Role of complement in a murine model of peanut-induced anaphylaxis

Immunobiology 218 (2013) 844–850

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Role of complement in a murine model of peanut-induced anaphylaxis Toshihisa Kodama, Hideharu Sekine ∗ , Minoru Takahashi, Daisuke Iwaki, Takeshi Machida, Kazuko Kanno, Yumi Ishida, Yuichi Endo, Teizo Fujita Department of Immunology, Fukushima Medical University, Fukushima 960-1295, Japan

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Article history: Received 11 July 2012 Accepted 11 October 2012 Available online 17 October 2012 Keywords: Anaphylaxis C4 Classical pathway Complement Lectin pathway MASP Peanut

a b s t r a c t Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice. © 2012 Elsevier GmbH. All rights reserved.

Introduction Food allergy is now recognized as a worldwide problem in westernized nations, and like other allergic disorders, it appears to be on the increase. Although food allergy has harmful effects on family economics, interpersonal relations, and health related quality of life (Bollinger et al. 2006; Perry et al. 2009), little is known about the cellular and molecular process in the pathogenesis of food allergy. Unlike other food allergies, peanut allergy is a severe disease that develops anaphylactic symptoms involving multiple organs, including the skin, gastrointestinal tract, and respiratory system in patients (Sampson 2000). In the most severe cases, the cardiovascular system is also involved and systemic shock develops. Moreover, peanut allergy in childhood usually persists into

Abbreviations: FCN, ficolin; IRF-4, interferon regulatory factor-4; MASP, mannose-binding lectin associated serine protease; MBL, mannose-binding lectin; PE, peanut extract. ∗ Corresponding author at: Department of Immunology, Fukushima Medical University, 1 Hikarigaoka, Fukushima city, Fukushima 960-1295, Japan. Tel.: +81 24 547 1148; fax: +81 24 548 6760. E-mail address: [email protected] (H. Sekine). 0171-2985/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.imbio.2012.10.003

adulthood and lasts for a lifetime, unlike most food allergens (Skolnick et al. 2001). Recent studies from the United Kingdom and the United States indicate that the prevalence of peanut allergy has doubled in young children during the past decade (Grundy et al. 2001; Sicherer et al. 2003). Despite an increasing number of patients, there are no proven effective therapies for peanut allergy. Type I food allergy is characterized by T helper 2 (Th2) associated responses, resulting in increased levels of allergen-specific IgE, and causing anaphylaxis involving the release of chemical mediators, such as histamine and platelet activating factor (PAF) from mast cells and other immune cells (Finkelman 2007). Previous animal studies suggested that peanut allergy was associated with the up-regulation of Th2-cytokines, B cells, and mast cells (Cardoso et al. 2008; Sun et al. 2007). Almost all studies focused on adaptive immunity, and there are few studies focused on innate immunity, such as toll-like receptors and complement proteins. The complement system, which consists of more than 30 plasma and cell-surface proteins, is known to be a highly sophisticated host-defense system. Once the complement system is activated by danger signals, it is able to respond to them directly by the lytic effect, but also indirectly by activating cellular innate and adaptive immune responses via several complement receptors. Complement is activated by 3 different pathways: the classical, lectin, and

