Surface Of Jurkat Cells Research Articles

Effect of HDAC Inhibitor Chidamide on PD-L1 Expression in Peripheral T-Cell Lymphoma

To explore the role of chidamide in the regulatory mechanism of PD-1/PD-L1 immune escape signaling pathway in peripheral T-cell lymphoma. Jurkat cell line was treated with different concentrations of chidamide. The changes of PD-L1 and JAK/STAT pathway gene mRNA expression and PD-L1 protein expression on cell surface were detected by fluorescence quantitative PCR and flow cytometry after treatment. Chidamide upregulated PD-L1 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.989). The mRNA expression of PD-L1 in 5.0 μmol/L group was 15.4 times higher than that in the control group. The proportion of PD-L1 positive cells in Jurkat cell line was less than 0.5%. Chidamide upregulated PD-L1 protein expression on Jurkat cell surface. Chidamide upregulated the mRNA expression of JAK2, STAT1 and STAT3 in Jurkat cell line. The level of up-regulation was obvious in high concentration group (5.0 μmol/L group). Meanwhile, the mRNA expression of SOCS1 and SOCS3, the negative regulatory genes upstream of the JAK/STA T pathway, were up-regulated. In peripheral T-cell lymphoma, chidamide may up-regulate the expression of cell surface PD-L1 and induce T-cell chemokines by upregulation of STAT1 expression, thus improving the reaction rate of PD-1 monoclonal antibody and T-cell toxicity.

Expression Level of SOCS3 in Acute Lymphoblastic Leukemia Cells Affects the Cytotoxicity of NK Cells

To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells. The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells. Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells. SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.

Generating and characterizing the anti-human CD45 monoclonal antibody

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis.
 Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity.
 Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay.
 Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

CD25 expression on the surface of Jurkat cells

Expression of the α-subunit of the interleukin-2 receptor (IL-2Rα) was assessed by quantifying CD25 upregulation in the Jurkat cell line (human acute T cell leukemia). It was found that phytohemagglutinin (PHA, 5 μmL) or PHA in combination with 12,13-phorbol dibutirate (PDBu, 10–8 M) increased the number of CD25-positive cells in the proliferating Jurkat culture. In 24 h, their number was 32.3 ± 3.4 (n = 11) and 44.8 ± 8.6% (n = 6), respectively. Interleukin-2 (IL-2, 200 U/mL), alone or together with PDBu, did not induce CD25 expression in Jurkat cells. The agents regulating normal T-cell proliferation did not affect Jurkat cell growth. It was found that, unlike normal human blood lymphocytes, tyrphostin WHI-P131, an inhibitor of JAK/STAT signaling from the IL-2 receptor to CD25, inhibited Jurkat cell growth and blocked the cell cycle in G2/M rather than decreasing the induced CD25+ cell number. It has been concluded that transformed IL-2-independent Jurkat T-cells do not lose the mechanism of IL-2Rα expression. WHI-P131, an inhibitor of IL-2 receptor-associated tyrosine kinase, JAK3 arrests the cell cycle at G2/M phase and has targets other than JAK3.

One-step synthesis of dual clickable nanospheres via ultrasonic-assisted click polymerization for biological applications.

Dual clickable nanospheres (DCNSs) were synthesized in one step using an efficient approach of ultrasonic-assisted azide-alkyne click polymerization, avoiding the need of surfactants. This novel approach presents a direct clickable monomer-to-nanosphere synthesis. Field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), and dynamic laser scattering (DLS) were used to characterize the synthesized DCNSs. Numerous terminal alkynyl and azide groups on the surface of DCNSs facilitate effective conjugation of multiple molecules or ligands onto a single nanocarrier platform under mild conditions. To exemplify the potential of DCNSs in biological applications, (1) multivalent glyconanoparticles (GNPs) were prepared by clicking DCNSs with azide-functionalized and alkyne-functionalized lactose sequentially for the determination of carbohydrate-galectin interactions with quartz crystal microbalance (QCM) biosensor. Using protein chip (purified galectin-3 coated on chip) and cell chip (Jurkat cells immobilized on chip), the QCM sensorgrams showed excellent binding activity of GNPs for galectins; (2) fluorescent GNPs were prepared by clicking DCNSs with azide-functionalized Rhodamine B and alkyne-functionalized lactose sequentially in order to target galectin, which is overexpressed on the surface of Jurkat cells. The fluorescent images obtained clearly showed the cellular internalization of fluorescent GNPs. This fluorescent probe could be easily adapted to drugs to construct lectin-targeted drug delivery systems. Thus, DCNSs prepared with our method may provide a wide range of potential applications in glycobiology and biomedicine.

