Besides maturation, the levels of antigen uptake by APCs have a large impact on subsequent T-cell activation. We found that Pyr-OVA and AGE-OVA were taken up by BMDCs to a stronger extent than native OVA. No enhanced uptake of CE-OVA, CM-OVA or MGO-OVA by BMDCs was observed. These results suggest that the enhanced uptake of Pyr-OVA and AGE-OVA by DCs lead to the enhanced T-cell activation by Pyr-OVA and AGE-OVA.
Recently, Rupa et al. showed that glycation of OVA with mannose (OVAMan) reduced the allergen uptake by DCs, whereas glycation with glucose (OVAGlu) did not alter the uptake level by DCs.(109) As a consequence, OVAMan induced less IL-2 production by naive CD4+ T-cells compared to native OVA or OVAGlu.(109) The findings were explained by a reduced α-helical configuration of OVAMan, but not of OVAGlu, compared to native OVA.(109) The partial denaturation might have destroyed receptor binding sites important for antigen uptake, since it has been shown that minor changes in the secondary structure of OVA influences protein function.(32, 109) In our measurements of the CD-spectra, the glycated OVAs retained a similar secondary structure as does native OVA. The discrepancy between our and their observations might be explained by differences in the levels and the profiles of glycation. AGE-OVA was prepared in phosphate buffer with glucose at 50 °C, whereas dry incubation at 100 °C for a shorter period was selected in the study of Rupa et al.(109) Around 69.8 % of lysine residues are modified in AGE-OVA in our study, in comparison to 95.4 % in OVAMan and 93.5 % in OVAGlu, respectively.(109) The different incubation conditions could generate different types of glycation structures. However, taken together both studies show that the uptake levels of glycated allergens by APCs influence the overall immunogenicity of the protein.
4.1.9.3.1 SR-AI/II mediates the enhanced uptake of Pyr-OVA and AGE-OVA by DCs
The identification of the receptors involved in the enhanced antigen uptake of Pyr-OVA and AGE-OVA was of particular interest, since SR-AI/II, CD36, SR-BI and galectin-3 are known AGE-binding receptors. We found that uptake of Pyr-OVA or AGE-OVA by SR-A-/- DCs was reduced compared to wt DCs showing that SR-AI/II mediates the uptake of Pyr-OVA and AGE-OVA. In contrast, the receptor deficiency did not impair the uptake of native OVA, CE-OVA, CM-OVA or MGO-OVA by DCs. The results suggest that SR-AI/II binds to pyrraline, but not to CEL, CML or MG-H1.
SR-AI/II is a trimer comprised of a transmembrane domain, a spacer region, a helical coiled-coil domain, a collagenous domain and a C-terminal cysteine-rich domain.(48) A variety of ligands, such as oxLDL or acLDL and chemically modified proteins including AGEs, bind to the collagenous domain of SR-AI/II.(48, 55) Previously, Nagai et al. showed that MGO-derived AGEs conjugated to BSA were not taken up by RAW264.7 cells, a murine macrophage-derived cell line expressing SR-AI/II.(84) Furthermore, no endocytosis of glyoxal-modified BSA, which contained high amounts of CML, by RAW264.7 cells was observed.(84) The results support the hypothesis that SR-AI/II does not bind the glycation structures CML and MG-H1.
SR-AI/II has been suggested to transfer its ligands to the MHC class II loading pathway for efficient T-cell activation.(110, 111) Consistent with this SR-AI/II function pyrraline modification enhances the CD4+ T-cell immunogenicity, but not the CD8+ T-cell immunogenicity of OVA. We could show that SR-AI/II is an important mediator of the enhanced CD4+ T-cell immunogenicity of Pyr-OVA and AGE-OVA. This result suggests that the association of SR-AI/II with glycated food allergens might have an influence on the sensitization phase of allergy, because TH2-cells, a subset of CD4+ T-cells,
are crucial for an efficient IgE production by B-cells.
4.1.9.3.2 The mannose receptor plays a role in the uptake of Pyr-OVA and AGE-OVA by DCs
It is well known that the MR binds to natural mannose residues in OVA. Blocking of the MR with mannan resulted in a reduced uptake of native OVA, Pyr-OVA and AGE-OVA by SR-A-/- DCs. At first, it was thought that the reduced DC uptake of the OVA samples by mannan treatment was only due to inhibition of the binding to the natural mannose residues. However, we found that the uptake of AGE-BSA by BMDCs was also reduced after blockage of the MR with mannan, although native BSA does not possess natural ligands of the MR. These results suggest that the MR might be an AGE-binding receptor and contributes to the uptake of Pyr-OVA and AGE-OVA by recognition of the
Results and Discussion 83 glycation structures. However, a previous study by Burgdorf et al. showed that the MR delivers its ligands to the MHC class I loading pathway for CD8+ T-cell activation.(59) Notably, only the CD4+ T-cell immunogenicity, but not the CD8+ T-cell immunogenicity, of OVA was enhanced by modification of OVA with pyrraline or AGEs. Moreover, the enhanced OVA-specific CD4+ T-cell activation by Pyr-OVA and AGE-OVA was significantly attenuated in co-culture with SR-A-/- BMDCs. These results suggest that SR-AI/II plays a crucial role in the uptake of Pyr-OVA and AGE-OVA by wt BMDCs, which lead to an efficient delivery to the MHC class II loading pathway for antigen presentation and therefore, this enhances OVA-specific CD4+ T-cell activation.
We also observed that mannan-treated SR-A-/- DCs took up Pyr-OVA, AGE-OVA and AGE-BSA to a small extent. The results suggest that another receptor could be involved in the endocytosis of the glycated samples by DCs, although it could only play a minor role in the uptake of Pyr-OVA and AGE-OVA. The receptors galectin-3, SR-BI or CD36, do not play a role, because their inhibition using specific inhibitors or blocking antibodies did not reduce the uptake of the OVA samples by SR-A-/- cells.
In summary, the strong uptake of Pyr-OVA and AGE-OVA is mediated by SR-AI/II and the MR, and another yet unknown receptor could be partially involved in the endocytosis of Pyr-OVA and AGE-OVA.