Spatial Separation of HLA-DM/HLA-DR Interactions within MIIC and Phagosome-Induced Immune Escape

SummaryMajor Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system

Rab27a regulates exocytosis of tertiary and specific granules in human neutrophils

The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinaseenriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca 2+ and GTP-γ-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation. Copyright © 2008 by The American Association of Immunologists, Inc.This work was supported in part by Grants SAF2005-04293 and SAF2008-00251 from Ministerio de Educación y Ciencia of Spain, Fundación de Investigación Médica Mutua Madrileña (FMM), Grant BM05-30-0) from Fundación “la Caixa”, Grants CSI04A05, CSI01A08, and SAN196/SA07/07 from Junta de Castilla y León, Grant FIS-FEDER 04/0843 from Fondo de Investigación Sanitaria and European Commission, and by Grant RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, Ministerio de Sanidad of Spain (to F.M.). This work is also supported by Grants 18022048, 18050038, 18057026, 18207015, and 19044003 from the Ministry of Education, Culture, Sports, and Technology of Japan, Naito Foundation, Takeda Science Foundation, Gushinkai Foundation, and Uehara Memorial Foundation (to M. F.).Peer Reviewe

Role of Vesicle-Associated Membrane Protein-2, Through Q-Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor/R-Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Interaction, in the Exocytosis of Specific and Tertiary Granules of Human Neutrophils

We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptobrevin- 2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca2 and GTP- -S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electropermeabilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that QSNARE/ R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosisThis work was supported by Grant FIS-01/1048 from the Fondo de Investigacio´n Sanitaria, Grants SA-087/01 and CSI-1/01 from Junta de Castilla y Leo´n, and Grant SAF2000-0169 from the Ministerio de Ciencia y Tecnologı´a of SpainPeer reviewe

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response. Copyright © 2007 by The American Association of Immunologists, Inc.This work was supported by grants from Ministerio de Ciencia y Tecnología of Spain (SAF02/1634) and Fondo de Investigación Sanitaria (FIS04/1275) (to F.D.-G.) and from Fondo de Investigación Sanitaria (FIS04/0843, FIS01/1048), Fundación de Investigación Médica Mutua Madrileña, Fundación “La Caixa” (BM05-30-0), Junta de Castilla y León (CSI04A05) (to F.M.) F.D.-G. was a recipient of a grant for Research Intensification from Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III. M.G.-G.’s work was supported by a grant from Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III (BEFI 99/0259) and M.D.-L.’s work was supported by a grant from Fundación Rafael Clavijo and Fundación Española de Reumatología.Peer Reviewe

Sorting of neutrophil-specific granule protein human cathelicidin, hCAP-18, when constitutively expressed in myeloid cells.

Neutrophil granulocytes carry storage organelles, e.g., azurophil and specific granules. Poorly understood are the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Therefore, we asked whether the specific granule protein human cathelicidin (hCAP-18) could be sorted for storage in other granules when the biosynthetic window is widened to allow this. We observed that hCAP-18 was targeted for storage in lysosome-related organelles when expressed constitutively in the rat basophilic leukemia and the mouse promyelocytic (MPRO) cell lines. In addition, premature release of the antibiotic C-terminal peptide LL-37 was observed. Retention of hCAP-18 was diminished by induction of differentiation of MPRO cells. In conclusion, a specific granule protein with native conformation may be sorted for storage in lysosome-related organelles of myeloid cells and converted prematurely to a supposedly biologically active form

Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.

Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents. (C) 2003 by The American Society of Hematology