Lysis of human cytomegalovirus infected fibroblasts by natural killer cells: demonstration of an interferon-independent component requiring expression of early viral proteins and characterization of effector cells

We investigated the susceptibility of cells infected with human cytomegalovirus (HCMV) to lysis by human natural killer (NK) cells, examining in particular its relationship to sequential viral protein expression, interferon (IFN), and the nature of the effector cells. HCMV-infected fibroblasts were lysed by peripheral blood mononuclear cells from normal seronegative individuals. The effector cells were large granular lymphocytes of Leu-7+, Leu-11+, and to a lesser extent Leu-7- phenotype. Depletion studies suggested they were the same population of NK cells that lyse uninfected fibroblasts, but a subset of NK cells that lyse K562 cells. HCMV-infected cells treated with phosphonoformate and cells infected for 16 hr that only express the nonstructural HCMV immediate early and early proteins and not the late (structural) proteins were susceptible to lysis by IFN-pretreated effector cells, whereas cells expressing immediate early antigens alone were not. This enhanced susceptibility to lysis was associated with increased effector:target binding in target cell binding assays, and was competitively inhibited by uninfected fibroblasts in cold target competition assays. It was independent of IFN release from the infected target cells or effector cells. These results suggest that the increased susceptibility to lysis by NK cells produced by a human herpes virus HCMV i) is manifest when early viral proteins are expressed, ii) is related to enhanced expression of a target structure likely to be present on uninfected fibroblasts, and iii) has a major component that is independent of IFN.     

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures

After a primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in myeloid lineage cells, and the virus has developed several mechanisms to avoid immune recognition and destruction of infected cells. In this study, we show that HCMV utilizes two different strategies to reduce the constitutive expression of HLA-DR, -DP, and -DQ on infected macrophages and that infected macrophages are unable to stimulate a specific CD4+ T-cell response. Downregulation of the HLA class II molecules was observed in 90% of the donor samples and occurred in two phases: at an early (1 day postinfection [dpi]) time point postinfection and at a late (4 dpi) time point postinfection. The early inhibition of HLA class II expression and antigen presentation was not dependent on active virus replication, since UV-inactivated virus induced downregulation of HLA-DR and inhibition of T-cell proliferation at 1 dpi. In contrast, the late effect required virus replication and was dependent on the expression of the HCMV unique short (US) genes US1 to -9 or US11 in 77% of the samples. HCMV-treated macrophages were completely devoid of T-cell stimulation capacity at 1 and 4 dpi. However, while downregulation of HLA class II expression was rather mild, a 66 to 90% reduction in proliferative T-cell response was observed. This discrepancy was due to undefined soluble factors produced in HCMV-infected cell cultures, which did not include interleukin-10 and transforming growth factor beta1. These results suggest that HCMV reduces expression of HLA class II molecules on HCMV-infected macrophages and inhibits T-cell proliferation by different distinct pathways.     

Human cytomegalovirus inhibits differentiation of monocytes into dendritic cells with the consequence of depressed immunological functions

Human cytomegalovirus (HCMV) infections in immunocompromised patients are associated with impaired immunological functions. Blood monocytes, which can differentiate into dendritic cells upon cytokine stimulation, play a central role in adequate immune reactivity and are believed to carry latent HCMV. We demonstrate here that HCMV infection of monocytes results in a block in the cytokine-induced differentiation of monocytes into functionally active CD1a-positive dendritic cells, which exhibited severely depressed immunological functions in vitro. The HCMV-infected cells exhibited a significantly reduced ability to endocytose fluorescein isothiocyanate-labeled dextran particles as well as a more than 90% reduced ability to migrate in response to the chemoattractant factors RANTES, MIP-1alpha, and MIP-3beta. Interestingly, HCMV-infected cells expressed high levels of the costimulatory molecule CD86, in contrast to the low levels of expression that was observed on uninfected monocytes and uninfected immature dendritic cells. Furthermore, HCMV-infected CD1a-negative cells were unable to induce a T-cell response. Thus, these observations suggest that HCMV infection of monocytes in vitro blocks cytokine-induced dendritic cell differentiation, and since dendritic cells play a central role in initiating immune responses, these findings suggest a powerful tactic to avoid immune recognition and to blunt the immune response at early phases of infection.     

Modulation of HLA-G expression in human neural cells after neurotropic viral infections

HLA-G is a nonclassical human major histocompatibility complex class I molecule. It may promote tolerance, leading to acceptance of the semiallogeneic fetus and tumor immune escape. We show here that two viruses-herpes simplex virus type 1 (HSV-1), a neuronotropic virus inducing acute infection and neuron latency; and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection-upregulate the neuronal expression of several HLA-G isoforms, including HLA-G1 and HLA-G5, the two main biologically active isoforms. RABV induces mostly HLA-G1, and HSV-1 induces mostly HLA-G3 and HLA-G5. HLA-G expression is upregulated in infected cells and neighboring uninfected cells. Soluble mediators, such as beta interferon (IFN-beta) and IFN-gamma, upregulate HLA-G expression in uninfected cells. The membrane-bound HLA-G1 isoform was detected on the surface of cultured RABV-infected neurons but not on the surface of HSV-1-infected cells. Thus, neuronotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially. HLA-G may therefore be involved in the escape of certain viruses from the immune response in the nervous system.     

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.     

Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.     

Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes

Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.     

Soluble interleukin-6 receptor alpha inhibits the cytokine-Induced fractalkine/CX3CL1 expression in human vascular endothelial cells in culture

Soluble form of IL-6 receptor alpha (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor beta chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma. IL-1alpha-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the adhesion to monolayers stimulated with IL-1alpha. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1alpha or IFN-gamma was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.     

Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Immunological aspects of human amniotic fluid cells: implication for normal pregnancy

Amniotic fluid cells (AFCs) are routinely obtained and expanded in vitro for prenatal diagnosis; nevertheless current knowledge about their properties is limited. The detailed mechanisms underlying normal pregnancies are yet to be discovered. The goal of this study was to identify the immunological aspects of AFCs including cytokine production and human leukocyte antigen (HLA) expression, and to discuss its implication for pregnancy. Eighty-six samples of AFCs were determined for HLA expression before and after culture. Cytokine production was measured with flow cytometry in AFC culture supernatants. Treatment of interferon (IFN)-gamma on induction of HLA-DR expression in cultured AFCs was also investigated. Data indicated that both fresh and cultured AFCs express HLA-I, HLA-G, but not HLA-DR, and the cultured AFCs predominately produce the cytokine interleukin (IL)-6. Importantly, we observed that IFN-gamma could induce HLA-DR expression on cultured AFCs in a dose-dependent manner. Taken together, our results indicated that AFCs are functionally active cells and are significant in pregnancy.     

Human cytomegalovirus (HCMV) infection in osteosarcoma cell line suppresses GM-CSF production by induction of TGF-beta

This study was performed to elucidate the possible mechanism of the disturbance of hemopoiesis by HCMV infection. Saos-2 cells constitutively express mRNA of GM-CSF, and its expression was profoundly decreased by HCMV infection, which required full replication of the virus and was mediated by soluble factors released from the HCMV-infected Saos-2 cells. TGF-beta1 production was statistically and significantly increased from one day after HCMV infection. Expression and production of GM-CSF in Saos-2 cells were restored when a culture supernatant of HCMV-infected Saos-2 cells was reacted with neutralizing anti-TGF-beta antibody. Conclusively, HCMV inhibits GM-CSF expression in Saos-2 cells partly by the increased production of TGF-beta1.     

HLA expression in hemopoietic development. Class I and II antigens are induced in the definitive erythroid lineage and differentially modulated by fetal liver cytokines.

We investigated the expression of HLA Ag on hemopoietic progenitors (burst-forming unit E, CFU-E, and CFU-granulocyte-macrophage) and precursors from human embryonic fetal liver (FL) and peripheral blood at 5 to 9 wk postconception. The expression on progenitors was evaluated by complement-mediated cytotoxicity followed by assay of residual progenitors in clonogenic culture; immunofluorescence and RIA were used for differentiated precursors. HLA Class I and II Ag are not expressed on the primitive erythroid lineage, i.e., on yolk sac-derived megaloblasts circulating in 5- to 6-wk peripheral blood. However, they are gradually induced on the definitive erythroid lineage in FL. Their expression on progenitors is first detected at 6 wk and shows a progressive increase through 9 wk, up to greater than or equal to 50% of adult values. A similar expression pattern is observed for FL erythroblasts. Incubation of 6-wk FL erythroid cells with IFN-alpha, -beta, or -gamma, or TNF-alpha induces a sharp rise of the expression of HLA class I, but not HLA class II Ag on both progenitors and precursors. In contrast, incubation with PHA-stimulated adult leukocyte-conditioned medium causes a marked increase of both HLA-ABC and HLA class II Ag expression. We hence investigated the effect of cytokines present in leukocyte-conditioned medium on the expression of HLA class II Ag: although IL-1 alpha, IL-2, and granulocyte-macrophage-CSF do not exert a significant action, IL-1 beta and IL-4 induce a marked increase of HLA class II, but not class I, Ag expression on 6-wk FL progenitors and precursors. Low amounts of IFN-gamma, TNF-alpha, and IL-1 beta were detected in the supernatants and extracts of 6-wk FL cells. The concentrations of these cytokines in both supernatants and extracts sharply increase in the 7- to 8-wk period, particularly for TNF-alpha and IL-1 beta, thus indicating a direct correlation with the rise of HLA Ag expression on FL erythroid cells in the same period. In conclusion, the expression of HLA class I and II Ag is not detected on primitive megaloblasts, but is gradually induced on definitive FL progenitors and precursors, possibly via production of specific cytokines in the FL microenvironment, i.e., IFN-gamma and TNF-alpha for class I Ag and IL-1 beta for class II Ag.     

c-myc down-regulates class I HLA expression in human melanomas

Expression of class I HLA antigen has been shown to be reduced in a number of human tumours. Here we show that in a panel of 11 melanoma cell lines with variable class I HLA expression an inverse correlation exists between the mRNA levels of c-myc and class I HLA. This suggests that high expression of the c-myc oncogene might inhibit the class I HLA expression. To test this hypothesis a melanoma cell line with a low c-myc and high class I HLA mRNA expression was transfected with a c-myc expression vector. All clones expressing the transfected c-myc gene show reduced class I HLA mRNA and beta 2-microglobulin mRNA expression. Reduced class I HLA mRNA levels result in a lowered class I protein expression on the cell surface. Treatment with gamma-interferon fully restores the class I HLA and beta 2-microglobulin expression in these cells. This effect is preceded by a transient decrease of the c-myc mRNA level. These results show that the class I HLA expression is modulated by the level of c-myc expression, thus opening up the possibility that high expression of this oncogene influences the interaction of melanoma cells with the immune system.     

Human cytomegalovirus-induced upregulation of intercellular cell adhesion molecule-1 on villous syncytiotrophoblasts

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.