The present study focused on two important issues—the structural characteristics of Ag required for efficient cross-presentation by DC and the involvement of HSP70 in cross-presentation. These two issues may be seemingly distinct; however, they share a common thread—antigen translocation from endosome to the cytosol.
We showed that paraformaldehyde-fixed OVA was significantly less efficient in translocation from endosome to cytosol following internalization, thus, in cross-presentation, compared to native OVA. As internalization efficiency was nearly comparable between two forms of the protein, the decreased ability of fixed OVA is, at least in part, likely caused by its inflexible structure, which makes it difficult to translocate through a putative translocon in the endosomal membrane. In other words, the pore structure of the translocon is too narrow for native proteins to pass through while keeping their original 3D structure, as previously suggested [1
]. Artificial creation of pores in the endosomal membranes with Endo-Porter allowed fixed OVA to translocate to the cytosol, whereas normal OVA translocation was unaffected (), suggesting that the translocation mechanism of fixed OVA may be different from that of native OVA. It is likely that translocation of fixed OVA depends simply on the pore size of the translocon. Diffusion to the cytosol in an Ag dose-dependent manner might be a main mechanism operating in this case. By contrast, unfolding of an internalized Ag occurs within the endosome in the case of native OVA, which might provide an opportunity for HSP90 or other molecules to capture and pull the molecule out to the cytosol through the narrow window of the translocon. This concept should be further examined in future's experiments.
We showed that an HSP70 inhibitor, VER, completely blocked cross-presentation as well as endogenous Ag presentation (). By contrast, the HSP90 inhibitor, radicicol, blocked cross-presentation completely but showed only partial inhibition of endogenous Ag presentation (, [19
]). These results suggest that the molecular mechanism inhibited by VER is distinct from that inhibited by radicicol. Radicicol mainly blocks translocation of Ag from endosome to cytosol [20
], whereas VER might block a mechanism common to both cross-presentation and endogenous Ag presentation. The common mechanism might be the transport of unfolded Ag to the proteasome. Thus, unfolded Ag emerging across the endosomal membrane or newly synthesized proteins on the ribosome that have become unfolded due to various reasons are captured by HSP70 and forwarded to the proteasome for degradation [26
]. Indeed, HSP70 was shown previously associated with Bag1- and HSP70-bound proteins to be degraded are transported to the proteasome [27
]. A postproteasomal event might also be a target of VER, since a similar HSP70 inhibitor deoxyspergualin (DSG) blocked MHCI antigen presentation due to dissociation of peptides chaperoned by HSP70 [28
In contrast to radicicol, VER facilitates translocation of AF647-labeled OVA to the cytosol, indicating that HSP70 has a suppressive effect on translocation of exogenous Ag to the cytosol. HSP70-dependent, constitutive suppression of Ag translocation would therefore result in downregulation of cross-presentation. This scenario may seem contraindicated in terms of effective host defense. However, blocking of Ag translocation into the cytosol results in Ag retention in endosome, which in turn stimulates fusion with lysosomes, eventually followed by enhanced MHCII antigen presentation. In this context, we recently observed that HSP90α
-deficient mice showed enhanced T-cell-dependent antibody production [29
], whereas T-cell-independent antibody production was unchanged [29
]. We believe that this phenotype is caused by a spontaneous increase in the knock-out mice of MHCII presentation because compared to DC of normal mice, splenic DC of HSP90α
-deficient mice showed increased ability to present Ag to CD4+
T cells after pulsing with graded doses of OVA [29
]. A model depicting these pathways is shown in .
Figure 7 Model illustrating how HSP70 and HSP90 regulate cross-presentation and endogenous Ag presentation. In cross-presentation, internalized exogenous Ag is unfolded within the endosome and translocated through a putative translocon Sec61 complex into the cytosol. (more ...)
In conclusion, extracellular Ag to be translocated from endosome to the cytosol needs some structural flexibility, and translocation of Ag to the cytosol is regulated by two distinct cytosolic molecular chaperones, HSP70 and HSP90. The dichotomy of Ag presentation (MHCI versus MHCII presentation of exogenous Ag) regulated by HSP70 and HSP90 might be associated with cross-priming and cross-tolerance, which would give rise to differential outcomes in the onset of autoimmune disease as well as in host defense against cancer and infectious disease.