Hepatitis C virus protects human B lymphocytes from Fas-mediated apoptosis via E2-CD81 engagement

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitt's lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production.     

The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis.

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.     

The T3 complex on human T lymphocytes involves four structurally distinct glycoproteins

Monoclonal antibodies allow for the detection of antigens which are specific for human thymus-derived lymphocytes. Among these antigens, the T3 complex is of particular interest since it is involved in several T cell functions. The main target antigen of the anti-T3 reagents is borne by a 20-kDa glycoprotein. In addition, glycoproteins of 25-28, 37, and 44 kDa are found in anti-T3 immunoprecipitates derived from surface-labeled cells. The four antigens appeared to be strongly associated with each other in detergent-containing solutions. Comparative studies of the four proteins, facilitated by the use of endo-beta-N-acetylglycosaminidase F, revealed that their polypeptide backbones have different molecular weights and pI values. Moreover, peptide maps of the 20-kDa T3 and the 25-28-kDa T3 were quite different. Metabolic labeling experiments suggested that the 25-28-kDa protein might become associated with the 20-kDa T3 antigen during biosynthesis. The 37-kDa and 44-kDa proteins could not, however, be detected and, therefore, might become associated with the 20-kDa T3 on the cell surface. Evidence has been found for the existence of a fifth member of the T3 complex, namely an unglycosylated 20-kDa T3 species.     

Genotoxic effects of 3 T magnetic resonance imaging in cultured human lymphocytes

The clinical and preclinical use of high-field intensity (HF, 3 T and above) magnetic resonance imaging (MRI) scanners have significantly increased in the past few years. However, potential health risks are implied in the MRI and especially HF MRI environment due to high-static magnetic fields, fast gradient magnetic fields, and strong radiofrequency electromagnetic fields. In this study, the genotoxic potential of 3 T clinical MRI scans in cultured human lymphocytes in vitro was investigated by analyzing chromosome aberrations (CA), micronuclei (MN), and single-cell gel electrophoresis. Human lymphocytes were exposed to electromagnetic fields generated during MRI scanning (clinical routine brain examination protocols: three-channel head coil) for 22, 45, 67, and 89 min. We observed a significant increase in the frequency of single-strand DNA breaks following exposure to a 3 T MRI. In addition, the frequency of both CAs and MN in exposed cells increased in a time-dependent manner. The frequencies of MN in lymphocytes exposed to complex electromagnetic fields for 0, 22, 45, 67, and 89 min were 9.67, 11.67, 14.67, 18.00, and 20.33 per 1000 cells, respectively. Similarly, the frequencies of CAs in lymphocytes exposed for 0, 45, 67, and 89 min were 1.33, 2.33, 3.67, and 4.67 per 200 cells, respectively. These results suggest that exposure to 3 T MRI induces genotoxic effects in human lymphocytes.     

Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis

Nitric oxide (NO) can either prevent or promote apoptosis, depending on cell type. In the present study, we tested the hypothesis that NO suppresses ultraviolet B radiation (UVB)-induced keratinocyte apoptosis both in vitro and in vivo. Irradiation with UVB or addition of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) increased apoptosis in the human keratinocyte cell line CCD 1106 KERTr, and apoptosis was greater when the two agents were given in combination. Addition of the chemical NO donor S-nitroso-N-acetyl-penicillamine (SNAP) immediately after UVB completely abrogated the rise in apoptosis induced by l-NAME. An adenoviral vector expressing human inducible NOS (AdiNOS) also reduced keratinocyte death after UVB. Caspase-3 activity, an indicator of apoptosis, doubled in keratinocytes incubated with l-NAME compared with the inactive isomer, d-NAME, and was reduced by SNAP. Apoptosis was also increased on addition of 1,H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Mice null for endothelial NOS (eNOS) exhibited significantly higher apoptosis than wild-type mice both in the dermis and epidermis, whereas mice null for inducible NOS (iNOS) exhibited more apoptosis than wild-type mice only in the dermis. These results demonstrate an antiapoptotic role for NO in keratinocytes, mediated by cGMP, and indicate an antiapoptotic role for both eNOS and iNOS in skin damage induced by UVB.     