T. Kodama et al. / Immunobiology 218 (2013) 844–850

alternative pathways (Fujita 2002). The classical pathway is activated by antibody–antigen complexes, whereas, the lectin pathway is activated by pattern-recognition molecules, such as mannose-binding lectin (MBL) and ficolin (FCN) which recognize carbohydrates, and the alternative pathway is activated by the covalent binding of C3. In human studies, elevated levels of complement anaphylatoxins have been observed in bronchoalveolar lavage fluid (BALF) of asthmatic patients (Humbles et al. 2000; Krug et al. 2001; van de Graaf et al. 1992), and complement anaphylatoxins were also reported to relate to drug induced anaphylaxis (Szebeni 2005), however, little is known about the role of complement in food allergy. In animal studies, Th2 responses in asthma (Drouin et al. 2001) and in skin eosinophilia (Yalcindag et al. 2006) were suppressed in C3 deficient mice, and C3a was also reported to cause cardiac anaphylaxis in guinea pigs (del Balzo et al. 1988). By contrast, C3aR deficient mice exhibited an exaggerated Th2 response to epicutaneous sensitization with ovalbumin (Kawamoto et al. 2004). Moreover, C5a causes severe cardiac abnormalities, a short-lived transient hypertension followed by massive hypotension in association with brad arrhythmia, in pigs (Szebeni et al. 2006). The relationship between the onset of allergy and the complement activation in animal studies is inconsistent. With regards to relationships between each complement pathway and allergy, the alternative pathway is concerned with murine allergic airway inflammation (Taube et al. 2006), however, the classical and lectin pathways remain unknown. In 2009, Khodoun et al. showed that peanuts could activate complement and generated C3a induced hypothermia in mice under conditions of high sensitivity to vasoactive mediators (Khodoun et al. 2009). C3a mediated histamine and PAF released from macrophages and basophils are suggested as causes of hypothermia, however, the role of each complement pathway in peanut allergy remains unclear. In this study, we investigated the role of complement activation by peanut extract (PE) in vitro using sera of mannose-binding lectin associated serine protease (MASP)-1/3-, MASP-2-, C4-, and immunoglobulin-deficient mice. We further investigated the role of complement activation in systemic anaphylaxis in vivo. Materials and methods Mice Wild-type (WT) mice, C57BL/6 (C57BL/6JJcl), were purchased from CLEA Japan Inc (Tokyo, Japan). MASP-1/3 deficient mice and MASP-2 deficient mice have previously been described (Iwaki et al. 2006; Takahashi et al. 2008). C4 deficient mice were purchased from the Jackson Laboratory (Fischer et al. 1996). Interferon regulatory factor-4 (IRF-4) deficient mice, which are known to lack serum immunoglobulin (Mittrucker et al. 1997), were used as a source of immunoglobulin deficient sera (Sekine et al. 2010). 8- to 12-week-old mice (C57BL/6 background) were used for the experiments. All animal protocols were approved by the Animal Care and Use Committee in accordance with the Guidelines for the Animal Experiments of Fukushima Medical University, Japanese Government Law Concerning the Protection and Control of Animals and Japanese Government Notification on Feeding and Safekeeping of Animals.

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4 h at room temperature and then the insoluble components were removed by means of centrifugation. Following dialysis, supernatants were fractionated by ammonium sulfate precipitation, with retention of the fraction that was soluble in 25% saturated ammonium sulfate and insoluble in 80% saturated ammonium sulfate. Protein concentration of PE was determined with Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). C3 deposition assay MaxiSorp microwells (Nunc, Roskilde, Denmark) were coated with PE in 0.1 M sodium bicarbonate buffer (pH 9.6) overnight at 4 ◦ C. Coated plates were blocked with 1% BSA/PBS for 2 h at room temperature. Murine sera were diluted with BBS buffer (4 mM barbital, 145 mM NaCl, 10 mM CaCl2 , and 10 mM MgCl2 ). Diluted samples were added to PE-coated microwells and incubated at 37 ◦ C for 30 min. After washing, C3 deposition was detected with HRP-conjugated anti-human C3c polyclonal antibodies which cross-react with mouse C3 (Dako Cytomation, Glostrup, Denmark). Following 1 h incubation at room temperature, the wells were washed and 3,3 ,5,5 -tetramethylbenzidine solution (TMB; KPL, Gaitherburg, MD) was added. After 15 min incubation, the enzymatic reaction was stopped with 1 M H3 PO4 and measured at 450 nm in a DTX880 plate reader (Beckman Coulter, Brea, CA). In Ca2+ free assay, murine serum was diluted with EGTA containing BBS buffer (4 mM barbital, 145 mM NaCl, 10 mM MgCl2 , and 10 mM EGTA). Inhibition of the classical pathway was performed with anti-C1q sera (Yonemasu and Sasaki 1981). Immunoglobulin and C1q, MBL, FCN deposition assay MaxiSorp microwells (Nunc) were coated with PE in 0.1 M sodium bicarbonate buffer (pH 9.6) overnight at 4 ◦ C. Plates were then blocked with 1% BSA/PBS for 2 h at room temperature. PBSdiluted murine sera were added to PE-coated microwells and incubated for 1 h at room temperature. Plates were washed and reacted with biotinylated goat anti-mouse IgG2b, IgG3, or anti-IgM antibody (Southern Biotech, Birmingham, AL). For C1q, MBL and FCN deposition assay, anti-C1q sera, anti-MBL-A (Hycult Biotechnology, Uden, Netherlands), anti-MBL-C (Hycult Biotechnology), or anti-FCN-A (Endo et al. 2010) was used. Deposition of Immunoglobulin, C1q, MBL, or FCN-A was then detected by using VECTASTAIN ABC system (Vector Laboratories Inc, Burlingame, Canada). PE-induced shock Mice were pretreated intravenously for 24 h with 1 ␮g of recombinant mouse IL-4 (Shenandoah Biotechnology, Warwick, PA) and 5 ␮g of BVD4-1D11 rat IgG2b anti-mouse IL-4 mAb (Invitrogen, Carlsbad, CA), and for 20 min with 100 ␮g of propranolol (Sigma–Aldrich, Saint-Louis, MO, USA). Mice were challenged intravenously with 500 ␮g of PE and rectal temperature was measured using thermista (Takara thermista, Tokyo, Japan). C3a ELISA Rat anti-mouse C3a antibody, biotinylated Rat anti-mouse C3a antibody, and mouse C3a protein purchased from BD Pharmingen (San Diego, CA) were used for sandwich ELISA to measure plasma C3a concentration.

