In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

We investigated epidermal cell suspensions prepared from lesional and nonlesional atopic eczema skin, other inflammatory skin conditions, and normal human skin for high-affinity IgE receptor (Fc epsilon RI) expression on dendritic CD1a cells by quantitative flow cytometric analysis. A single CD1a bright/CD1b neg/Fc epsilon RI dim/CD23 neg/CD32 dim/HLA-DR bright/CD36 neg population was found in normal skin. In contrast, lesional skin of atopic eczema and other inflammatory skin diseases harbored variable proportions of two distinct CD1a populations. Both populations exhibited typical ultrastructural features of Langerhans cells, but the second one lacked Birbeck granules and was unreactive to the Birbeck granule-specific LAG antibody. Both populations differed phenotypically: classical Langerhans cells were CD1a bright/CD1b neg/Fc epsilon RI dim/CD23 neg/CD32 dim/HLA-DR bright/CD36 dim, while the second population was CD1a dim/CD1b dim/Fc epsilon RI bright/CD23 dim/CD32 dim/HLA-DR bright/CD36 bright. The highest Fc epsilon RI expression was found on the second CD1a population in lesional atopic eczema skin. Furthermore, Fc epsilon RI expression on CD1a cells correlated significantly with the serum IgE level of the patients. Thus, a distinct population of CD1a inflammatory dendritic epidermal cells different from classical Langerhans cells appears in the epidermis of lesional skin and is subjected to specific signals leading to the upregulation of Fc epsilon RI in atopic eczema skin.

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.