Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella—zoster virus antigen

Fas antigen and bcl-2 expression on peripheral blood mononuclear cells (PBMC) cultured with cytomegalovirus (CMV) and varicella—zoster virus (VZV) antigen was analyzed by three-color flow cytometry. Expression of Fas antigen increased significantly in CD45RO⁺ populations of both CD4⁺ and CD8⁺ cells obtained from CMV and VZV seropositive donors after culture with CMV and VZV antigen for 6 days. Fas antigen expression did not increase in PBMC cultured with control antigen. In contrast, bcl-2 expression decreased both in CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ cells from the same donors. Fas antigen and bcl-2 expression in CD45RA⁺ populations did not change. Cell viability of cultured cells with CMV and VZV antigen decreased after treatment with anti-Fas antibody and DNA fragments indicating apoptosis were detected in the cell lysate of these cultured cells after treatment with anti-Fas antibody. These data suggest that Fas antigen and bcl-2 protein may interact in regulating the cell death process of activated memory lymphocytes and eliminating lymphocytes activated by viral infection.     

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

Varicella-zoster virus infection of human dendritic cells and transmission to T cells: implications for virus dissemination in the host

During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% +/- 6.6% (mean +/- SEM) of CD1a(+) cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a(+) dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3(+) T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.     

Investigation of varicella-zoster virus DNA in lymphocyte subpopulations by quantitative PCR assay

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real-time quantitative polymerase chain reaction assay. Varicella-zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.     

Analysis of immune function in herpes zoster patients: demonstration and characterization of suppressor cells

We sought to identify imbalances of immune regulatory cells that might contribute to the depression of cell-mediated immunity that occurs during an episode of herpes zoster. Peripheral blood mononuclear cells (PBMC) were obtained from patients with herpes zoster during the acute (less than 7 days after disease onset) and convalescent (more than 10 days after disease onset) phases of illness and from healthy seropositive donors. The PBMC were analyzed for: lymphoproliferative responses to varicella-zoster virus (VZV) antigens, Leu-3 (helper/inducer):Leu-2 (cytotoxic/suppressor) ratios, and percentages of suppressor cells as defined by coexpression of the Leu-2 and OKM1 antigens. Significantly depressed proliferative responses of VZV antigens and Leu-3:Leu-2 ratios, and increased percentages of Leu-2+ OKM1+ suppressor cells were observed in PBMC of acute phase herpes zoster patients as compared with the PBMC of convalescent patients or healthy donors. These differences were also observed in individual patients sequentially studied during both phases of disease. Cryopreserved acute phase PBMC suppressed the proliferative response of autologous convalescent phase PBMC to VZV antigens, but not to herpes simplex virus (HSV) antigens. The acute phase PBMC suppressor cell was radiation sensitive and was identified as a Leu-2+ cell by fluorescence-activated cell sorting. Thus, depression of cell-mediated immunity during the acute phase of herpes zoster was associated with a relative increase of lymphocytes expressing a suppressor cell phenotype and the activation of a radiosensitive Leu-2+ suppressor cell with some degree of antigen specificity.     

Investigation of varicella-zoster virus infection of lymphocytes by in situ hybridization.

Peripheral blood mononuclear cells harboring viral gene sequences were detected during primary varicella-zoster virus (VZV) infection of the human host and the strain 2 guinea pig by in situ hybridization with a 3H-labeled VZV DNA probe. Activated T lymphocytes were permissive for VZV infection at low frequency in vitro.     

Varicella-zoster virus retains major histocompatibility complex class I proteins in the Golgi compartment of infected cells

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene ORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.     

Varicella-zoster virus-infected human sensory neurons are resistant to apoptosis, yet human foreskin fibroblasts are susceptible: evidence for a cell-type-specific apoptotic response

The induction of apoptosis or programmed cell death in virus-infected cells is an important antiviral defense mechanism of the host, and some herpesviruses have evolved strategies to modulate apoptosis in order to enhance their survival and spread. In this study, we examined the ability of varicella-zoster virus (VZV) to induce apoptosis in primary human dorsal root ganglion neurons and primary human foreskin fibroblasts (HFFs). Three independent methods (annexin V, TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] staining, and electron microscopy) were used to assess apoptosis in these cells on days 1, 2, and 4 postinoculation. By all three methods, apoptosis was readily detected in VZV-infected HFFs. In stark contrast, apoptosis was not detected during productive VZV infection of neurons. The low-passage clinical isolate Schenke and the tissue culture-adapted ROka strain both induced apoptosis in HFFs but not in neurons, suggesting that this cell-type-specific apoptotic phenotype was not VZV strain specific. These data show that the regulation of apoptosis differs markedly between HFFs and neurons during productive VZV infection. Inhibition of apoptosis during infection of neurons may play a significant role in the establishment, maintenance, and reactivation of latent infection by promoting survival of these postmitotic cells.     

