Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells

BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.     

Human cytomegalovirus infection in a retinoblastoma cell line in vitro

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.     

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.     

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.     

Down-regulation of the surface expression of class I MHC antigens by human cytomegalovirus

Class I major histocompatibility complex (MHC) antigens of higher eukaryotes play a crucial role in the recognition of human cytomegalovirus (HCMV)-infected cells by cytotoxic T lymphocytes. In the present study, we have demonstrated that HCMV infection resulted in a marked reduction in the surface expression of class I MHC antigens on human embryonic lung fibroblasts (HEL). Even when HCMV-infected HEL were cultured in the presence of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG), the reduction was observed to the same extent, indicating that HCMV DNA synthesis was not required for this phenomenon. However, immunoprecipitation studies have shown that there was no significant reduction in the synthesis of either the heavy chain or the light chain of class I MHC antigens after HCMV infection. In addition, Western blotting studies have revealed that the total amount of the antigens was almost unaltered after HCMV infection. These results suggest that the reduction in the cell surface expression of class I MHC antigens is due to some defect occurring post-translationally, most likely at the level of the correct complex formation and/or the intracellular transport of class I MHC antigens.     

Effects of dietary supplementation with alpha-linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerel from 24 to 72 weeks of age

The physiopathology of experimental cerebral malaria (CM), an acute neurological complication of Plasmodium berghei ANKA (PbA) infection, involves interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), two cytokines that are known to modulate major histocompatibility complex (MHC) molecule expression. The aim of this study was to evaluate whether the genetic susceptibility to CM is related to the constitutive or IFN-gamma-induced expression of MHC molecules on brain microvessels. To this end, brain microvascular endothelial cells (B-MVEC) were isolated from CM-susceptible (CM-S, CBA/J) and resistant (CM-R, BALB/c) mice. By flow cytometry, we found that less than 5% of CM-S B-MVEC constitutively expressed MHC class I molecules, in contrast to up to 90% of CM-R B-MVEC. Upon stimulation with IFN-gamma, the percentage of positive cells for MHC class I molecules in CM-S B-MVEC became comparable to CM-R B-MVEC, but a higher fluorescence intensity existed on CM-S B-MVEC compared with CM-R B-MVEC. MHC class II molecules were not constitutively expressed on B-MVEC from either strain. IFN-gamma-induced expression of MHC class II (I-A, I-E) molecules was significantly higher in CM-S than CM-R B-MVEC both in percentage of positive cells and fluorescence intensity. These data demonstrate that absent or low MHC class I and higher inducibility of MHC class II expression on B-MVEC are associated with the genetic susceptibility to CM.     

Increased number of T cells committed to IL-5 production after respiratory syncytial virus (RSV) infection of human mononuclear cells in vitro

We examined changes in the cytokine profile of T cells induced by in vitro infection with RSV. Isolated mononuclear cells from 27 healthy adults and six infants were infected with RSV at a concentration of 3 MOI (multiplicity of infection). After 48 h cells were restimulated with phorbol ester and ionomycin in the presence of monensin for 5 h. The intracellular expression of viral antigen, the cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-gamma), and the expression of surface markers were assessed by immunofluorescent staining and flow cytometry. There was a significant (P<0.001) rise of IL-5 expression in RSV-infected cultures in comparison with uninfected cultures from the same individuals, whereas there were no changes in the expression of the other lymphokines. The increase in IL-5 generation depended on viable infectious RSV rather than inactivated virus. RSV infection as well as IL-5 production in T cells were confined to the CD8 subpopulation. However, there was no simultaneous expression of RSV antigen and IL-5. Purified T cells did not show any increase in IL-5 generation. However, when the rate of RSV infection was enhanced in monocytes by means of a specific monoclonal antibody, co-cultured T cells displayed an increase of IL-5 production compared with samples with ordinary low rate RSV infection. It is therefore likely that the increased commitment of lymphocytes to produce IL-5 after RSV infection in vitro is mediated by monocytes or other antigen-presenting cells.     

Morphology and structure of photosensitive dye J-aggregates adsorbed on AgBr microcrystals grown in gelatin

Our purpose was to determine the expression of the major histocompatibility complex (MHC) class I and class II gene products as well as the costimulatory molecules B7-1 and B7-2 on cervical epithelial cells, and to determine to what extent inflammatory cytokines regulate their expression. Immunohistology and flow cytometry techniques were used to identify and quantify MHC class I and class II molecules, and the costimulatory molecules B7.1 and B7.2, on sections and primary epithelial cell cultures of human endo- and ectocervix. MHC class I but not class II molecules were constitutively expressed on tissue sections and primary epithelial cell cultures derived from endo- and ectocervix. Expression of MHC class I and class II was upregulated in vitro by IFN-gamma in a time and dose dependent fashion. The induction of class II expression was more pronounced on ectocervical cells than on endocervical cells. MHC class I but not class II expression was also enhanced by IFN-alpha as well as TNF-alpha. TNF-alpha and TGF-beta1 inhibited the IFN-gamma induced MHC class II expression. Expression of the costimulatory molecules B7-1 and B7-2 were not detected in tissue sections or on resting or cytokine-treated cervical epithelial cells in vitro. The present results support the concept that endo- and ectocervical epithelial cells, like their counterparts at other mucosal sites. constitutively express MHC class I molecules and can express MHC class II upon cytokine stimulation, indicating that they are capable of presenting antigens to T-cells.     

Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells

Human cytomegalovirus (HCMV)-infected cells persist in the presence of anti-HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus-encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host-encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV-1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up-regulation of CD55 suppressed the activity of cell-associated C3 convertases on HCMV-infected cells. Furthermore, increased CD55 expression protected infected cells from complement-mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host-encoded complement regulator proteins may provide protection of HCMV-infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement-mediated lysis and decreasing the deposition of C3-derived products on the cell surface.     

Infection of cells with varicella-zoster virus down-regulates surface expression of class I major histocompatibility complex antigens

In vitro assays indicate that both major histocompatibility complex (MHC) class I and II-restricted cytotoxic T lymphocytes are important for recognition of varicella-zoster virus (VZV)-infected cells. This study demonstrates that infection of human fibroblasts with wild-type or recombinant-derived strain Oka VZV results in down-regulation of surface expression of class I MHC heavy chains. Radioactive labeling of infected cells indicated that the amount of newly synthesized class I antigen was similar in uninfected and VZV-infected cells. In addition, immunoblotting showed that the amount of total cellular class I MHC heavy chains was unaffected by VZV infection. These results suggest that the reduction of class I heavy chains on the surface of VZV-infected cells is due to a defect in posttranslational processing. The down-regulation of class I MHC antigens in VZV-infected cells may provide a mechanism for the virus to escape the host immune response.     

An important role for major histocompatibility complex class I-restricted T cells, and a limited role for gamma interferon, in protection of mice against lethal herpes simplex virus infection

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-gamma) has been proposed. To address the roles of MHC class I and of IFN-gamma, C57BL/6 mice deficient in MHC class I expression (beta2 microglobulin knockout [beta2KO] mice), in IFN-gamma expression (IFN-gammaKO mice), or in both (IFN-gammaKO/beta2KO mice) were infected with HSV by footpad inoculation. beta2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-gamma appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-gamma.     

Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes

Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.     

Changes in the leukocyte phenotype profile of goats infected with the caprine arthritis encephalitis virus

The proportions of different sub-populations of leukocytes in five healthy goats and five goats infected with the caprine arthritis encephalitis virus (CAEV) were examined using immunofluorescence and flow cytometry. A panel of monoclonal antibodies that identified a monocytegranulocyte marker (GMI); the CD4, CD8, IgM, MHC Class I, MHC Class II and T19 antigens, and the gamma delta (gamma delta) T cell receptor was used. We observed a significant (P = 0.016) reduction in the proportion of monocytes in the peripheral blood of infected (5.98%) compared with healthy control goats (9.92%). There was also a decrease in the proportion of CD4+ T lymphocytes that approached significance (P = 0.076) accompanied by a slight increase in the proportion of CD8+ T lymphocytes, in infected compared with uninfected animals. Consequently, three of the five infected animals had lower CD4:CD8 ratios than any of the healthy animals and two of these three ratios were inverted. Approximately 14% of T cells in the peripheral blood of healthy goats was identified as gamma delta T cells and all expressed the T19 antigen. A significantly elevated level of gamma delta T cells (P = 0.030) and an elevated level of T19 cells were observed in infected, compared with healthy animals. The proportion of leukocytes expressing surface IgM (B cells) was also elevated, although not significantly, in CAEV-infected compared to healthy controls. The changes in peripheral blood leukocyte subsets in infected goats suggest that immune responses to the infection are probably altered in these animals with eventual progression to severe disease and death.     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.     

Expression of human leukocyte antigens class I and class II on cultured biliary epithelial cells after cytomegalovirus infection

The influence of cytomegalovirus (CMV) infection on the HLA expression on cultured biliary epithelial cells (BEC) was investigated. CMV-infection augmented expression of HLA class I but not of HLA class II. CMV reduced the IFN-gamma-mediated induction of the de novo expression of HLA class II while the stimulated expression of HLA class I was not impaired. Autologous but not allogeneic PBL responded to CMV-infected BEC. This response resulted in upregulation of HLA class I on BEC which was significantly higher compared with the expression on infected BEC alone or on uninfected BEC cocultured with autologous PBL. The results suggest that CMV modulates the immunogenic potential of BEC, which is important for the HLA and CMV-mediated pathomechanisms in vivo.     

A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3

Human cytomegalovirus (HCMV) infection can result in neurological symptoms. In vitro replication of the HCMV was studied in primary cultures of microglial cells from the central nervous systems (CNS) of human embryos. The microglial cells were infected with various amounts of either the AD169 laboratory HCMV strain or a clinical HCMV isolate. A specific cytopathic effect occurred within 24 h and persisted for two months. Immunocytochemical tests for immediate early and late viral antigens done one and three days after the infection demonstrated that 60% to 80% of the microglial cells were infected and that 3% to 8% were the site of viral DNA replication. Kinetic studies showed accumulation of viral particles in the supernatant during the first two weeks after the infection. Prestimulation of the cells by PMA 24 h before the infection was associated with increased release of viral particles and with an increased percentage of cells expressing late viral antigens. The microglial cells of the human embryonic CNS are fully permissive targets for the HCMV. The in vitro HCMV model used in this study may prove useful for investigating the pathophysiology of HCMV encephalitis, in particular after mother-to-fetus transmission of the virus.     

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.