Peanut extract (PE)

Statistical analysis

Commercial roasted peanuts were ground by Omni TH115 tissue homogenizer (Omni International, Kennesaw, GA, USA) in 0.1 M ammonium bicarbonate (pH 9.0). Homogenates were incubated for

All data are given as means ± SEMs. Comparison of the means of 2 groups was performed by student’s t test. Comparison of more than 2 groups was performed by one-way ANOVA. When the

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Time (min) Fig. 1. PE activates complement in vitro. (A) Influence of coating doses of PE. Sera (5 ␮l) in BBS (95 ␮l) were added to microtiter wells coated with 0.01, 0.1, 1, 10, or 100 ␮g of each PE and to control wells. Plates were then incubated at 37 ◦ C for 30 min. (B) Influence of serum dilution with BBS buffer. (C) Kinetics of C3 deposition. C3 deposition was detected with HRP-conjugated anti-human C3c polyclonal antibodies which cross-react with mouse C3. Data represents the means and SEMs for 3 separate experiments.

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Serum (%) Fig. 2. PE activates complement via C4 but not MASP-1/3 and MASP-2 in vitro. (A) C3 deposition assay in Ca2+ free conditions. Sera were diluted with BBS in the presence or absence of EGTA to chelate Ca2+ . C3 deposition assay in MASP-1/3 deficient sera and MASP-2 deficient sera (B) and C4 deficient sera (C). Plates were coated with 10 ␮g of PE. C3 deposition was measured after 30 min incubation. Data represent the means and SEMs for 3 separate experiments.

PE activates complement via C4 but not MASP-1/3 and MASP-2 in vitro mean values of the groups showed a significant difference, pairwise comparison was performed using the Bonferroni/Dunnett’s test or Turkey/Kramer test. A P < 0.05 was considered to be statistically significant.

Results PE activates complement in vitro First, we examined whether PE could activate complement by C3 deposition assay in vitro. Diluted murine sera were added to PEcoated microwells, and the amount of C3 deposited on the wells was measured. As shown in Fig. 1, increased levels of C3 deposition were observed depending on coating doses of PE (Fig. 1A), serum concentrations (Fig. 1B), and incubation time (Fig. 1C). These results show that PE has a potential to activate serum complement. The following C3 deposition assays were performed under conditions of 10 ␮g/well PE and 30 min incubation.

To assess the contribution of the alternative pathway for serum complement activation by PE, we examined C3 deposition assay in Ca2+ free conditions. There is no Ca2+ requirement associated with the alternative pathway activation, however Mg2+ is required for the interaction between Factor B and C3b. In this study, we used EGTA, which chelates Ca2+ , to specifically block the classical and lectin pathway activation. When C3 deposition assay was performed in the presence of EGTA, C3 deposition on PE was completely abolished (Fig. 2A). Next, we determined whether the alternative pathway or lectin pathway contributed to complement activation on PE using MASP-1/3 deficient sera or MASP-2 deficient sera. As previously reported, MASP-1/3 deficient sera lack the alternative pathway activation (Takahashi et al. 2010), while MASP-2 deficient sera lack the lectin pathway activation (Iwaki et al. 2006). As shown in Fig. 2B, C3 deposition on PE occurred in MASP-1/3 deficient sera and MASP-2 deficient sera, and there was no statistically significant difference in the levels of C3 deposition among WT, MASP-1/3 deficient and MASP-2 deficient sera. We further determined whether the classical pathway contributed to complement

T. Kodama et al. / Immunobiology 218 (2013) 844–850

0.2

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0.15 0.1 0.05

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anti-C1q serum (μg/mL) 2

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10 Fig. 4. C3 deposition on PE was inhibited by anti-C1q sera. Anti-C1q sera were added to diluted sera before incubation to achieve final concentrations to 3, 10, or 30 ␮g/mL. Plates were coated with 10 ␮g of PE, and C3 deposition was measured after 30 min incubation. Data represent the means and SEMs for 3 separate experiments (** P < 0.01, compared with control samples).

