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Notes: Alveolar macrophages and mast cells isolated from guinea pig lung were passively sensitized with IgG1, IgG2, or serum obtained from guinea pigs actively sensitized with ovalbumin. The release of histamine by mast cells and of thromboxane A2 by alveolar macrophages upon ovalbumin challenge indicated that both antibodies and serum were capable of sensitizing these cells with similar effectiveness. Heating the scrum at 56°C for 4 h to inactivate IgE did not modify the antigen-dependent response of lung cells. These results suggest a predominant role for IgG in the allergic response of the guinea pig through the activation of different cell types such as lung mast cells and alveolar macrophages.

Notes: Background Mouse models of allergy are used to study the mechanisms of induction and perpetuation of bronchopulmonary hyper-reactivity (BHR) as related to eosinophils and specific IgE.Objective Our aim was to adapt the current model for the study of bovine β-lactoglobulin (BLG), a major cow's milk allergen, and to further analyse the mechanisms of the acute and late allergic reaction.Methods Female Balb/c mice were sensitized intraperitoneally with BLG and the influence of the adjuvant and of the BLG dose on the IgE response was analysed, IgE and IgG1 epitopes being characterized. Once optimized, this model was applied to the study of the active phase of allergy in the respiratory tract after a single airway challenge using native or denatured BLG, which contains only linear epitopes.Results An immediate allergic reaction was characterized by the rapid release of histamine into the bronchoalveolar lavage fluids. Prostaglandin (PG)D2 was only present when the standard histamine-releasing agent compound 48/80 or denatured BLG were used as triggers, whereas native BLG induced leukotriene release. Twenty-four hours after challenge, BHR, eosinophil influx, IL-4 and IL-5 production, plasma exudation and mucus production were very much increased, differently depending on the allergen structure, and indicated the occurrence of the late allergic reaction. Our results show that the murine model can be used to study the mechanisms of allergy to clinically relevant antigens, such as those contained in cow's milk. The acute allergic reaction, which depends on the structural feature of the allergen, is composed of two distinct pathways characterized by peptido-leukotrienes or PGD2 production, which may result from distinct activation intensities of mast cells, leading to distinct late reactions.Conclusion This study thus demonstrates a clear link between the structural feature of a protein, and the physiopathology of the experimental asthmatic reaction.

Notes: Background Antigen-induced bronchopulmonary hyper-reactivity (BHR) is generally associated with eosinophilia. It involves cytokines produced by Th2 lymphocytes, including IL-4, IL-5 and IL-13, which are implicated in IgE production, eosinophil differentiation and attraction, and related events relevant to allergic inflammation, whose mechanisms remain unclear.Objective To investigate the mechanisms by which Th2 cytokines mediate eosinophilia and subsequent BHR using ovalbumin (OVA)-immunized and OVA-challenged IL-4Rα–/– and IL-4–/– mice, which fail to transduce and/or to produce IL-4 and IgE as compared with wild type (WT) mice, and specific neutralizing antibodies.Methods On days 0 and 7, mice were immunized subcutaneously (s.c.) with OVA. At day 14, anti-IL-5 or anti-IL-13 antibodies were administered intranasally and/or intravenously before allergenic challenge. Different functional and cellular parameters were studied in vivo and cytokine production was followed with a newly described ex vivo procedure using lung explants.Results IL-4Rα–/– and IL-4–/– mice developed BHR and pulmonary eosinophilia, even though eosinophil recruitment to the bronchoalveolar liquid lavage (BALF) was reduced. In vivo, IL-4–/– and IL-4Rα–/– mice produced, respectively, no or reduced amounts of IL-5 in the BALF/serum as compared with WT mice, whereas no IL-13 in the BALF was detected. By contrast, ex vivo, surviving lung explants from WT and IL-4–/– or IL-4Rα–/– mice produced IL-13 and large amounts of IL-5. The neutralization of IL-5 in vivo (BALF and serum) and ex vivo (from lung explant) in IL-4Rα–/– and WT mice failed to suppress BHR and lung eosinophilia, and to modify IL-13 production ex vivo. In addition, neutralization of IL-13 in vivo from lung explant also failed to abrogate BHR and lung eosinophilia, whereas IL-5 was unchanged.Conclusion Antigen-induced BHR can develop independently from IL-4, IL-5 or IL-13 and from the IL-4α receptor chain, suggesting a possible novel IL-4, IL-5 and IL-13-independent pathway for the development of BHR in allergic BALB/c mice. The failure of IL-5 or IL-13 antibodies to prevent BHR in IL-4Rα–/– mice suggests that neither is indispensable for BHR but does not exclude a role for lung tissue eosinophilia.

Notes: Background Alveolar macrophages (AM) may participate in brochopulmonary hyperreactivity by secreting cytokines that recruit mature eosinophils, or induce eosinophil production from recruited circulating progenitors.Objective To define whether AM products can contribute to lung eosinophil production in immunized guinea pigs (GP), by analysing the effect of AM culture supernatatits (AM-SN) on in vitro eosinophilopoiesis.Methods Liquid and semi-solid bone marrow (BM) cultures were seeded witb SN from 95% pure AM exposed to LPS.Results AM-SN increased very significantly the long-term viability, cell proliferation and eosinophil production in liquid culture and supported formation of eosinophil-bearing mixed colonies, by acting on progenitors depleted of mature eosinophils. The effect on eosinophil production was not duplicated by natural or recombinant sources of GM-CSF (which nevertheless supported GM colony formation by GP BM), not by rhIL-8 (which was active on GP cells) and was not due to residual LPS. FPLC separation of active AM SN yielded a peak of apparent m.w. 43 kDa, active on both liquid and semi-solid cultures. The active moiety was heat- and trypsin-resistant. Neutralizing monoclonal antibodies to hGM-CSF, mGM-CSF, hIL-3 and mIL-3 failed to deplete the activity in AM-SN. Ovalbumin immunization induced its production by AM even without LPS challenge.Conclusions The lack of T lymphocytes among factor-producing AM, the properties of the active material, the inability of GM-CSF to reproduce these effects, and the failure of MoAbs to GM-CSF and to IL-3 to neutralize the activity indicate it is not due to the major eosinopoietic factors GM-CSF, IL-3 or IL-5.

Notes: Alveolar macrophages from guinea-pigs sensitized by different amounts of ovalbumin, administered either by subcutaneous injection or aerosol exposure, liberate increased amounts of arachidonic acid and thromboxanc B2 when challenged in vitro with ovalbumin. This antigen-dependent activation of macrophages was immunospecific. The comparison between different sensitization procedures showed that the aerosol exposure was the most efficient with respect to the activation of macrophages, as cells from guinea-pigs sensitized subcutaneously were poorly activated by the antigen unless high doses were used for sensitization. The antigen-dependent activation of macrophages was affected by acid and neutral washings, suggesting the involvement of a loosely bound antibody that could not be identified. These observations suggest that, as mast cells and basophils, alveolar macrophages from actively sensitized guinea-pigs contribute to the allergic reaction by an antibody-mediated mechanism.