Photoprotective Properties of the Fluorescent Europium Complex in UV-Irradiated Skin

In this study, we compared the UV-protective abilities of the europium complex compared to titanium dioxide, which represents the most common physical filter for ultraviolet light in the broad-band spectral range. The UV absorption and light transformative capacities of the europium complex were evaluated using a spectrometer with a double-integrating sphere showing that the europium complex does not only absorb and reflect UV light, but transforms it into red and infrared light. It was found that the europium complex binds to the surface of Jurkat cells in vitro. Cells incubated with the europium complex showed a significantly higher viability after UVA and UVB irradiation as compared to untreated cells and cells incubated with titanium dioxide pointing out its photoprotective properties. The europium complex and titanium dioxide show similar penetration capacities into the stratum corneum as tested in human and porcine skin using tape stripping analysis. The europium complex has proved to be an efficient UV filter with a low cyto- and phototoxic profile and therefore represents a potential candidate for use in sunscreen formulations.

Macrophage Recognition of Thiol-Group Oxidized Cells: Recognition of Carbohydrate Chains by Macrophage Surface Nucleolin as Apoptotic Cells

The mechanism was investigated for macrophage recognition of cells oxidized by diamide, a thiol group-specific oxidizing reagent. Jurkat cells exposed to various concentrations of diamide were recognized by macrophages, the cells exposed to 25 µM diamide being best recognized. CD43, a major glycoprotein on the Jurkat cell surface, tended to form clusters upon diamide oxidization, and pretreating Jurkat cells with the anti-CD43 antibody inhibited macrophage binding. This indicates that macrophages appeared to recognize CD43. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and a Western blot analysis of CD43 of the diamide-oxidized cells showed no increase in the amount of cross-linked CD43 compared with control cells, indicating that cross-linking of CD43 by a disulphide bond was not involved in the clustering. Both CD43 clustering and binding of the oxidized cells to macrophages was prevented by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), suggesting that the oxidized and macrophage-bound cells were undergoing apoptosis. A closer examination revealed that the caspase-3 activity, chromatin condensation, and DNA fragmentation in Jurkat cells were all increased by oxidation. The macrophage receptor involved in the binding appeared to be the cell-surface protein, nucleolin; an anti-nucleolin antibody treatment inhibited the binding. These results suggest that thiol group-oxidized cells underwent early apoptosis and were recognized by nucleolin on macrophages as early apoptotic cells.

Architecture effects on L-selectin shedding induced by polypeptide-based multivalent ligands

Multivalent interactions between selectins and their ligands play key roles in mediating the rolling and tethering of leukocytes in the early steps of the inflammatory response, as well as in lymphocyte circulation. L-selectin shedding, which is the proteolytic cleavage of L-selectin, can be induced by L-selectin clustering through the binding of multivalent ligands to multiple L-selectin molecules, and it has been shown to regulate leukocyte rolling and subsequent integrin activation for firm adhesion. In this paper, we report the production of homogenous glycopolypeptides modified with a 3,6-disulfo-galactopyranoside equipped with a caproyl linker. The saccharide residue was chemically attached to various polypeptide backbones of differing architectures; the composition and purity of the sulfated glycopolypeptides was confirmed via1H-NMR spectroscopy, amino acid analysis (AAA), and electrophoretic analysis. The retention of the conformation of the polypeptide backbone was confirmed via circular dichroic spectroscopy. The shedding of l-selectin from the surface of Jurkat cells induced by these sulfated glycopolypeptides, determined via ELISA-based methods, varied based on differences in the architectures of the polypeptide scaffolds, suggesting opportunities for these strategies in probing cell-surface receptor arrays and directing cell signaling events.

N-terminal negatively charged residues in CD3ɛ chains as a phylogenetically conserved trait potentially yielding isoforms with different isoelectric points: Analysis of human CD3ɛ chains

CD3ɛ chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3ɛ our previous data in mouse CD3ɛ showing that, in T cells, proteolytic processing of the acidic N-terminal sequence of CD3ɛ chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3ɛ (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3ɛ chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes. Comparison of CD3ɛ chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3ɛ chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3ɛ chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds. Interestingly, CD3ɛ chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains.

Altered expression of glycoproteins on the cell surface of Jurkat cells during etoposide-induced apoptosis: Shedding and intracellular translocation of glycoproteins

Background The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells. Methods We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface. Results The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells. Conclusions These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.