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Serum (%) Fig. 3. PE activates complement via immunoglobulin and C1q in vitro. (A) C3 deposition assay in IRF-4 deficient sera. Plates were coated with 10 ␮g of PE, and C3 deposition was measured after 30 min incubation. (B) Deposition of IgM, IgG2b, IgG3 and C1q on PE. Plates were coated with 10 ␮g of PE, and deposition of immunoglobulin isotypes and C1q was measured after 30 min incubation. Data represent the means and SEMs for 3 separate experiments.

activation on PE by using C4 deficient sera which lack both the lectin and classical pathway activation. Unlike WT sera, no C3 deposition on PE was observed in C4 deficient sera (Fig. 2C). PE activates complement via immunoglobulin and C1q in vitro To assess the involvement of immunoglobulin in complement activation on PE, we examined C3 deposition assay in sera from IRF-4 deficient mice. Consistent with the previous report, the IRF-4 deficient mice exhibited a profound reduction in serum immunoglobulin concentrations (more than 100 times reduction compared to WT sera; data not shown) (Mittrucker et al. 1997). As shown in Fig. 3A, C3 deposition on PE was abrogated in sera from IRF-4 deficient mice compared to that from WT mice. We also examined whether IgM, IgG2b, IgG3 and C1q could deposit on PE. Diluted murine sera were added to PE-coated microwells, and the amount of IgM, IgG2b, IgG3 and C1q deposited on the wells was measured. The results showed that IgM, IgG2b, IgG3 and C1q deposited on PE depending on serum concentrations (Fig. 3B). Levels of IgM and C1q deposition were higher than those of IgG2b and IgG3. These results suggest that antibodies binding to PE are critical for the complement activation on PE. To confirm the above results, we determined whether anti-C1q sera could inhibit C3 deposition on PE. Anti-mouse C1q sera were added to diluted sera before incubation in PE-coated microwells. The sera inhibited C3 deposition on PE in a dose-dependent manner, and the inhibition with 30 ␮g/mL of anti-C1q sera showed statistically significant reduction of C3 deposition compared to the control (Fig. 4). These in vitro results suggested that PE activates complement via the classical pathway not the lectin or alternative pathway.

Although the lectin pathway was not involved in PE-dependent complement activation in vitro, we examined whether pattern recognition molecules in the lectin pathway could bind to PE. Diluted murine sera were added to mannan (5 ␮g/well) or PE (10 ␮g/well) -coated microwells, and the amount of MBL-A and MBL-C deposited on the wells was measured. Both MBL-A and MBL-C deposited on mannan as expected. However, these pattern recognition molecules did not deposit on PE (Fig. 5A and B). Furthermore, we determined whether FCN-A could deposit on PE. As shown in Fig. 5C and D, FCN-A bound to PE depending on serum concentration, and its binding was inhibited by the addition of N-acetylglucosamine (GlcNAc) in a dose-dependent manner. PE activates complement in vivo We injected PE to WT mice intravenously and measured C3a levels in sequentially sampled plasma to determine whether PE could activate complement in vivo. Whereas an injection of saline caused no changes in plasma C3a levels, an injection of PE intravenously at a dose of 1000 ␮g per mouse caused a rapid increase in plasma C3a levels that was statistically significant as soon as 5 min after injection (Fig. 6). Plasma concentration of C3a peaked at 5 min after PE injection and decreased slowly thereafter. Complement activation by PE was attenuated in MASP-2 deficient mice and C4 deficient mice We injected PE to MASP-1/3 deficient mice, MASP-2 deficient mice, and C4 deficient mice to clarify the complement activation pathway by PE in vivo. Levels of C3a were determined in plasma that was collected 5 min after injection. The results showed that the generation of C3a occurred in MASP-1/3 deficient mice as well as in WT mice, whereas the generation was significantly decreased in MASP-2 deficient mice and C4 deficient mice (Fig. 7). Anaphylaxis induced by PE required C4, and partially MASP-2 Finally, we determined the contribution of C4 and MASP-2 in complement mediated PE anaphylaxis model (Khodoun et al. 2009). Mice were pretreated with a long acting form of IL-4 and propranolol to increase sensitivity (Finkelman et al. 1993; Strait et al. 2003; Toogood 1987), and were subsequently injected with 500 ␮g of PE intravenously. Rectal temperature was measured for 90 min after injection of PE. In this model, we observed that PE injection induced hypothermia in a dose-dependent manner (data not shown). As shown in Fig. 8A, WT mice developed severe hypothermia after the injection of PE, and rectal temperature continuously

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Fig. 6. PE activates complement in vivo. 1000 ␮g of PE was intravenously injected in WT mice. Plasma samples were collected at various time points from tail vein and assayed for C3a levels by ELISA. Data represent the means and SEMs for 4 separate experiments (** P < 0.01, * P < 0.05, compared with saline injected mice).

