Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing.
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Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum. (+info)
Nonvectorial surface transport, endocytosis via a Di-leucine-based motif, and bidirectional transcytosis of chimera encoding the cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells.
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Transfer of passive immunity from the mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to major histocompatibility class I antigens. Here, we analyzed the intracellular routing of chimera encoding the rat FcRn tail fused to the ecto- and transmembrane domain of the macrophage FcgammaRIIb. Newly synthesized chimera were delivered in a nonvectorial manner to the apical and basolateral cell surface, from where the chimera were able to internalize and transcytose. Apical to basolateral and basolateral to apical transcytosis were differently regulated. This intracellular routing of the chimera is similar to that of the native FcRn, indicating that the cytosolic tail of the receptor is necessary and sufficient to endow an unrelated FcR with the intracellular transport behavior of FcRn. Furthermore, the di-leucine motif in the cytosolic domain of FcRn was required for rapid and efficient endocytosis but not for basolateral sorting of the chimera. (+info)
The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells.
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HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE. (+info)
Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils.
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Differential display-polymerase chain reaction (DD-PCR) was used to evaluate changes in mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) treated human neutrophils to better understand how this cytokine affects the functions of neutrophils at the molecular level. Although a variety of cDNA fragments were identified as modulated by GM-CSF with the use of DD-PCR, one fragment in particular, NGS-17 (neutrophil GM-CSF-stimulated fragment #17), was characterized. The NGS-17 fragment hybridized to a 3.8-kh mRNA that encodes for a protein of a predicted molecular mass of 47.6 kDa. After cloning and sequencing, this gene was found to code for the recently sequenced tapasin or TAP-A protein. Immunoprecipitation and immunoblotting studies using anti-tapasin antibodies showed that tapasin is expressed in neutrophils and is associated with the MHC class I-TAP complex. Moreover, tapasin expression was found to be induced by dimethyl sulfoxide and by retinoic acid in HL-60 cells. This is the first report on the expression of tapasin in human neutrophils. It provides novel information, at the molecular level, on how GM-CSF enhances the functions of these cells. (+info)
Lipopolysaccharide can block the potential of monocytes to differentiate into dendritic cells.
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We examined whether priming monocytes (MO) with lipopolysaccharide (LPS) influenced their further differentiation into either macrophages (Mphi) or dendritic cells (DC). LPS-primed MO differentiated into Mphi when cultured further with Mphi colony-stimulating factor (M-CSF) but, if cultured then with granulocyte/Mphi (GM)-CSF and IL-4 (interleukin-4), only about 30% of the cells differentiated into CD1a+ CD14- DC and half became CD1a- CD14+ Mphi. Cytokines present during LPS priming could affect subsequent MO differentiation. Relative to priming with LPS alone, adding M-CSF to LPS did not modify differentiation of MO to Mphi in further culture with M-CSF, nor did it change the way of differentiation of MO into DC was altered if culture was later switched to GM-CSF/IL-4. Using GM-CSF/IL-4 plus LPS upon priming did not modify differentiation of MO to Mphi in further culture with M-CSF, as compared to priming with GM-CSF/IL-4 alone, but it counteracted the effect of LPS on the differentiation of MO to DC in further culture with GM-CSF/IL-4: about 75% of cells then became DC. Alternatively, despite activation by LPS, mature M-CSF-induced Mphi preserved the potential to differentiate into DC on subsequent culture with GM-CSF/IL-4. Thus, LPS, a bacterial product known to sustain maturation of MO/Mphi as well as of DC, may block the differentiation of MO into DC, except if signal triggering DC differentiation is delivered concomitantly, and modulate in this manner the induction of adaptive immune responses to infection. (+info)
Multicentric origin of hemochromatosis gene (HFE) mutations.
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Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends +info)
Demonstration of bovine CD8+ T-cell responses to foot-and-mouth disease virus.
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The aim of this study was to investigate the importance of cellular immunity in foot-and-mouth disease in cattle, in particular to determine whether a CD8+ T-cell response could be detected, as these cells may play a role in both immunity and virus persistence. As attempts to characterize classical cytotoxic T cells had yielded non-reproducible results, largely due to high backgrounds in control cultures, a proliferation assay was developed that was demonstrated to detect antigen-specific, MHC class I-restricted bovine CD8+ cells responding to foot-and-mouth disease virus (FMDV). Proliferative CD8+ T-cell responses were detected consistently from 10 to 14 days following infection with FMDV and typically lasted 3-4 weeks. The role of CD8+ T cells in control of the disease, in particular their relevance for the establishment of persistence, may now be investigated. (+info)
In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins.
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Heat shock proteins (HSP) Hsp70 and gp96 prime class I-restricted cytotoxic T cells against Ags present in the cells from which they were isolated. The immunization capacity of HSPs is believed to rely on their ability to bind antigenic peptides. In this study, we employed the well-established OVA and beta-galactosidase (beta-gal) antigenic model systems. We show that in vitro long-term established OVA and beta-gal-specific CTL clones release TNF-alpha and IFN-gamma when incubated with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides, HSP-mediated secretion of TNF-alpha and IFN-gamma requires cell contact of the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70 molecules purified from Ag-negative tissue, e.g., negative for antigenic peptide, are able to activate T cells in vivo, leading to significant higher frequencies in OVA-specific CD8+ T cells. In unprimed animals, these T cells lyse OVA-transfected cell lines and produce TNF-alpha and IFN-gamma after Ag stimulus. Taken together our data show that, besides the well-established HSP/peptide-specific CTL induction and activation, a second mechanism exists by which Hsp70 and gp96 molecules activate T cells in vivo and in vitro. (+info)