Activated peripheral T lymphocytes undergo apoptosis when cultured with monocytes activated by HLA class II ligation

We treated PBMC with anti-MHC class II mAb known to inhibit T lymphocyte proliferation. Adherent cells from mAb-treated PBMC showed increased metabolic activity by the MTS assay that was not due to cell proliferation. PBMC cultured with solid-phase anti-class II mAb in chamber inserts inhibited, across a membrane, the proliferation of PBMC cultured with soluble anti-CD3 mAb. PBMC treated with both soluble mAb underwent apoptosis as shown by nucleosomal DNA fragmentation. The monocytes formed multinucleated giant cells as shown by fluorescent microscopy, and contained apoptotic bodies as shown by the TUNEL method and by electron microscopy. The apoptotic cells were identified as T cells by double-staining with anti-CD4/CD8-PE and annexin-V-FITC. Thus, MHC class II ligation stimulates monocytes to increase their metabolic activity, induce apoptosis of activated T lymphocytes, and phagocytize the apoptotic cells. TCR-mediated ligation of MHC class II may play a role in the downregulation of immune responses.     

Varicella-zoster virus DNA in cells isolated from human trigeminal ganglia

To determine the type of cell(s) that contain latent varicella-zoster virus (VZV) DNA, we prepared pure populations of neurons and satellite cells from trigeminal ganglia of 18 humans who had previously had a VZV infection. VZV DNA was present in 34 of 2,226 neurons (1.5%) and in none of 20,700 satellite cells. There was an average of 4.7 (range of 2 to 9) copies of VZV DNA per latently infected neuron. Latent VZV DNA was primarily present in large neurons, whereas the size distribution of herpes simplex virus DNA was markedly different.     

Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice.

To investigate the cell tropism and pathogenicity of varicella-zoster virus (VZV) strains, we analyzed VZV replication by using SCID-hu mice that carry human fetal thymus/liver implants under the kidney capsule or as subcutaneous fetal skin implants. MRC-5 cells infected with wild-type VZV or the Oka strain, used in the live attenuated varicella vaccine, were injected into the implants. The implants were surgically removed 2, 7, 14, and 21 days postinfection. The VZV titer from infected thymus/liver implants peaked on day 7 for the wild-type strain and on day 14 for the Oka strain. Histological analysis showed necrotic areas characterized by thymocyte depletion and fibrosis. VZV protein synthesis was detectable by immunohistochemical staining in the necrotic areas and in distant regions that did not show cytopathic changes, and VZV DNA was detected by in situ hybridization in the same distribution. Fluorescence-activated cell sorting analysis of thymocytes harvested at day 7 postinfection showed that VZV proteins were expressed in CD4+, CD8+, and CD4+ CD8+ T cells; VZV was cultured from each T-cell subpopulation. The Oka strain had tropism for human cell types similar to that of wild-type VZV. T lymphocytes released infectious VZV, which is a novel and important observation about the replication of this otherwise highly cell associated virus. VZV-infected skin implants exhibited microscopic epidermal lesions that were indistinguishable histologically from the characteristic lesions of varicella. These experiments demonstrate a unique tropism of VZV for human T lymphocytes, explaining its capacity to cause viremia in natural disease, and demonstrate the value of the SCID-hu model for studies of VZV pathogenesis.     

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.     

