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J Salamero

Publications and source records attributed to J Salamero.

At least 19 recordsLinked to original sources
PubMed2001

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.

The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

Animals↗
PubMed2001

Unstimulated human CD4 lymphocytes express a cytoplasmic immature form of the common cytokine receptor gamma-chain.

As a component of various cytokine receptors, common cytokine receptor gamma-chain (gamma(c)) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of gamma(c) (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding gamma(c) gene. This apparent paradox was solved by the demonstration that polyclonal anti-gamma(c) Abs detected endoglycosidase-H-sensitive immature forms of gamma(c) (54-58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature gamma(c) is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature gamma(c) is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of gamma(c) is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of gamma(c) may require intracellular interactions with lineage- or subset-specific molecular partners.

Animals↗
PubMed2001

CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

Antigens, CD1↗
PubMed2001

In vitro production of dendritic cells from human blood monocytes for therapeutic use.

Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient's serum or AB serum) or in a serum-free medium (AIM V). The characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.

Adjuvants, Immunologic↗
PubMed2001

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.

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.

Cell Membrane↗
PubMed2001

Intracellular retention of the two isoforms of the D(2) dopamine receptor promotes endoplasmic reticulum disruption.

The dopamine D(2) receptor exists as a long (D(2a)) and a short (D(2b)) isoform generated by alternative splicing of the corresponding transcript, which modifies the length of the third cytoplasmic loop implicated in heterotrimeric G-protein-coupling. Anatomical data suggested that this segment regulates the intracellular traffic and localization of the receptor. To directly address this question we used a combination of tagging procedures and immunocytochemical techniques to detect each of the two D(2) receptor isoforms. Surprisingly, most of the newly synthesized receptors accumulate in large intracellular compartments, the plasma membrane being only weakly labeled, without significant difference between the two receptor isoforms. Double labeling experiments showed that this localization corresponded neither to endosomal compartments nor to the Golgi apparatus. The D(2) receptor is mostly retained in the endoplasmic reticulum (ER), the long isoform more efficiently than the short one. It is accompanied by a striking vacuolization of the ER, roughly proportional to the expression levels of the two receptor isoforms. This phenomenon is partly overcome by treatment with pertussis toxin. In addition, an intrinsic activity of the D(2) receptor isoforms is revealed by [(35)S]-GTP gamma S binding and cAMP assay, which suggested that expression of weakly but constitutively active D(2) receptors promotes activation of heterotrimeric G protein inside the secretory pathway. This mechanism may participate in the regulation of the cellular traffic of the D(2) receptors isoforms.

Animals↗
PubMed2001

Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

Antigens, CD↗
PubMed2000

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network.

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

Cell Compartmentation↗
PubMed2000

Expression of low-affinity Fc gamma receptor by a human metastatic melanoma line.

The class IIa of low-affinity receptors for the Fc region of IgG, Fc gamma RIIa, are expressed on immune cells. The cross-linking of Fc gamma RIIa by complexed IgG triggers activation of protein tyrosine kinase and internalization of immune complexes. In this report, we demonstrate the expression of Fc gamma RIIa by a human melanoma cell line (VIO) derived from a metastasis of a patient with regressive melanoma. The analysis of Fc gamma RIIa functions was performed in VIO cells and Fc gamma RlIa- or Fc gamma RIlb-transfected human melanoma cells (A375). The Fc gamma RIIa cross-linking induced protein tyrosine phosphorylation, including Fc gamma RIIa phosphorylation, and led to its internalization in a clathrin-independent way in human melanoma cells. Moreover, we showed that a part of internalized Fc gamma RIIa migrates in late endosomes, lysosomes and class II-containing compartments. These results suggest that melanoma cells can express functional Fc gamma RII, which might play a role in tumor-host relationships.

Blotting, Western↗
PubMed2000

Characterization of CD1e, a third type of CD1 molecule expressed in dendritic cells.

Dendritic cells express several alternatively spliced CD1e mRNAs. These molecules encode proteins characterized by the presence of either one, two, or three alpha domains and either a 51- or 63-amino acid cytoplasmic domain. Moreover, mRNAs encoding isoforms lacking the transmembrane domain are observed. Several of these CD1e isoforms were expressed in transfected cells, and two of them, with three alpha domains, displayed a particular processing pathway. These latter isoforms slowly leave the endoplasmic reticulum due to the presence of atypical dilysine motifs in the cytoplasmic tail. These molecules are associated with the beta(2)-microglobulin and accumulate in late Golgi and late endosomal compartments. In the latter compartments, they are cleaved into soluble forms that appear to be stable. In dendritic cells, these isoforms are mainly located in the Golgi apparatus, and upon maturation they are redistributed to late endosomal compartments. This work demonstrates the existence of CD1e molecules. As compared with other CD1 molecules, CD1e displays fundamentally different properties and therefore may represent a third type of CD1 molecules.

Alternative Splicing↗
PubMed2000

In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.

Antigen Presentation↗
PubMed1999

Functional and phenotypic analysis of thymic CD34+CD1a- progenitor-derived dendritic cells: predominance of CD1a+ differentiation pathway.

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.

Antigens, CD↗
PubMed1999

A novel model to study the dorsolateral migration of melanoblasts.

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermomyotome. To understand the role of specific proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line deficient for beta1 integrin subunits, proteins involved in cell-extracellular interactions, had a severely impaired migratory behavior. Interestingly, ES cells deficient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus, grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular mechanisms controlling dorsolateral migration.

Animals↗
PubMed1998

Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

Adaptor Protein Complex gamma Subunits↗
PubMed1998

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells.

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 +/- 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these alphabeta Ii complexes are converted into alphabeta-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the alphabeta Ii complexes. More than 80% of alphabeta dimers originating from internalized alphabeta Ii complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface alphbeta Ii into SDS-stable and -unstable alphabeta dimers. Altogether, these data suggest that newly liberated class II alphabeta heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.

Cell Membrane↗
PubMed1998

A tyrosine-based signal present in Ig alpha mediates B cell receptor constitutive internalization.

B lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig alpha/Ig beta heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig alpha/Ig beta components in BCR constitutive endocytosis. The role of Ig alpha subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig alpha, surface BCR lacking Ig alpha cytoplasmic domain were not constitutively internalized. The respective roles of Ig alpha and Ig beta subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig alpha cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig alpha chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig alpha, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.

Amino Acid Sequence↗
PubMed1998

[Radiation-induced DNA fragmentation evaluated by anti-poly(ADP-ribose)immunolabeling in CHO cells. Standardization with pulsed-field electrophoresis].

The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate gamma-ray-induced DNA fragmentation at the level of isolated cells after gamma-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immunolabelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after gamma-irradiation.

Animals↗