Cytolethal Distending Toxin-induced Cell Cycle Arrest of Lymphocytes Is Dependent upon Recognition and Binding to Cholesterol

Induction of cell cycle arrest in lymphocytes after exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. In this study we further demonstrate that the association of Cdt with lymphocyte plasma membranes is dependent upon binding to cholesterol. Depletion of cholesterol resulted in reduced toxin binding, whereas repletion of cholesterol-depleted cells restored binding. We employed fluorescence resonance energy transfer and surface plasmon resonance to demonstrate that toxin association with model membranes is dependent upon the concentration of cholesterol; moreover, these interactions were cholesterol-specific as the toxin failed to interact with model membranes containing stigmasterol, ergosterol, or lanosterol. Further analysis of the toxin indicated that the CdtC subunit contains a cholesterol recognition/interaction amino acid consensus (CRAC) region. Mutation of the CRAC site resulted in decreased binding of the holotoxin to cholesterol-containing model membranes as well as to the surface of Jurkat cells. The mutant toxin also exhibited reduced capacity for intracellular transfer of the active toxin subunit, CdtB, as well as reduced toxicity. Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for Cdt and that this association can be blocked by either depleting membranes of cholesterol or mutation of the CRAC site.

Contribution of α 4β 1 integrin to the antiallergic effect of levocabastine

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin α 4β 1 is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC 50 406 μM), but not the first-generation antihistamine chlorpheniramine, displaced 125I-FN binding to human integrin α 4β 1 and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin α 4 expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the α 4β 1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects α Lβ 2/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of α 4β 1 integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

SARS-CoV accessory protein 7a directly interacts with human LFA-1

The SARS-CoV accessory protein 7a is a type I membrane protein with an extracellular domain of 81 amino acid residues. It is described to be expressed during infection and to be a component of the virus particle surface. In this study, we demonstrate that protein 7a binds directly and specifically to human lymphocyte function-associated antigen 1 (LFA-1) on the cell surface of Jurkat cells. The binding is increased upon artificial cell activation with phorbol ester. These observations are confirmed by direct in vitro binding of recombinant protein 7a to the wild type and mutant K287C/K294C I domain showing that the I domain is the 7a binding site in the alpha(L) chain of LFA-1. Consequences of the LFA-1 interaction with 7a are discussed. In particular, our data suggest LFA-1 to be an attachment factor or the receptor for SARS-CoV on human leukocytes.

Correlation between soluble Fas level and apoptosis of T cells in ovarian carcinoma

Objectives The objectives were to examine the correlation between soluble Fas (sFas) level and apoptosis of T cells in peripheral blood and peritoneal fluid of patients with ovarian carcinoma and to investigate the possible sFas effect on T cell apoptosis. Study design Patients with stages I–II ovarian carcinoma ( n = 10) and patients with stages III–IV ovarian carcinoma ( n = 22), as well as ovarian benign tumors ( n = 8), were enrolled in the study. Apoptosis of and Fas expression on T cells from peripheral blood and peritoneal fluids were assessed by flow cytometry. Soluble Fas level was assayed using an ELISA kit. The effects of peritoneal fluid on Jurkat cell apoptosis with or without depletion of sFas were evaluated and compared in vitro. Results The sFas level and apoptosis of T cells in peripheral blood and peritoneal fluid from stages III–IV ovarian carcinoma were significantly higher than those from stages I–II ovarian carcinoma ( p < 0.01 in all instances) and benign ovarian tumor ( p < 0.01 in all instances). In peritoneal fluid, the sFas level and apoptosis of T cells from stages I–II ovarian carcinoma were significantly higher than those from benign ovarian tumor ( p < 0.01 in all instances), and the Fas expression on T cells from ovarian carcinoma were higher than those from benign ovarian tumor ( p < 0.05 in all instances). There was a positive correlation between the sFas level and the apoptosis of T cells in peritoneal fluids from stages III–IV ovarian carcinoma ( r = 0.647, p = 0.001). Peritoneal fluid of ovarian carcinoma could induce significant Jurkat cell apoptosis. The blocking of Fas expression on the Jurkat cell surface, but not the deletion of sFas, may remarkably restrain the apoptosis level. Conclusions Elevated sFas is correlated with apoptosis of T cells in peripheral blood and peritoneal fluid from ovarian carcinoma. Soluble Fas evidently does not affect T cell apoptosis, which is probably due to elevated Fas expression on T cells.

Enhanced Expression of Membrane-Associated Sialidase Neu3 Decreases GD3 and Increases GM3 on the Surface of Jurkat Cells during Etoposide-Induced Apoptosis

