De novo expression of the class-A macrophage scavenger receptor conferring resistance to apoptosis in differentiated human THP-1 monocytic cells. (41/7876)

The class-A macrophage scavenger receptor (MSR) is a trimeric multifunctional protein expressed selectively in differentiated monomyeloid phagocytes which mediates uptake of chemically modified lipoproteins and bacterial products. This study investigated whether MSR plays a role in the regulation of apoptosis, a model of genetically programmed cell death. De novo expression of MSR occurred in human THP-1 monocytic cells differentiated with phorbol esters, which activated a nuclear transcription factor binding to the Ap1/ets-like domain of the MSR promoter. The phorbol ester-stimulated THP-1 cells also expressed increased levels of the pro-apoptotic gene products, caspase-3 and Fas ligand, but the cells exhibited no change in apoptosis. Global activation of GTP-binding proteins with fluoride anions triggered apoptosis of THP-1 cells in a time- and concentration-dependent manner, demonstrated by nuclear shrinkage and fragmentation and internucleosomal DNA fragmentation. However, the MSR-expressing THP-1 macrophage-like cells showed a significant reduction in apoptosis compared to undifferentiated control THP-1 cells, which produce MSR at undetectable levels. Fluoride stimulation also triggered apoptosis of human Jurkat T cells. Stimulation with phorbol ester made no difference in apoptosis between treated and untreated Jurkat cells. Finally, Chinese hamster ovary (CHO) cells overexpressing the class-A MSR type I by cDNA transfection showed markedly increased resistance to G-protein-coupled apoptosis. Thus, de novo expression of MSR associated with monocyte maturation into macrophages appears to confer the resistance of macrophages to apoptotic stimulation by G-protein activation.  (+info)

Triggering of effector functions on a CD8+ T cell clone upon the aggregation of an activatory CD94/kp39 heterodimer. (42/7876)

Some T lymphocytes express the CD94 Ag, which is known to form heterodimers with members of the NKG2 family. We have studied the expression pattern and function of CD94 heterodimers in different alphabeta or gammadelta T cell clones. Most of the CD94+NKG2A- T cells have a low to intermediate expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer in one of these CD8 alphabeta CD94+NKG2A- T cell clones (K14B06) was able to: 1) increase the intracellular concentration of Ca2+, 2) induce the up-regulation of CD25 Ag expression and the secretion of IFN-gamma, and 3) trigger redirected cytotoxicity in a TCR-independent manner. This activatory property was not shared by any other costimulatory molecule expressed by the K14B06 T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The immunoprecipitation of CD94 heterodimer showed a 39-kDa band with a similar m.w. to the activatory heterodimer found on some NK clones. A novel form of the NKG2 family (NKG2H) was identified in K14B06. NKG2H protein represents an alternative spliced form of the NKG2E gene, displaying a charged residue in the transmembrane portion and a cytoplasmic tail that lacks immunoreceptor tyrosine-based inhibitory motifs. The expression of NKG2H in the cell membrane through its association to CD94 and DAP-12 molecules supports that it could form part of the activatory CD94/Kp39 heterodimer present on K14B06 cells.  (+info)

Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line. (43/7876)

NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.  (+info)

Lipopolysaccharide-coated erythrocytes activate human neutrophils via CD14 while subsequent binding is through CD11b/CD18. (44/7876)

Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.  (+info)

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors. (45/7876)

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.  (+info)

The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae. (46/7876)

The uptake of cholesterol esters from high density lipoproteins (HDLs) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool. Cholesterol esters are subsequently internalized to a nonreversible pool. Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective. The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin-rich microdomains called caveolae. Caveolae are directly involved in cholesterol trafficking. Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE). Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible plasma membrane pool of CE. Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.  (+info)

Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells. (47/7876)

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.  (+info)

Does formyl-methionyl-leucyl-phenylalanine exert a physiological role in labor in women? (48/7876)

The classical chemotactic receptor for N-formyl peptides has traditionally been associated with polymorphonuclear and mononuclear phagocytes; however, several recent reports indicate that this receptor is also expressed in non-myeloid cells. In this study we have investigated the presence of binding sites for formyl-methionyl-leucyl-phenylalanine (fMLP) in human amniotic membranes of laboring and nonlaboring women; we have also evaluated the effect of the peptide on prostaglandin E (PGE) release from the same tissue. Our results demonstrate the presence of specific, saturable binding sites for 3H-fMLP; Scatchard plot analysis suggests the presence of both high- and low-affinity binding sites in laboring amnion, while only the low-affinity receptors were evident in nonlaboring tissue. N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (Boc-MLP), a formyl peptide receptor antagonist, inhibited 3H-fMLP binding in both preparations. In addition, fMLP was able to significantly increase PGE synthesis in perifused amnion fragments from laboring and nonlaboring women. This effect was counteracted by Boc-MLP treatment. The presence of specific binding sites for fMLP in amniotic tissue and their differing expression in laboring versus nonlaboring membranes, together with the action of the peptide on PGE synthesis, all suggest a physiological role for fMLP in labor.  (+info)