Direct stimulation of T cells by membrane vesicles from antigen-presenting cells

Activation of na?ve T cells generally requires T cell receptor-mediated contact with MHC-bound peptides on viable antigen-presenting cells such as dendritic cells (DC). Here evidence is presented that dissociated cell membrane fragments from a DC line can be used as an effective substitute for viable DC. Ultracentrifuged material derived from sonicates of IFN-gamma-matured DC is enriched in small membrane vesicles that closely resemble exosomes. When complexed with MHC class I-restricted specific peptide, vesicles from DC sonicates generate strong responses by purified na?ve CD8(+) cells in vitro in the absence of normal antigen-presenting cells and can also efficiently prime T cells for tumor rejection in vivo. Both in terms of total yields from DC and relative immunogenicity, membrane vesicles from DC sonicates are much more effective than classic exosomes and may be a valuable tool for tumor immunotherapy.     

New protein kinase from plasma membrane of Ehrlich ascites tumor cells activated by natural polypeptides.

A polypeptide-dependent protein kinase was purified about 80-fold from an extract of plasma membranes of Ehrlich ascites tumor cells. The membranes were extracted with Nonidet P-40, and the extract was purified by ammonium sulfate fractionation and hydroxylapatite and affinity chromatography. The activity was stimulated 10-fold or more by polypeptide preparations from a variety of tissues, including placenta and hypothalamus. Polypeptide-dependent protein kinase had a pH optimum of about 7.5 and required Mg2+ for activity. Mn2+ at low concentrations (200 microM) stimulated enzyme activity somewhat but inhibited activity strongly at higher concentrations. The best available substrate for polypeptide-dependent protein kinase was beta-casein, and little or no phosphorylation was observed with alpha-casein, kappa-casein, phosvitin, alpha-lactalbumin, alpha-lactoglobulin, and histone. However, several endogenous substrates from plasma membranes of Ehrlich ascites tumor cells were phosphorylated. Polypeptide-dependent protein kinase activity was not inhibited by 10 mM N-ethylmaleimide, and this resistance was useful in differentiating this protein kinase from other protein kinases that were present in crude fractions and sensitive to the inhibitor.     

Thiamine transport by basolateral rat liver plasma membrane vesicles.

Hepatic thiamine transport is thought to be a saturable, Na(+)- and energy-dependent process. However, the transport of this organic cation has not been examined in experimental models that allow direct characterization of carrier-mediated processes. Recently, a sinusoidal organic cation/H+ antiport was identified, using N1-methylnicotinamide as a marker. To determine whether thiamine is a substrate for this antiport, the characteristics of thiamine uptake were examined in rat liver basolateral membrane vesicles. An inwardly directed Na+ gradient had no effect on thiamine uptake as compared with an identical K+ gradient. An outwardly directed H+ gradient stimulated thiamine uptake as compared with pH-equilibrated conditions, and H(+)-dependent uptake was not the result of an H+ diffusion potential. Identical pH gradients stimulated uptake under voltage-clamped conditions, consistent with electroneutral thiamine/H+ exchange. Unlabeled intravesicular thiamine trans-stimulated [3H]thiamine uptake. Choline and imipramine cis-inhibited thiamine/H+ exchange; a series of other organic cations and thiamine analogues had no effect. Carrier-mediated [3H]thiamine uptake showed two saturable systems. In conclusion, a thiamine/H+ antiport is present on the sinusoidal membrane, distinct from Na+/H+ and NMN+/H+ exchange.     

Taurocholate transport by basolateral plasma membrane vesicles isolated from human liver

