differed significantly between these mouse strains. Levels of IgA and IgG2a were highest in C3H/HeOuJ mice while the levels of IgG1 and IgE were significantly higher in BALB/c mice. The small increase in IgG2a in C57BL/6 mice is likely due to cross reactivity of the used antibodies, since these mice do not express IgG2a but IgG2c instead (figure 1). To investigate the underlying T cell responses in the used mouse strains after oral sensitization, spleen and MLN cells were re-stimulated with PE (figure 2). This led to an induction of the Th2 cytokines IL-4, IL-5 and IL-13 in both spleen and MLN cells of C57BL/6 and BALB/c mice, but much less in C3H/HeOuJ mice. This induction was highest in BALB/c mice and significantly less pronounced in C3H/HeOuJ mice. IFN-c was induced equally in all mouse strains in MLN cultures but was induced significantly higher in spleen cultures of BALB/c mice. This level of Th1 (IFNc) cytokine induction after restimulation reflects a mixed ex vivo Th1/Th2 response in all strains in our model. Overall, oral sensitization and challenge with PE led to increased levels of PE-specific antibody and T cell responses, including IgE and Th2 cytokine production. Nonethe- less, the level of these allergic parameters differed significantly among the used mouse strains. The degree of systemic anaphylaxis and ear swelling is mouse strain dependent Next, we assessed whether the differences in antibody and T cell responses in different mouse strains would lead to differences in clinical manifestations of food allergy as well. Therefore, we compared mast cell degranulation and systemic anaphylaxis after intra-gastric and systemic challenge with PE and observed that both challenges in PE-sensitized mice led to a significant release of, mucosal mast cell-derived, Mouse Mast Cell Protease-1 (MMCP-I) in the serum of C3H/HeOuJ and BALB/c, but not C57BL/6 mice (figure 3a). However, MMCP-I is not produced by connective tissue mast cells and to investigate the in vivo response of this type of mast cells, we measured mast cell-mediated ear swelling after intradermal injection of PE in the ears of sensitized mice. This showed that C3H/HeOuJ, C57BL/6 and BALB/c mice developed significant ear swelling upon challenge with PE (figure 3b). C3H/HeOuJ mice showed a significantly higher ear swelling compared to C57BL/6 and BALB/c mice. Hereafter, we studied the development of systemic anaphylaxis after PE oral sensitization and systemic (i.p.) challenge. We measured an unambiguous anaphylactic response, reflected by a strong drop in temperature and increased clinical score, in C3H/ HeOuJ and to lesser extent in C57BL/6 mice (figure 3c). Surprisingly, BALB/c mice only showed a very minimal anaphylactic response upon challenge. In addition to many previous attempts, oral challenge with PE of sensitized mice did not lead to any anaphylactic responses in all three mouse strains (data not shown). Together, these data shows a clear discrepancy between mucosal and connective tissue mast cell degranulation and development of systemic anaphylaxis to PE in different mouse strains. C3H/HeOuJ mice displayed mucosal mast cell degranu- lation and the strongest level of systemic anaphylaxis. C57BL/6 mice did not show mucosal mast cell degranulation and exhibited less pronounced mast cell-mediated ear swelling and systemic anaphylaxis when compared to C3H/HeOuJ. PE-induced systemic anaphylaxis is FcRc- mediated Subsequently, to assess whether the observed PE-induced systemic anaphylaxis in C57BL/6 mice was IgG and IgE mediated, we used FcRc2/2 knockout C57BL/6 mice in our model. The Fc associated c chain is a key component of FccRI, III Effector Pathways in Peanut-Induced Anaphylaxis and IV and FceRI but not of FccRIIb and FceRII (CD23). Therefore, in FcRc2/2 mice both IgG and IgE cannot bind with high affinity and/or activate responsive receptors. Remarkably, FcRc2/2 mice showed normal levels of IgE, IgG1 and IgG2a antibodies after oral sensitization in comparison to WT positive control mice (figure 4a), but did not develop systemic anaphylaxis upon PE challenge (figure 4b). This shows that PE-induced systemic anaphylaxis but not oral PE sensitization is FcRc- mediated and dependent on activation via IgE or IgG. The role of basophils, neutrophils, monocytes/ macrophages and mast cells in PE-induced systemic anaphylaxis The occurrence of antibody mediated anaphylactic responses to PE despite no obvious mucosal mast cell response and a lower connective tissue mast cell response in C57BL/6 mice led us to explore alternative pathways of anaphylaxis in these mice compared to C3H/HeOuJ mice. It has been suggested that basophils, neutrophils or macrophages can contribute to or cause anaphylactic responses as well. Importantly, a comparison of the number of platelets, basophils, monocytes/macrophages and neutrophils showed no differences in the number of these cell types in spleen or peritoneal cavity of C3H/HeOuJ and C57BL/6 mice (figure S1). Therefore, we first investigated whether basophils influenced the development of systemic anaphylaxis in C57BL/6 mice and C3H/HeOuJ mice. Basophil depletion with the antibody BA103 did not influence PE oral sensitization as measured by levels of PE-specific IgG1, IgG2a and IgE (figure 5a) or systemic anaphylactic responses of C3H/HeOuJ and C57BL/6 mice (figure 5b). As a control, MMCP-I levels after PE challenge were equal among control and basophil-depleted mice (figure 5c), showing that mast cell responses were unaffected by BA103 treatment. In addition, neutrophil depletion using Gr-1 did not influence systemic anaphylactic responses of C3H/HeOuJ and C57BL/6 mice (figure S2). Hereafter, we investigated whether monocytes/macrophages play a role in PE-induced systemic anaphylaxis. Remarkably, selective depletion of monocytes/ macrophages using clodronate liposomes did not affect the drop in temperature in C3H/HeOuJ mice, but it significantly lowered the anaphylactic response in C57BL/6 mice (figure 5d). The induction of systemic anaphylaxis in the absence of mucosal mast cell degranulation was further examined in mast cell-deficient C57BL/6W-sh/W-sh mice. Importantly, mast cell deficiency did not lead to differences in IgE, IgG1 or IgG2a levels compared to littermates (figure 6a). Interestingly, in contrast to C57BL/6 littermates C57BL/6W-sh/W-sh mice did not develop any anaphylactic manifestations (figure 6b). Thus, basophils do not play a role in PE-induced oral sensitization and systemic anaphylaxis. In contrast, monocytes/macrophages do play a significant role in PE-induced systemic anaphylaxis in C57BL/6 mice, while playing no role in C3H/HeOuJ mice. This would suggest that parts of the alternative pathway of anaphylaxis, dependent on connective tissue mast cells, are mediating peanut- induced systemic anaphylaxis in C57BL/6 mice. PAF and platelets mediate PE-induced systemic anaphylaxis, depending on mouse strain PAF is considered an important mediator in the alternative pathway of anaphylaxis. For that reason, we looked whether inhibition of the PAF pathway would affect PE-induced systemic anaphylaxis. However, treatment before challenge with the PAFR inhibitor WEB2086 did not significantly inhibit systemic anaphy- laxis responses in C3H/HeOuJ mice although the observed PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28917