We previously reported that, in Jurkat human T cells, the topoisomerase II inhibitor etoposide enhances sialidase activity and reduces cell surface sialic acid levels at an early stage of apoptosis and that the decreases in sialic acid are suppressed by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [Azuma Y., et al., Glycoconj. J., 17, 301-306 (2000)]. In the current studies, we treated Jurkat cells with etoposide and examined the changes in the cell surface levels of gangliosides GM1, GM2, GM3, GD1a, and GD3 at physiological pH using anti-ganglioside antibodies. We also examined the sialidase activity on the cell surface using 4-methylumbelliferyl N-acetylneuraminic acid and measured the mRNA expression of the plasma membrane-associated sialidase Neu3 and the lysozomal Neu1 using real-time PCR. We found an increase in GM3 and a decrease in GD3 during the early stage (4 h) of etoposide-induced apoptosis that preceded the increase in cell surface exposure of phosphatidylserine (4 to 6 h). The caspase 3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde significantly suppressed changes in GM3 and GD3 and blocked the enhanced cell surface sialidase activity. Furthermore, etoposide caused a gradual up-regulation of Neu3 mRNA expression but not Neu1 mRNA expression. Enhanced Neu3 mRNA expression was suppressed in the presence of caspase 3 inhibitor. These results indicate that Neu3 is up-regulated in Jurkat cells undergoing etoposide-induced apoptosis through intracellular signaling events downstream of caspase 3 activation and that enhanced Neu3 activity is closely related to the changes of cell surface ganglioside composition.

A novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction, and its application for testing inhibitory peptides

The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2–CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2–CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 μM of BCECF-AM for 45 min at 37 °C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2–CD58 interaction.

Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria.

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.

Sialic acids linked to glycoconjugates of Fas regulate the caspase-9-dependent and mitochondria-mediated pathway of Fas-induced apoptosis in Jurkat T cell lymphoma

To clarify the functions of sialic acids linked to glycoconjugates of Fas in Fas-induced apoptosis, Jurkat T cells, untreated and treated with neuraminidase, were incubated with anti-Fas monoclonal antibody, CH11. Apoptosis of Jurkat T cells induced by incubation with CH11 was enhanced by the pre-treatment with neuraminidase. By flow cytometry sialylated glycoconjugates were detected on the cell surface of Jurkat T cells using LFA lectin, which specifically reacts with sialic acid, and pre-treatment with Vibrio Cholerae neuraminidase resulted in desialylation of Jurkat cell surface glycoconjugates. The enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was inhibited by z-VAD-fmk, a broad caspase inhibitor, and Ac-LEHD-CHO, an inhibitor of caspase-9, but not by Ac-IETD-CHO an inhibitor of caspase-8 or 6, imipramine, an inhibitor of acidic sphingomyelinase, glutathione, an inhibitor of neutral sphingomyelinase and Fumonisin B1, an inhibitor of ceramide synthase. Mitochondrial membrane potentials (Deltapsim) measured with a Mitocapture assay kit demonstrated that the loss of Deltapsim involved in Fas-induced apoptosis was enhanced by pre-treatment with neuraminidase. Furthermore, Western blot analysis using polyclonal antibody (C-20) against Fas detected Fas at about 45 kDa, and pre-treatment with neuraminidase resulted in a reduction of the molecular weight of Fas of about 8 kDa. These data suggest that the enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was mediated by a caspase-9 dependent pathway closely associated with the loss of Deltapsim, not by activation of caspase-8, -6 or acidic and neutral sphingomyelinases, and that sialic acid linked to glycoconjugates of Fas may regulate Fas-induced apoptosis in human T cell lymphoma.

TCR dynamics on the surface of living T cells.

T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell-antigen-presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in defined three-dimensional structures. To better understand how TCR are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCR on living Jurkat cell surface. We show that: (i) surface-expressed TCR exhibit an intrinsic, actin cytoskeleton-independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within approximately 60 min and (ii) non-stimulated TCR rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation.

Apoptotic effects in primary human umbilical vein endothelial cell cultures caused by exposure to virion-associated and cell membrane-associated HIV-1 gp120.

During the course of HIV-1 infection, free virus, infected cells, and free HIV-1 proteins circulate within the host, exposing the host endothelium to these viral factors. We have previously presented evidence showing that soluble HIV-1 gp120 protein interacts with chemokine receptors on primary human endothelium and (through those interactions) induces apoptosis as well as other intracellular effects. The current study examines the effect of exposure of vascular endothelium to gp120 IIIb expressed on the surface of Jurkat cells and in the context of viral particles. Apoptosis was observed in human umbilical vein endothelial cell (HUVEC) cultures exposed to gp160-transfected Jurkat cells as well as to virion particles with gp120 on their surface. Additional experiments show that this apoptotic effect was caused by gp120 protein acting through chemokine receptors on the HUVEC surface, primarily the CXCR4 receptor. At higher concentrations of gp120, this lymphotrophic variant, which has been shown to interact predominantly with CXCR4, seems to interact with and induce apoptosis through the CCR5 receptor. Finally, this apoptotic effect in HUVEC cultures occurs at low levels of the inducing agent, gp120, on cell membranes or on virion particles. These results demonstrate that HIV-1 gp120 is capable of interacting with and killing vascular endothelial cells in multiple in vivo contexts.