Transport of taurocholate into the hepatocyte against unfavorable chemical and electrical gradients occurs via a sodium-dependent, carrier-mediated transport system. Although this cotransporter has been characterized in the rodent, it has not been demonstrated in man. Therefore, we utilized human liver, obtained via multiorgan donation but not used for transplantation, to prepare basolateral (sinusoidal) liver plasma membrane vesicles by a Percoll gradient method. Na+,K+-ATPase, a marker enzyme for the basolateral domain, was enriched 28.9-fold in the final membrane fraction compared with homogenate, whereas the bile canalicular membrane enzymes Mg++-ATPase and alkaline phosphatase were enriched only 3.4- and 6.4-fold, respectively. Marker enzyme activities for endoplasmic reticulum, lysosomes and mitochondria were not enriched compared with homogenate. Integrity of the membrane vesicles was confirmed by the demonstration of Na+-dependent concentrative uptake of the amino acid L-alanine (estimated intravesicular volume of 0.59 microliter per mg protein). An inwardly directed 100 mM Na+ gradient stimulated the initial rate of 2.5 microM taurocholate uptake and energized a transient 2-fold accumulation of the bile acid above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot occurred with a K+ gradient. A negative intravesicular potential, created by altering accompanying anions or by valinomycin-induced K+ diffusion potentials, did not enhance taurocholate uptake, suggesting an electroneutral cotransport mechanism. Chloride as the accompanying anion stimulated the initial rate of uptake compared with anions of lesser or greater lipid permeability. Na+-dependent taurocholate (4 microM) uptake was significantly inhibited by 250 microM cholate, taurocholate, glycocholate, taurochenodeoxycholate and bromsulfophthalein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-stimulated alpha-aminoisobutyric acid transport by membrane vesicles from simian virus-transformed mouse cells.

Uptake of alpha-aminoisobutyric acid, by membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by simian virus 40, is stimulated by sodium chloride. Both in the presence and absence of Na+ uptake is time-dependent and osmotically sensitive. The Na+-stimulated uptake is inhibited by other amino acids. The kinetics of transport of alpha-aminoisobutyric acid are shown to be biphasic both in whole cells and in the membrane vesicles. Only the high affinity system is stimulated by sodium in the membrane vesicles. These results demonstrate that observations made on living cells correlate with observations made on isolated membrane vesicles, and indicate that these membrane vesicles have retained the cellular amino acid transport system functionally intact.     

Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro. A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 x 10(4) cells be stirred with 1.35 x 10(8) platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.     

Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro . A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 2 10 4 cells be stirred with 1.35 2 10 8 platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.     

Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.

Senescent human diploid cells (HDC) were fused to replicative transformed cells of different types, and DNA synthesis was monitored in the resulting heterodikaryons. Human cells transformed by simian virus 40 or adenovirus serotype 5 were able to induce DNA synthesis in senescent HDC nuclei in heterodikaryons. In contrast, carcinogen-transformed cells were not able to induce DNA synthesis in senescent HDC nuclei; rather, the transformed nuclei in these heterodikaryons were inhibited from entering S phase. Cells transformed by Rous sarcoma virus and most human tumor cells tested are similarly inhibited by fusion to senescent HDC. These results suggest that the mechanism for transformation by DNA tumor viruses may be fundamentally different from that of other viruses and carcinogens and from that of most human tumor cells. A simple model to explain these results is that (i) senescent HDC contain an inhibitor of entry into S phase; (ii) cells transformed by DNA tumor viruses have gained a transforming factor, perhaps large tumor antigen, that is capable of overriding the normal inhibitor; and (iii) cells transformed by carcinogens or RNA viruses have lost or altered the mechanism for expression of the normal inhibitor yet are still sensitive to it. We propose that this inhibitor is produced in normal cells when they experience conditions that are inadequate for proliferation and that it plays a role in putting the cells into a distinct quiescent state with long-term viability. The override of this inhibitor function in simian virus 40-transformed HDC can explain why they have low viability in plateau-phase cultures and why they die during crisis.     

Preferential digestion of basement membrane collagen by an enzyme derived from a metastatic murine tumor.

The specificity of human skin collagenase and of an enzyme from an invasive tumor were studied by using types I, II, III, IV, and V (AB) collagen as substrates. Human skin collagenase degraded types I, II, and III collagen, producing the characteristic 3/4 and 1/4 cleavage products, but failed to degrade type IV or V collagen. Collagenase prepared from the invasive tumors showed maximal activity after trypsin treatment. The tumor enzyme degraded type IV (basement membrane) collagen, producing fragments consistent with a single cleavage site but did not attack types I, II, III, and V collagen. Because type IV collagen prepared by pepsinization of placenta was also digested, it is likely that cleavage of type IV collagen by the tumor collagenase occurs within a largely helical domain. A type IV collagenase could play a significant role in tumor metastases and in normal tissues where basement membrane turnover takes place.     