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Fig. 7. Complement activation by PE was attenuated in MASP-2 deficient mice and C4 deficient mice. 1000 ␮g of PE was intravenously injected in WT mice (n = 4), MASP-1/3 deficient mice (n = 4), MASP-2 deficient mice (n = 3), and C4 deficient mice (n = 3). Plasma samples were collected 5 min after PE injection from tail vein and assayed for C3a levels by ELISA. Data represent the means and SEMs (** P < 0.01, compared with WT mice).

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dropped during the measurement. Notably, PE-induced hypothermia was partially prevented in MASP-2 deficient mice and almost completely prevented in C4 deficient mice. We further measured plasma C3a levels collected 5 min after PE injection in the anaphylaxis model. As shown in Fig. 8B, plasma C3a levels in C4 deficient mice and MASP-2 deficient mice were significantly lower than that in WT mice. Consistent with the severity of their PE-induced hypothermia, plasma C3a levels were significantly lower in C4 deficient mice compared to MASP-2 deficient mice. Thus, these results show that complement activation is a critical factor in inducing hypothermia in this anaphylaxis model, and PE activates both the lectin and classical pathways in vivo. Discussion

0 0

0.1

0.3

1

GlcNAc (M) Fig. 5. FCN-A but not MBL binds to PE. Diluted sera were added to microtiter wells coated with mannan (5 ␮g/well) or PE (10 ␮g/well), and the amount of MBL-A (A) and MBL-C (B) on the wells was measured. (C) Diluted sera were added to PE coated wells and FCN-A deposition was measured. (D) Dose-dependent inhibition of GlcNAc in binding of FCN-A to PE was analyzed. GlcNAc was added to diluted sera, and FCNA deposition on PE was detected. Data represent the means and SEMs for 3 separate experiments (** P < 0.01, compared with control samples).

Although previous studies have shown an important role of complement activation in murine PE-induced anaphylaxis model, until now it has been unclear which pathway is involved in complement activation. In this study, using a murine anaphylaxis model, we demonstrated that peanuts could activate complement and induce hypothermia shock via both the lectin and classical pathways in vivo. Although Khodoun et al. showed that PE activates complement in vitro by using murine and human plasma, their studies did not reveal the details of PE-induced complement activation, such as the contribution of each complement pathway and recognition molecules. In our in vitro studies, we demonstrated that

T. Kodama et al. / Immunobiology 218 (2013) 844–850

Rectal temperature (ºC)

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Fig. 8. Anaphylaxis induced by PE required C4, and partially MASP-2. (A) WT (n = 6), MASP-2 deficient (n = 4), and C4 deficient mice (n = 6), which were pretreated with long acting form of IL-4 and propranolol, were injected intravenously with 500 ␮g of PE, and followed for 90 min with rectal thermometry. (B) Plasma samples were collected 5 min after PE injection and assayed for C3a levels by ELISA. Data represent the means and SEMs (** P < 0.01, compared with WT mice. # P < 0.05, compared with MASP-2 deficient mice).

immunoglobulin and C1q bound to PE (Fig. 3B), and PE caused complement activation via Ca2+ dependent manner (Fig. 2A), serum immunoglobulin (Fig. 3A), and C4 (Fig. 2C). Furthermore, we showed that PE-induced C3 deposition was inhibited by an addition of anti-C1q sera (Fig. 4). Whereas C3 deposition was completely abolished in C4 deficient sera, we found no difference in C3 deposition among WT sera, MASP-1/3 deficient sera or MASP-2 deficient sera in vitro. These results show that PE activates complement via the classical pathway only, and not the alternative or lectin pathway in vitro. Nevertheless, the levels of C3 deposition on PE did not decrease in MASP-2 deficient sera, we demonstrated that the elevation of plasma C3a concentration caused by PE injection was attenuated not only in C4 deficient mice but also in MASP-2 deficient mice (Fig. 7). Moreover, PE-induced anaphylactic shock was also prevented in C4 deficient mice and partially in MASP-2 deficient mice consistent with decreased plasma C3a levels (Fig. 8). Furthermore, we showed that FCN-A bound to PE (Fig. 5C). Taken together, our data suggest that PE activates complement via the lectin and classical pathways in vivo. Despite not being able to show the role of the lectin pathway in our in vitro C3 deposition study, we showed the involvement of the lectin pathway in PE-induced anaphylaxis in vivo. Though C3 deposition assay on plate-coated antigen is a common method to evaluate complement activation, there are some limitations. Since, we used PE as coated antigen in the in vitro system, it dropped some types of antigen, especially low molecular mass proteins that could not effectively bound to plate. Alternatively, there were no coagulation factors and no cells in the in vitro system because we used sera. It leads to a lack of interaction between coagulation factors and cell-associated complement regulatory factors, such as fibrinogen,

