Depletion of monocytes/macrophages with clodronate liposomes significantly reduced the hypothermia response

Depletion of monocytes/macrophages with clodronate liposomes significantly reduced the hypothermia response. in this model. These features were reduced in mice lacking the IgE receptor FcRI, the IgG receptor FcRIII or the common -chain FcR. FcRIV blockade resulted in a partial reduction of inflammation without any effect on hypothermia. Depletion of monocytes/macrophages with clodronate liposomes significantly reduced the hypothermia response. By contrast, depletion of neutrophils or basophils experienced no significant effects in this ASA model. Both the hypothermia and inflammation were dependent on platelet-activating factor (PAF) and histamine and were reduced in two types of mast cell (MC)-deficient mice. Finally, engraftment of MC-deficient mice with bone marrow-derived cultured MCs significantly exacerbated the hypothermia response, and restored inflammation to levels much like those observed in wild-type mice. Conclusion Components of the classical and option pathways contribute to anaphylaxis in this adjuvant-free model, with key functions for mast cells and monocytes/macrophages. and/or MC-deficient mice9, 13, 20. Depending on the mouse strain and ASA model used, basophils have been shown either to contribute to the systemic response13, 19 or to play little to no significant role17, 21. Similarly, depletion of monocytes/macrophages or antibody-mediated neutrophil depletion reduces anaphylaxis in some but not all ASA models11, 17, 19, 21. We hypothesize that such conflicting results in ASA models might reflect, at least in part, the choice of adjuvant used during sensitization, as numerous adjuvants might differentially influence the production of individual antibody isotypes. Moreover, in humans who develop anaphylactic reactivity, sensitization to antigen generally occurs in the absence of an artificial adjuvant. We therefore developed an adjuvant-free mouse model of ASA and assessed the contributions of components of the classical and alternate pathways of anaphylaxis in this model. Methods Mice C57BL/6J (WT) mice were purchased from Jackson Laboratories (Bar Harbor, Me) or Charles River (France). mice (B6.129P2-mice (B6.129P2 mice backcrossed to C57BL/6 for 12 generations) were from Taconic (New York, NY). C57BL/6-mice were originally provided by Peter Besmer (Memorial Sloan-Kettering Malignancy Center, NY, USA); we then backcrossed these mice to C57BL/6J mice for more than 11 generations22. mice7 (FcRI alpha chain-deficient mice backcrossed to C57BL/6 for more than 8 generations and kindly provided by Jean-Pierre Kinet, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA), mice (backcrossed to C57BL/6 for at least 10 generations)23, and mice (backcrossed to C57BL/6 for at least Pasireotide 9 generations)24 were bred and maintained at the Stanford University or college Research Animal Facility. Aged-matched male mice were used in all experiments. Experiments in Fig 2 used C57BL/6J WT mice as controls. We used littermate controls in all other experiments. All Pasireotide animal care and experimentation was conducted in Rabbit Polyclonal to HER2 (phospho-Tyr1112) compliance with the guidelines of the National Institutes of Health and with the specific approval of the Institutional Animal Care and Use Committee of Stanford University or college and of the Animal Ethics committee CETEA (Institut Pasteur, Paris, France) registered under #C2EA-89. Open in a separate window Physique 2 Functions of Fc receptors in OVA-induced ASA(ACB) OVA-induced hypothermia in OVA-sensitized WT, and mice (A), or WT mice treated with an anti-FcRIV antibody or an isotype control (B). (CCH) Numbers of leukocytes in the PLF 3 days after challenge. Data are pooled from two (Isotype Pasireotide and -FcRIV; 0.05, 0.01 or 0.001. OVA-induced adjuvant-free model of active anaphylaxis Six to 8-week-old mice were sensitized intraperitoneally (i.p.) with 10 g of endotoxin-free ovalbumin (Endograde OVA; BioVendor; 0.01 EU endotoxin per injection) in 100 L of PBS once a week for 6 weeks. Two weeks after the last i.p. sensitization, mice were challenged i.p. with 500 g of OVA. Rectal measurements of body temperature were performed immediately before (time 0) and at different time points for up to 2 h after Pasireotide challenge. Mice were sacrificed at numerous time points after challenge (as indicated) for assessment of inflammatory cell figures in the peritoneal cavity and histology. Other methods Please observe this articles Online Repository at for the methods for circulation cytometry, peritoneal lavage and differential cell counts, depletion of basophils, monocytes/macrophages and neutrophils, blockade of FcRIV, histologic analysis, measurement of serum OVA-specific IgG1 and IgG2c antibodies, IgE-mediated PSA, ASA with adjuvant, treatment with an H1 anti-histamine, a PAF receptor antagonist, quantification of histamine and PAF-AH, and generation and adoptive transfer of bone marrow-derived cultured mast cells (BMCMCs). Statistical analyses Results represent mean SEM or mean + SEM, with values for individual mice represented for quantifications of histamine, PAF-AH and leukocytes. We used an unpaired Students test (body temperature) or an unpaired Mann-Whitney test (all other data) to assess the significance of differences between two units of data. values 0.05 are considered statistically significant. Results Development of an adjuvant-free model of ASA in C57BL/6 mice We developed an adjuvant-free mouse model of ASA consisting of performing i.p. sensitizations with endotoxin-free.