Effector Pathways in Peanut-Induced Anaphylaxis Figure 2. T cell responses after PE oral sensitization in different mouse strains. C3H/HeOuJ (C3H), C57BL/6 and BALB/c mice were exposed to PBS+CT (cont) or PE+CT (PE) during a 4 week period, as described in material and methods. Spleens and MLN were isolated and single cell suspensions of these tissues were incubated in the absence (medium) or presence of sterilized peanut extract (PE) in complete RPMI1640. Graphs depict levels of IL-4, IL-5, IL-13 and IFN-c 96 h after stimulation. Data are represented as mean 6 SEM of 6 mice. *: p,0.05 compared to control. #: p,0.05 compared to C3H/HeOuJ PE group. ‘: p,0.05 compared to C57BL/6 PE group. doi:10.1371/journal.pone.0028917.g002 temperature drop did seem less severe at later time points after PAF inhibition (figure 7a). In contrast, PAF inhibition significantly lowered anaphylactic responses in C57BL/6 mice. Using another PAF R inhibitor, CV3988 gave the same results (data not shown). Antibody-mediated depletion of platelets did significantly inhibit systemic anaphylaxis in both C3H/HeOuJ and C57BL/6 mice (figure 7b). Therefore, although PE induced systemic anaphylaxis involves platelets in both C3H/HeOuJ and C57BL/6 it only significantly involves PAF in C57BL/6 mice. Again suggesting that parts of the alternative pathway of anaphylaxis are mediating peanut-induced systemic anaphylaxis in C57BL/6 mice Discussion Manifestations of food allergy and food induced anaphylaxis may vary widely among individuals, even among patients reacting to the same responsible allergen. In addition, the underlying immunological mechanisms of these differences may be the reason why a classical diagnosis of food allergy and/or anaphylaxis is not always conclusive. In an attempt to reveal possible mechanisms of this pleiomorphy, we set out to investigate and compare parameters and manifestations of food allergy in different well- known mouse strains, i.e. C3H/HeOuJ, C57BL/6 and BALB/c mice. We observed that oral sensitization to PE, when comparing IgE and Th2 cytokine levels, was most effective in BALB/c mice, followed by C57BL/6 mice, and least effective in C3H/HeOuJ mice. Remarkably, the degree of systemic anaphylaxis was strain- dependent but, in contrast to oral sensitization, consistently most severe in C3H/HeOuJ, less pronounced in C57BL/6 and very low or minimal in BALB/c mice. This observed differences obviously point to variable genetic susceptibility to food sensitiza- tion in mice, as in human subjects. The parameters of food sensitization in BALB/c mice in our model were different from some observations in literature, reporting lower levels of IgE and Th2 responses in these mice [14,20]. Notably, anaphylaxis in BALB/c mice is clearly allergen- dependent as evidenced by the findings that less complex allergens such as ovalbumin or ß-lactoglobuline [11,18] induce anaphylactic responses in these mice. Furthermore, the challenge dose and route of exposure is very important in this model. We as well as others [19] were not able to observe anaphylactic responses after various intragastric doses of PE in any mouse strain. But orally induced anaphylaxis could be demonstrated to very high amounts of protein (200 mg protein/mouse), comparable to 700 grams or more than 1000 peanuts in a human situation [17]. The occurrence of anaphylaxis after either systemic exposure, or after extreme oral doses or to single purified and relatively small allergens is in line with a recent study showing that ingested allergens must be absorbed systemically to induce anaphylaxis [21]. Notwithstanding, the anaphylactic responses measured in our studies after oral sensitization and systemic challenge are strong and consistent, resulting in a robust model and a notable experimental advantage. In this model, BALB/c mice did not show anaphylaxis despite a relative high level of expression of Th2 cytokines and IgE after oral sensitization. In addition, despite high levels of IgE and the occurrence of systemic anaphylaxis, C57BL/ 6 mice did not show increased mucosal mast cell degranulation upon intra-gastric or systemic PE challenge. This clearly points to multiple mechanisms involved in susceptibility to peanut-induced anaphylaxis. Classically, the mast cell has always been the central mediator of allergy-mediated anaphylaxis. Two types of mast cells have been described in the mouse: mucosal mast cells expressing MMCP-I and connective tissue mast cells [22,23,24]. To investigate the responses of connective tissue mast cells, we measured acute ear swelling, which occurred in C57BL/6 mice and in BALB/c mice but clearly less than C3H/HeOuJ mice. This suggests that the observed differences in systemic anaphylactic symptoms between the mouse strains may be due to differential involvement of either mucosal or connective tissue mast cell subsets. In addition, the apparent lack of involvement of mucosal mast cells in C57BL/6 led us to investigate the role of the classical versus alternative pathways of anaphylaxis in these mice compared to C3H/HeOuJ mice. The role of the alternative pathway of anaphylaxis involving IgG, macrophages, basophils and PAF in food allergy and food- induced anaphylaxis has already been suggested [6]. Recently, studies by Jo¨nsson suggested a novel alternative pathway in which neutrophils, activated via IgG contributed to systemic anaphylactic shock in mice as well [7].In our experiments, peanut-induced systemic anaphylaxis in C57BL/6 mice was shown to be entirely dependent on FcRc and mast cells and partially dependent on PAF, platelets and monocytes/macrophages. Systemic anaphylaxis in C3H/HeOuJ was also dependent in platelets, but not on PAF or monocytes/macrophages. Despite earlier reports on the role of basophils in other allergic diseases [25], basophils did not play a role in oral sensitization and systemic anaphylaxis in any of the strains. Moreover, neutrophil depletion using Gr-1 did not influence systemic anaphylactic responses of C3H/HeOuJ and C57BL/6 mice in our studies. This suggests that the involvement of components of the alternative anaphylaxis pathway, i.e. macrophages and PAF, in food allergy is strain-dependent but still dependent on mast cell activation. The earlier mentioned studies investigating alternative pathways were performed in mouse models using passive cutaneous or systemic anaphylaxis, penicillin-, or TNP-OVA-mediated anaphy- laxis [6,7,11,12]. Moreover, these experiments were done with substantial immunization protocols (such as Freund’s adjuvant) followed by injection of large amounts of antigen, which may not recapitulate the development of hypersensitivity in food allergy. More relevant to food allergy, it has been shown that in mice preconditioned for responsiveness to vasoactive mediators, peanuts could directly induce anaphylaxis by an IgE-independent but alternative pathway-dependent mechanism [10]. In addition, in a classical mouse model of food allergy using C57BL/6, Arias et al showed that a PAF-antagonist, but not a histamine antagonist inhibited systemic PE anaphylaxis [9].Recently in the same model, very high amounts (3.75 mg) of peanut injected intravenously induced anaphylaxis in mast cell deficient mice, which was dependent on macrophages and to lesser extent on basophils [26]. Again, this shows that probably the dose and route of exposure of allergen is crucial in which pathway of anaphylaxis will be involved. A very high dose or systemic exposure of allergen may involve the alternative pathway of anaphylaxis, as suggested by others [11]. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28917