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Crry and decay-accelerating factor (Endo et al. 2010; Li et al. 1993; Sjoberg et al. 2009). These factors may have been involved in the discordance between in vitro and in vivo studies. There is a study of complement in adenovirus infection, in which in vitro studies did not agree with in vivo studies, and it pointed out the difficulty of reproducing the complement activation in vitro in the same manner as observed in vivo (Tian et al. 2009). Our in vivo data suggest that PE activates complement via both the classical pathway and the lectin pathway. Other investigators suggested that PE might activate complement via the alternative or lectin pathway, because PE induced shock in Rag1 deficient mice and ␮MT mice (Khodoun et al. 2009). We investigated the role of the alternative pathway in PE-induced complement activation by using MASP-1/3 deficient mice lacking the alternative pathway activation (Takahashi et al. 2010) (Fig. 7). After PE injection, MASP1/3 deficient mice showed elevated levels of plasma C3a similar to WT mice. In contrast, both MASP-2 deficient and C4 deficient mice showed significantly low levels of plasma C3a compared to WT mice. These results indicate that the alternative pathway is not involved in PE-induced complement activation. To date, there are some studies about the relationship between complement pathways and allergy, but almost all studies pointed out the concern of the alternative pathway, not the lectin or classical pathway. There are only a few studies about the lectin and classical pathways in allergy. Though house dust allergens were reported to activate both pathways in vitro (Beukelman et al. 1986; Varga et al. 2003), there were no in vivo data. Another study also investigated the involvement of the lectin pathway in murine anaphylaxis. In their study, MBL-A and MBL-C bound to ovalbumin, however MBL deficient mice and H2-Bf/C2 deficient mice, which lack all three routes of complement activation, showed no improvement in ovalbumin induced anaphylaxis (Windbichler et al. 2006). Thus, the roles of the lectin and classical pathways to allergy remain unclear, and our study is the first report to show the roles of the lectin and classical pathways in an allergy model. In this study, we demonstrated that PE both in vitro and in vivo activated complement in mice, however the PE-antigen, which activates complement, remains unknown. Natural antibodies consisting of IgM are known to recognize carbohydrates. Similarly, recognition molecules of the lectin pathway, such as MBLs and FCNs, also recognize carbohydrates. Because peanuts are rich in glycoprotein, carbohydrate chains on peanut glycoprotein in PE may be recognized by natural antibodies (i.e., IgM) and lectins followed by complement activation. Indeed, the binding of IgM (Fig. 3B) and FCN-A (Fig. 5C) on PE was clearly observed in our study. Further studies are required to identify the PE antigen(s) that are recognized by IgM/FCN-A and activate complement. In conclusion, we show that PE activates complement via both the lectin and classical pathways in vivo, and the complement activation has a potential to cause anaphylaxis in mice. In our PEinduced shock model, PE injection alone was not enough to induce anaphylactic shock, and pre-treatment with IL-4, anti-IL-4 Ab, and propranolol was required. Therefore, we think that PE-induced complement activation acts synergistically with other factors, such as IgE dependent mast cell degranulation to cause anaphylaxis. Moreover, the C3 component attached to the antigen works as adjuvant (Dempsey et al. 1996), and followed by immunoglobulin production against PE antigens. A deeper understanding of PE-induced complement activation would suggest strategies to prevent severe anaphylaxis and sensitization.

References Beukelman, C.J., Rademaker, P.M., van Dijk, H., Aerts, P.C., Berrens, L., Willers, J.M., 1986. House dust allergen activates the classical complement pathway in mouse serum. Immunol. Lett. 13, 159–164.