Isolation of virus-free Herpesvirus saimiri antigen-positive plasma membrane vesicles.

Experiments were undertaken to determine whether virus-free, antigen-positive plasma membrane vesicles could be isolated from owl monkey kidney cells infected with Herpesvirus saimiri. The results demonstrate that vesicles can be produced by using a vesiculation fluid containing 25 mM formaldehyde/2 mM dithiothreitol. Electron microscopy revealed that these vesicles were free of detectable virus particles. Vesicles prepared from the infected cells contained virus-induced membrane antigens as shown by membrane immunofluorescence and by inhibition of antibody-dependent lymphocyte cytotoxicity. Nonhuman primates immunized with vesicles produced antibodies to these membrane antigens, late cytoplasmic antigens, and neutralizing antibodies. Infectious virus was not demonstrated in these vesicles by cocultivation with owl monkey kidney cells or by the inoculation of cottontop marmosets. Furthermore, no DNA could be demonstrated in vesicles prepared from Herpesvirus-infected owl monkey kidney cells. The implication of these findings in relation to the question of a virus-free membrane vaccine against Herpesvirus infections is discussed.     

Sodium ion-coupled uptake of taurocholate by rat-liver plasma membrane vesicles

ABSTRACT— Uptake of taurocholate into plasma membrane vesicles isolated from rat liver was investigated. In the presence of an extra- to intravesicular gradient of Na+ ions, a typical “overshoot” phenomenon in the accumulation pattern was observed. Osmotic manipulation of the incubation medium indicated that the transport of this bile acid occurs into an osmotically active intravesicular space. Uptake of taurocholate as measured after 1 min was specifically stimulated by Na+ ions: NaNO₃ and NaCl were capable of supporting accumulation, whereas KNO₃ was not. Na+-coupled uptake of taurocholate showed saturation kinetics and was inhibited by other bile acids or by preloading the vesicles with Na+. Our observations support the idea of a carrier-mediated bile-acid uptake system, as suggested previously for the intact rat liver and isolated rat hepatocytes. When the electrical potential difference across the vesicle membrane was changed by inducing different diffusion potentials (anion replacement), a more negative potential inside stimulated Na+-dependent taurocholate transport. The results demonstrate that rat-liver plasma membrane vesicles possess an electrogenic Na+-coupled transport system for taurocholate.     

Active transport of calcium in Neurospora plasma membrane vesicles.

Functionally inverted plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa catalyze Mg2+/ATP-dependent Ca2+ uptake. Inhibitors induced efflux studies and isotope-exchange experiments indicate that the Ca2+ is accumulated inside the vesicles against a concentration gradient of about 40-fold, and that the majority of the transported Ca2+ is present essentially in free solution. Comparisons of Mg2+/ATP-driven 45Ca2+ uptake and [14C]SCN-uptake with respect to the Mg2+/ATP concentration dependence, the effects of inhibitors, and the nucleotide and divalent cation specificities indicate that the energy for Ca2+ accumulation is derived from ATP hydrolysis catalyzed by the electrogenic plasma membrane ATPase. Energized Ca2+ uptake is stimulated by the permeant anion SCN- to a degree that varies reciprocally with the ability of this anion to dissipate the membrane potential, and is inhibited by K+ in the presence of nigericin. All of these data point to the conclusion that the active transport of Ca2+ across the Neurospora plasma membrane takes place via a Ca2+/H+ antiporter, which functions to pump Ca2+ out of the intact cell.     

Platelets in tumor metastasis: generation of adenosine diphosphate by tumor cells is specific but unrelated to metastatic potential

