Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40
Konstantina AntoniouFanny EnderTillman VollbrandtYves LaumonnierFranziska RathmannChandrashekhar PasareHarinder SinghJörg Köhl
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Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1- but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1- cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.Disulfide-reduced and carboxymethylated ovalbumin was treated at pH 9.9 and 55 degrees C for 24 h as a specific condition for preparation of S-ovalbumin. The stability and conformation of the product were investigated. Such alkaline treatment converted native protein to S-ovalbumin, but this modified ovalbumin was not stabilized, according to results of calorimetric analysis. Instead, it had lost its native like conformation; the magnitude of CD spectra decreased. The conformation after alkaline treatment was not clear, but the possibility of aggregation was excluded by electrophoretic analysis. These observations showed that the transformation of native ovalbumin into S-ovalbumin requires the presence of the disulfide bond.
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Intrinsic viscosity, Stokes radius and the hydrophobic coefficient of Keshavarz and Nakai [Biochim. Biophys. Acta, 576, 269 (1979)] were measured to compare the shape and surface hydrophobicity of ovalbumin and s-ovalbumin. Both the intrinsic viscosity and Stokes radius of s-ovalbumin were smaller than those of ovalbumin, which suggests that the configuration of s-ovalbumin became more compact during the ovalbumin-s-ovalbumin transformation. The hydrophobic coefficient of s-ovalbumin was larger than that of ovalbumin, which suggests that the surface hydrophobicity of s-ovalbumin was larger than that of ovalbumin. Further, these properties were measured for ovalbumin samples obtained at various stages of ovalbumin-s-ovalbumin transformation. Changes in the shape and surface hydrophobicity of ovalbumin were not found in the first stage of ovalbumin-s-ovalbumin transformation. They changed rapidly in the last stage of the ovalbumin-5-ovalbumin transformation.
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Ovalbumin gene Y has been known as a member of the ovalbumin gene family since 1982, when its encoding gene was sequenced. In the present study, ovalbumin gene Y has been demonstrated as a new minor protein of hen egg white. This protein has been isolated by isoelectrofocalization and two-dimensional polyacrylamide gel electrophoresis and has been characterized using peptide mass fingerprinting. The concentration ratio of ovalbumin gene Y:ovalbumin is about 13:100. Unlike ovalbumin, ovalbumin gene Y is not phosphorylated, but like ovalbumin, this protein is glycosylated. Ovalbumin gene Y exists as a mixture of three molecular species, which differ in their isoelectric points. The polymorphism of this protein cannot be explained by various glycosylation levels.
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Titration curves of ovalbumin and s-ovalbumin were compared in studies of changes in properties of ovalbumin on conversion to s-ovalbumin. Although there are 49 carboxyl groups in ovalbumin, two were not titrated in 0.25 M KCl but on conversion to s-ovalbumin. A similar change was also noted in the two carboxyl groups of ovalbumin in denaturation with guanidine hydrochloride. Liberation ofcarboxyl groups was noted in ovalbumin samples which were assumed to contain various amounts of intermediate and the isoelectric focusing patterns of these samples also changed. No difference was noted in the both amounts of ionizable amino groups and phenolic hydroxyl groups between ovalbumin and s-ovalbumin. This seems to show that these two groups are not concerned with the liberation of carboxyl groups during ovalbumin-s-ovalbumin transformation.
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Both ovalbumin and s-ovalbumin gave maximumgel strength at both sides of the isoelectric point. Maximumgel forming pHs of s-ovalbumin were almost the same as those of ovalbumin, but maximumgel strength values of s-ovalbumin were much smaller than those of ovalbumin. Although the gel strength of both proteins increased with increased heating temperature, the gel strength of s-ovalbumin was much smaller than that of ovalbumin at every heating temperature. About the results of creep experiments, all heat-induced gels were analyzed as a four-element model and the magnitude of all the parameters of both s-ovalbumin and intermediate was smaller than that of ovalbumin. Scanning electron microscopic studies showedthat the structure of ovalbumin gels was very fine comparing those of s-ovalbumin and the intermediate.
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To study the mechanism of the formation of the heat-stable form of ovalbumin (s-ovalbumin), comparisons were made about the properties of ovalbumin and s-ovalbumin in native state. Although gross structural difference could not be found, some minor differences were clearly noted in some cases such as the DEAE-cellulose chromatographic elution pattern, the isoelectric focusing and the titration curve of both proteins. All these results seem to show that changes in the surface charge occur during ovalbumin-s-ovalbumin transformation.
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Abstract We examined the limited proteolysis of ovalbumin by pepsin and its effect on the functional properties of the ovalbumin. Pepsin hydrolyzed only the single peptide bond of ovalbumin between His‐22 and Ala‐23. This provided a large intermediate (MW 42,500), P‐ovalbumin. A P‐ovalbumin solution gave a transparent gel when heated. Under the same conditions, an ovalbumin solution gave a turbid gel. We studied the physicochemical properties of P‐ovalbumin and the formation of the transparent gel.
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Pepsin
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Ovalbumin is changed to a more stable form (S-ovalbumin) during the storage of shell eggs. Conversion also occurs in an isolated ovalbumin solution, the rate increasing with pH and temperature. First order rate constants for the reaction have been measured at pH 9-10 and at temperatures between 20 and 50�C. The reaction is apparently irreversible and does not appear to involve loss of" amino acids or small peptides.
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Titration curves of ovalbumin and s-ovalbumin were compared in studies of changes in properties of ovalbumin on conversion to s-ovalbumin. Although there are 49 carboxyl groups in ovalbumin, two were not titrated in 0.25 M KCl but on conversion to s-ovalbumin. A similar change was also noted in the two carboxyl groups of ovalbumin in denaturation with guanidine hydrochloride. Liberation of carboxyl groups was noted in ovalbumin samples which were assumed to contain various amounts of intermediate and the isoelectric focusing patterns of these samples also changed. No difference was noted in the both amounts of ionizable amino groups and phenolic hydroxyl groups between ovalbumin and s-ovalbumin. This seems to show that these two groups are not concerned with the liberation of carboxyl groups during ovalbumin-s-ovalbumin transformation.
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Hydrochloride
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