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Bollinger, M.E., Dahlquist, L.M., Mudd, K., Sonntag, C., Dillinger, L., McKenna, K., 2006. The impact of food allergy on the daily activities of children and their families. Ann. Allergy Asthma Immunol. 96, 415–421. Cardoso, C.R., Provinciatto, P.R., Godoi, D.F., Vieira, T.S., Ferreira, B.R., Teixeira, G., Rossi, M.A., Cunha, F.Q., Silva, J.S., 2008. B cells are involved in the modulation of pathogenic gut immune response in food-allergic enteropathy. Clin. Exp. Immunol. 154, 153–161. del Balzo, U., Polley, M.J., Levi, R., 1988. Activation of the third complement component (C3) and C3a generation in cardiac anaphylaxis: histamine release and associated inotropic and chronotropic effects. J. Pharmacol. Exp. Ther. 246, 911–916. Dempsey, P.W., Allison, M.E., Akkaraju, S., Goodnow, C.C., Fearon, D.T., 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271, 348–350. Drouin, S.M., Corry, D.B., Kildsgaard, J., Wetsel, R.A., 2001. Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy. J. Immunol. 167, 4141–4145. Endo, Y., Nakazawa, N., Iwaki, D., Takahashi, M., Matsushita, M., Fujita, T., 2010. Interactions of ficolin and mannose-binding lectin with fibrinogen/fibrin augment the lectin complement pathway. J. Innate Immunol. 2, 33–42. Finkelman, F.D., 2007. Anaphylaxis: lessons from mouse models. J. Allergy Clin. Immunol. 120, 506–515. Finkelman, F.D., Madden, K.B., Morris, S.C., Holmes, J.M., Boiani, N., Katona, I.M., Maliszewski, C.R., 1993. Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine–anti-cytokine antibody complexes. J. Immunol. 151, 1235–1244. Fischer, M.B., Ma, M., Goerg, S., Zhou, X., Xia, J., Finco, O., Han, S., Kelsoe, G., Howard, R.G., Rothstein, T.L., et al., 1996. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157, 549–556. Fujita, T., 2002. Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2, 346–353. Grundy, J., Bateman, B.J., Gant, C., Matthews, S.M., Dean, T.P., Arshad, S., 2001. Peanut allergy in three year old children a population based study. J. Allergy Clin. Immunol. 107, S231. Humbles, A.A., Lu, B., Nilsson, C.A., Lilly, C., Israel, E., Fujiwara, Y., Gerard, N.P., Gerard, C., 2000. A role for the C3a anaphylatoxin receptor in the effector phase of asthma. Nature 406, 998–1001. Iwaki, D., Kanno, K., Takahashi, M., Endo, Y., Lynch, N.J., Schwaeble, W.J., Matsushita, M., Okabe, M., Fujita, T., 2006. Small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway. J. Immunol. 177, 8626–8632. Kawamoto, S., Yalcindag, A., Laouini, D., Brodeur, S., Bryce, P., Lu, B., Humbles, A.A., Oettgen, H., Gerard, C., Geha, R.S., 2004. The anaphylatoxin C3a downregulates the Th2 response to epicutaneously introduced antigen. J. Clin. Invest. 114, 399–407. Khodoun, M., Strait, R., Orekov, T., Hogan, S., Karasuyama, H., Herbert, D.R., Kohl, J., Finkelman, F.D., 2009. Peanuts can contribute to anaphylactic shock by activating complement. J. Allergy Clin. Immunol. 123, 342–351. Krug, N., Tschernig, T., Erpenbeck, V.J., Hohlfeld, J.M., Kohl, J., 2001. Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma. Am. J. Respir. Crit. Care Med. 164, 1841–1843. Li, B., Sallee, C., Dehoff, M., Foley, S., Molina, H., Holers, V.M., 1993. Mouse Crry/p65. Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF. J. Immunol. 151, 4295–4305. Mittrucker, H.W., Matsuyama, T., Grossman, A., Kundig, T.M., Potter, J., Shahinian, A., Wakeham, A., Patterson, B., Ohashi, P.S., Mak, T.W., 1997. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275, 540–543.

