutrophils were depleted one day before challenge by i.p. injection of 500 mg/mouse Gr-1 antibody [33]. Measurement of PE-specific antibodies and MMCP-I in serum PE-specific IgG1, IgG2a, IgG2c and IgE in serum of day 36 were measured by ELISA as described previously [34]. This PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28917