Apolipoprotein E protects astrocytes from hypoxia and glutamate-induced apoptosis

Apolipoprotein E (apoE) is predominantly synthesized by astrocytes in the brain. In this study, we investigated the role of apoE in astrocyte apoptosis. We demonstrated that apoE protects astrocytes from hypoxia-induced apoptosis in a dose-dependent manner. Glutamate release from astrocytic cultures is significantly lower from WT mice than from apoE knockout mice. Furthermore, the protective effect of apoE is mimicked by an NMDA receptor antagonist, MK-801. Finally, the apoE activator T0901317 significantly reduced the effect of glutamate-induced apoptosis of astrocytes. These results suggest that apoE protects astrocytes from hypoxia-induced apoptosis associated to NMDA receptor activation. Approaches that elevate apoE secretion in astrocytes might provide a novel strategy in the protection of neuronal ischemic injury.     

The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptosis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of the FADD death effector domain. E6 is produced in two forms as follows: a full-length version of approximately 16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.     

Chromosomal proteins in human B and T lymphocytes.

Histones and non-histone chromosomal proteins were characterized in B and T human lymphocytes by means of polyacrylamide disc gel electrophoresis. It was found that while histones do not present appreciable differences in the two examined populations, non-histone chromosomal proteins exhibit distinct electrophoretic profiles. Low molecular weight proteins predominate in B lymphocytes whereas high and intermediate proteins are largely represented in T lymphocytes. The latter proteins may be related to the capability of these resting cells to proliferate under appropriate antigenic stimuli.     

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.     

Novel role for E4 region genes in protection of adenovirus vectors from lysis by cytotoxic T lymphocytes

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.     

IL-2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death

In the IL-2-dependent T cell clone CTLL-2, dexamethasone, a synthetic glucocorticoid, induces a suicide program characterized by the early degradation of chromatin in oligonucleosome-length fragments which precedes the loss of cell viability by 2 to 4 h. These effects are most likely mediated through the interaction with a specific glucocorticoid receptor as suggested by the structure-activity relationship of the various steroids tested. Incubation of nuclei of glucocorticoid-untreated cells in the presence of calcium and magnesium ions induces the cleavage of DNA in the linker region between nucleosomes, suggesting that fragmentation of chromatin in intact cells by glucocorticoids may involve the activation of a preexisting endonuclease. Interestingly, the presence of a saturating dose of IL-2 during the treatment of CTLL-2 cells with glucocorticoids completely blocks the cell death program.     

Antigen recognition by human T lymphocytes is linked to surface expression of the T3 molecular complex

Four distinct surface molecules on human T cells are defined by the monoclonal antibodies anti-T1, anti-T3 (anti-T3_A), anti-T11 and anti-T12. Following cell binding, anti-T3 (anti-T3_A) and anti-T1 induce independent modulation of their respective ligands, whereas anti-T11 and anti-T12 do not. To explore the biological consequences of this modulation, we used cloned populations of T4 and T8 cytotoxic T lymphocytes. Anti-T3 (anti-T3_A), but not anti-T1, inhibits cytotoxic T lymphocyte effector function by T4 and T8 clones as well as antigen-specific T cell recognition. The latter is not secondary to a generalized inhibitory effect since responsiveness to interleukin 2 is maintained. Moreover, after modulation, cytotoxic T lymphocytes recover cytolytic function in parallel with reexpression of surface T3 molecules. We provide evidence for a direct linkage between antigen recognition by T lymphocytes and surface expression of the T3 molecular complex.     

Survival of human T and B lymphocytes after X-irradiation.

The survival of unstimulated human T and B lymphocytes after X-irradiation in vitro was measured by Trypan Blue dye exclusion over a period of four days. B cell numbers were observed to decline rapidly even after relatively low doses, but T cell numbers fell much more slowly. A comparison of the percentage survival 96 hours after irradiation shows that in this system T cells are between approximately 2 and 5 times more resistant than B cells. Data for interphase death after 48 hours are compared with cytogenetic data for interphase loss of PHA-stimulated human lymphocytes and are shown to be in broad agreement at radiation doses below 400 rad. It is suggested that at higher doses mitotic delay may be increasingly important leading to selection of non-irradiated cells at 48 hours.