Human 253J urinary carcinoma cells and the F1 (low-metastatic) and F10 (high-metastatic) variants of the B16 murine melanoma cell line have been shown to activate heparinized human platelets by an adenosine diphosphate (ADP)-dependent mechanism based on inhibition by creatine phosphate/creatine phosphokinase and the identification of aggregating concentrations (1 to 2 mumol/L) of ADP in cell-free culture supernatants by high-performance liquid chromatography. Aggregation did not occur in citrated samples, and hirudin was without effect. Studies were carried out to determine whether extracellular ADP arose from nonspecific cell damage during cell isolation and manipulation or was a specific process under control of the tumor cells themselves. Tumor cell damage during harvesting was shown not to be a factor because the amounts of ADP produced by the three cell lines (a) were inversely related to the appearance of lactic dehydrogenase in the culture supernatants and (b) were similar when measured in confluent monolayers, either in tumor cells after detachment and resuspension or after crossover studies involving culture in, alternatively, Hanks' balanced salt solution and minimal essential medium. Metabolic control of ADP production was indicated by the fact that (a) it was not dependent on cell number, which suggests feedback inhibition; (b) it was reduced 60% when tumor cells were treated with p- chloromercuribenzene sulfonate; and (c) it was completely abolished in those treated with iodoacetic acid, which might be expected to increase nonspecific leakage. These studies indicate that ADP production by these three lines does not arise due to leakage induced by nonspecific membrane damage during cell harvesting and manipulation but is a discrete process under metabolic control of the tumor cells. Moreover, in B16 murine melanoma cells the ability to produce ADP and to support platelet aggregation appears to be unrelated to metastatic potential insofar as identical results were obtained with the F1 and F10 variants.     

Two pharmacologically distinct sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporters are present in plasma membrane vesicles and reconstituted preparations from rat brain.

Electrogenic sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transport in crude synaptosomal membrane vesicles is partly inhibited by saturating levels of either of the substrate analogues cis-3-aminocyclohexanecarboxylic acid (ACHC) or beta-alanine. However, both of them together potently and fully inhibit the process. Transport of beta-alanine, which exhibits an apparent Km of about 44 microM, is also electrogenic and sodium and chloride dependent and competitively inhibited by GABA with a Ki of about 3 microM. This value is very similar to the Km of 2-4 microM found for GABA transport. On the other hand, ACHC does not inhibit beta-alanine transport at all. Upon solubilization of the membrane proteins with cholate and fractionation with ammonium sulfate, a fraction is obtained which upon reconstitution into proteoliposomes exhibits 4- to 10-fold-increased GABA transport. This activity is fully inhibited by low concentrations of ACHC and is not sensitive at all to beta-alanine. GABA transport in this preparation exhibits an apparent Km of about 2.5 microM and it is competitively inhibited by ACHC (Ki approximately 7 microM). These data indicate the presence of two GABA transporter subtypes in the membrane vesicles: the A type, sensitive to ACHC, and the B type, sensitive to beta-alanine.     

Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages.

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Clathrin-coated vesicles have been purified from CHO cells infected with vesicular stomatitis virus and shown to contain G protein in amounts nearly stoichiometric with clathrin. Pulse-chase experiments have demonstrated that this G protein is a transit form and have revealed that G is transported to the cell surface in two successive waves of coated vesicles. The oligosaccharides of G1 protein carried in the early wave are of the "high-mannose" variety which can be cleaved by the enzyme endoglycosidase H; the oligosaccharides of G2 protein in the second, later wave are resistant to endoglycosidase H. The early wave is therefore proposed to correspond to transport of G protein in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, where the oligosaccharides are processed and resistance to endoglycosidase H is conferred; the succeeding wave would represent transport from the Golgi apparatus to the plasma membrane.     

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli -LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli -LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli -LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. Conclusion: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.     

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli-LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli-LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli-LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. CONCLUSION: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.     

Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane proteins.

We have developed a cell-free assay that reproduces vesicular budding during exit from the Golgi complex. The starting preparation for the in vitro system was a rat liver stacked Golgi fraction immobilized on a magnetic solid support by means of an antibody against the cytoplasmic domain of the polymeric IgA receptor. Vesicular budding was ATP, cytosol, and temperature dependent and was inhibited by 1 mM N-ethylmaleimide. Budding was maximum within 10 min and originated preferentially from the trans-Golgi. Exocytic transport vesicles immunoisolated from the total budded population were enriched in the mature forms of secretory and membrane proteins destined to the basolateral plasma membrane and were depleted in lysosomal enzymes and galactosyl-transferase activity. The finding that a major proportion (greater than 70%) of newly synthesized, siaylated secretory and transmembrane proteins is contained in a single population of post-Golgi transport vesicles implies that, in a constitutively secreting cell, basolaterally destined proteins are sorted and packaged together into the same exocytic transport vesicles.