Perry, T.T., Scurlock, A.M., Jones, S.M., Tilford, J.M., 2009. Economic and caregiver impacts of food allergy. In: Annual Meeting of the American Academy of Allergy, Asthma, and Immunology, vol. 123, p. S113. Sampson, H.A., 2000. Food anaphylaxis. Br. Med. Bull. 56, 925–935. Sekine, H., Machida, T., Suzuki, E., Avila, E.M., Ruiz, P., Reilly, C.M., Gilkeson, G.S., 2010. IRF4-deficient lupus-prone MRL/lpr mice lack serum Ig/auto-Abs and Th17 cells but develop non-immune complex-mediated diffuse proliferative glomerulonephritis and dermatitis/ear necrosis. Arthritis Rheum. 62, 435. Sicherer, S.H., Munoz-Furlong, A., Sampson, H.A., 2003. Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: a 5-year follow-up study. J. Allergy Clin. Immunol. 112, 1203–1207. Sjoberg, A.P., Trouw, L.A., Blom, A.M., 2009. Complement activation and inhibition: a delicate balance. Trends Immunol. 30, 83–90. Skolnick, H.S., Conover-Walker, M.K., Koerner, C.B., Sampson, H.A., Burks, W., Wood, R.A., 2001. The natural history of peanut allergy. J. Allergy Clin. Immunol. 107, 367–374. Strait, R.T., Morris, S.C., Smiley, K., Urban Jr., J.F., Finkelman, F.D., 2003. IL-4 exacerbates anaphylaxis. J. Immunol. 170, 3835–3842. Sun, J., Arias, K., Alvarez, D., Fattouh, R., Walker, T., Goncharova, S., Kim, B., Waserman, S., Reed, J., Coyle, A.J., et al., 2007. Impact of CD40 ligand, B cells, and mast cells in peanut-induced anaphylactic responses. J. Immunol. 179, 6696–6703. Szebeni, J., 2005. Complement activation-related pseudoallergy: a new class of druginduced acute immune toxicity. Toxicology 216, 106–121. Szebeni, J., Baranyi, L., Savay, S., Bodo, M., Milosevits, J., Alving, C.R., Bunger, R., 2006. Complement activation-related cardiac anaphylaxis in pigs: role of C5a anaphylatoxin and adenosine in liposome-induced abnormalities in ECG and heart function. Am. J. Physiol. Heart Circ. Physiol. 290, H1050–H1058. Takahashi, M., Ishida, Y., Iwaki, D., Kanno, K., Suzuki, T., Endo, Y., Homma, Y., Fujita, T., 2010. Essential role of mannose-binding lectin-associated serine protease-1 in activation of the complement factor D. J. Exp. Med. 207, 29–37. Takahashi, M., Iwaki, D., Kanno, K., Ishida, Y., Xiong, J., Matsushita, M., Endo, Y., Miura, S., Ishii, N., Sugamura, K., et al., 2008. Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway. J. Immunol. 180, 6132–6138. Taube, C., Thurman, J.M., Takeda, K., Joetham, A., Miyahara, N., Carroll, M.C., Dakhama, A., Giclas, P.C., Holers, V.M., Gelfand, E.W., 2006. Factor B of the alternative complement pathway regulates development of airway hyperresponsiveness and inflammation. Proc. Natl. Acad. Sci. U. S. A. 103, 8084–8089. Tian, J., Xu, Z., Smith, J.S., Hofherr, S.E., Barry, M.A., Byrnes, A.P., 2009. Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo. J. Virol. 83, 5648–5658. Toogood, J.H., 1987. Beta-blocker therapy and the risk of anaphylaxis. CMAJ 136, 929–933. van de Graaf, E.A., Jansen, H.M., Bakker, M.M., Alberts, C., Eeftinck Schattenkerk, J.K., Out, T.A., 1992. ELISA of complement C3a in bronchoalveolar lavage fluid. J. Immunol. Methods 147, 241–250. Varga, L., Szilagyi, K., Lorincz, Z., Berrens, L., Thiel, S., Zavodszky, P., Daha, M.R., Thielens, N.M., Arlaud, G.J., Nagy, K., et al., 2003. Studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement. Mol. Immunol. 39, 839–846. Windbichler, M., Echtenacher, B., Takahashi, K., Ezekowitz, R.A., Schwaeble, W.J., Jenseniuis, J.C., Mannel, D.N., 2006. Investigations on the involvement of the lectin pathway of complement activation in anaphylaxis. Int. Arch. Allergy Immunol. 141, 11–23. Yalcindag, A., He, R., Laouini, D., Alenius, H., Carroll, M., Oettgen, H.C., Geha, R.S., 2006. The complement component C3 plays a critical role in both Th1 and Th2 responses to antigen. J. Allergy Clin. Immunol. 117, 1455–1461. Yonemasu, K., Sasaki, T., 1981. Purification and characterization of subcomponent C1q of the first component of mouse complement. Biochem. J. 193, 621–629.