Mink cell focus-forming murine leukemia virus killing of mink cells involves apoptosis and superinfection

Induction of apoptosis by different types of pathogenic retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To further study the ability of MCF MLVs to induce apoptosis and the role of this process in viral pathogenesis, we have developed an in vitro system of virus-induced apoptosis. MCF13 MLV infection of mink epithelial cells resulted in the production of cytopathic foci. In contrast, infection of mink cells with the 4070A amphotropic MLV did not produce any cytopathic effects. Staining of MCF13 MLV-infected cells with propidium iodide and annexin V-fluorescein isothiocyanate indicated that virus-induced cell death was due to apoptosis. At 6 days postinfection, the percentage of apoptotic MCF13 MLV-infected cells was 27% compared with 2 to 3% for mock- or amphotropic MLV-infected cells, representing a 9- to 14-fold difference. Assays for caspase-3 activation confirmed the detection by flow cytometry of apoptosis of MCF13 MLV-infected cells. Large amounts of unintegrated linear viral DNA were detectable by Southern blot analysis during the acute phase of infection, which indicated that MCF13 MLV is able to superinfect mink cells. Unintegrated viral DNA of only the linear form was detectable in thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the preleukemic period. These results indicated that the ability of MCF13 MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.     

Differential cell tropism of feline immunodeficiency virus molecular clones in vivo

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4(+) and CD8(+) lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4(+) lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4(+) and CD8(+) lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.     

Rotavirus infection alters peripheral T-cell homeostasis in children with acute diarrhea

The patterns of gene expression and the phenotypes of lymphocytes in peripheral blood mononuclear cells (PBMC) from children with diarrhea caused by rotavirus and healthy children were compared by using DNA microarray, quantitative PCR, and flow cytometry. We observed increased expression of a number of genes encoding proinflammatory cytokines and interferon or interferon-stimulated proteins and demonstrated activation of some genes involved in the differentiation, maturation, activation, and survival of B lymphocytes in PBMC of patients with rotavirus infection. In contrast, we observed a consistent pattern of lower mRNA levels for an array of genes involved in the various stages of T-cell development and demonstrated a reduction in total lymphocyte populations and in the proportions of CD4 and CD8 T lymphocytes from PBMC of patients. This decreased frequency of T lymphocytes was transient, since the proportions of T lymphocytes recovered to almost normal levels in convalescent-phase PBMC from most patients. Finally, rotavirus infection induced the activation and expression of the early activation markers CD83 and CD69 on a fraction of CD19 B cells and the remaining CD4 and CD8 T lymphocytes in acute-phase PBMC of patients; the expression of CD83 continued to be elevated and was predominantly exhibited on CD4 T lymphocytes in convalescent-phase PBMC. On the basis of these findings at the molecular, phenotypic, and physiologic levels in acute-phase PBMC, we conclude that rotavirus infection induces robust proinflammatory and antiviral responses and B-cell activation but alters peripheral T-cell homeostasis in children.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Differential Expression of Candidate Virus Receptors in Human T Lymphocytes Prone or Resistant to Infection with Patient-Derived Hepatitis C Virus

Accumulated evidence implies that hepatitis C virus (HCV) infects not only the liver but also the immune system. A lymphocyte-specific CD5 molecule was recently identified as essential for infection of T cells with native, patient-derived HCV. To assess whether the proposed hepatocyte receptors may also contribute to HCV lymphotropism, expression of scavenger receptor-class B type 1 (SR-B1), claudin-1 (CLDN-1), claudin-6 (CLDN-6), occludin (OCLN), CD5 and CD81 was examined by real-time RT-PCR and the respective proteins quantified by immunoblotting in HCV-prone and resistant T cell lines, peripheral blood mononuclear cells (PBMC), primary T cells and their subsets, and compared to hepatoma Huh7.5 and HepG2 cells. SR-B1 protein was found in T and hepatoma cell lines but not in PBMC or primary T lymphocytes, CLDN-1 in HCV-resistant PM1 T cell line and hepatoma cells only, while CLDN-6 equally in the cells investigated. OCLN protein occurred in HCV-susceptible Molt4 and Jurkat T cells and its traces in primary T cells, but not in PBMC. CD5 was displayed by HCV-prone T cell lines, primary T cells and PBMC, but not by non-susceptible T and hepatoma cell lines, while CD81 in all cell types except HepG2. Knocking-down OCLN in virus-prone T cell line inhibited HCV infection, while de novo infection downregulated OCLN and CD81, and upregulated CD5 without modifying SR-B1 expression. Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells. This study narrowed the range of factors potentially utilized by HCV to infect T lymphocytes amongst those uncovered using laboratory HCV and Huh7.5 cells. Together with the demonstrated role for CD5 in HCV lymphotropism, the findings indicate that virus utilizes different molecules to enter hepatocytes and lymphocytes.