Murine cytomegalovirus infection down-regulates MHC class II expression on macrophages by induction of IL-10.

Herpesviruses utilize many strategies for weakening the host immune response. For CMV, this includes avoidance of NK clearance and inhibition of MHC class I and class II presentation pathways. In this study, we report that mouse CMV (MCMV) specifically causes a premature and transient activation of host IL-10 very early in the course of infection, resulting in a dramatic and selective reduction in MHC class II surface expression. The expression of IL-10 is normally late in the immune response to a pathogen, serving to dampen the response by suppression of the production of inflammatory cytokines. In infection of macrophages, we show that MCMV induces the production of IL-10, leading to an early and selective reduction in the expression of MHC class II on the surface of the cells. Inhibition of MHC class II expression was not observed in the presence of neutralizing Abs to IL-10 or in macrophages from IL-10-deficient mice. Moreover, MCMV-infected IL-10-deficient mice developed an early and significantly more robust macrophage MHC class II induction than normal mice. Altogether, our results demonstrate that viral induction of an IL-10 autocrine pathway plays an essential early role in selectively reducing MHC class II expression on the surface of APC prior to stimulation by IFN-gamma.     

Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A

Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor α/β (ERα/β)-dependent mechanism with the evidence of ERα/β antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor κB (NF-κB) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health.     

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.     

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells,astrocytes and monocyte-derived macrophages

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Identification of specific and cross-reactive antigens of Leishmania donovani chagasi by human infection sera

Cloned Leishmania donovani chagasi (Ldc) promastigotes were analyzed by SDS-PAGE separation and immunoblotting with human infection sera. The patterns of antigen reactivity were compared by using sera from individuals with Ldc, Leishmania mexicana amazonensis (Lma), Trypanosoma cruzi, Mycobacterium tuberculosis, or Mycobacterium leprae infections. Sera from individuals with these infections recognized Ldc antigens in several m.w. ranges. Reactivity was due to recognition of Ldc molecules and not to Ldc culture medium components, as shown by comparing Ldc promastigotes grown in the presence or absence of fetal bovine serum (FBS), by immunoblotting of FBS, and by [35S]methionine labeling. The major findings of the study were as follows. Immunoblots with Ldc promastigotes could be used to distinguish individuals with Ldc infections from those with Lma infections. Persons with Ldc infections had antibodies to a Ldc antigen of approximately 32 to 35 kd not recognized by persons with Lma infections. Individuals cured of acute Ldc infection did not develop antibodies that differed in specificity to those present during their acute phase of infection. Ldc antigens in the 62 to 66 kd region were recognized by all individuals with Ldc or Lma infections but were not recognized by individuals in the other disease groups or by control sera. This region was found to contain at least four distinct bands, one of which appeared to be glycosylated as indicated by periodic acid-Schiff staining and concanavalin A labeling; an apparently nonglycosylated protein of 62 to 63 kd was eluted from SDS-PAGE gels and was used to diagnose Ldc infection by the ELISA. Whereas crude Ldc antigen gave false positive results with T. cruzi and mycobacteria infection sera, the eluted 62 to 63 kd protein was 100% specific and sensitive in the diagnosis of Ldc infection.     

Susceptibility of peritoneal macrophages to rat cytomegalovirus infection

Abstract Peritoneal macrophages from Lewis (Lew) and Brown Norway (BN) rats did not support rat cytomegalovirus (RCMV) replication. Intraperitoneal (i.p.) inoculation of virus into the rats resulted in a rapid clearance of virus from the peritoneal lavage fluid and an uptake of virus in the macrophages. The virus did not persist in the peritoneal macrophages of the rats.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.     

HLA-G in the human placenta: expression and potential functions

HLA-G is a non-classical class I molecule specifically expressed in the placenta, suggesting that it might have a physiological function at the materno-foetal interface. The structural characteristics of HLA-G, the placental pattern of expression and the functional properties of this class Ib glycoprotein in vitro are described and evaluated in the context of pregnancy. The possible anti-viral function of HLA-G, its modulatory role of natural killer cell activity and its likely non-immunological functions are discussed.     

Early induction of autophagy in human fibroblasts after infection with human cytomegalovirus or herpes simplex virus 1

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.     

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies.     

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens. Presented in part at the 71st Congress of the German Pathological Society, Salzburg, June 1987     

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens.     

Differential tolerance induction by lipoarabinomannan and lipopolysaccharide in human macrophages

Various bacterial cell wall components have been shown to induce hyporesponsiveness in macrophages (MAC). Here, mycobacterial glycolipids were employed to determine whether they induce a state of 'tolerance/hyporesponsiveness' in MAC in vitro in order to assess whether mycobacterial components negatively affect the immune response to Mycobacterium tuberculosis. Arabinosylated lipoarabinomannan (ARA-LAM) stimulated hyporesponsiveness by reducing TNF-alpha, GM-CSF, G-CSF, IL-10, and IL-6 release similarly to LPS, but caused no changes in IL-8 secretion. Mannose-capped LAM (MAN-LAM) acted in a different way in that TNF-alpha, GM-CSF, and IL-10 were upregulated after restimulation of MAC. Blocking experiments by mannan suggest mannose-receptor involvement in MAN-LAM activation only. Cross-stimulation experiments demonstrated a hierarchy of signaling, with LPS being the most potent stimulator and mediating abrogation of ARA-LAM-stimulated tolerance but not vice versa. MAN-LAM was the least potent stimulator of either MAC activation and induction of hyporesponsiveness. Similarly to LPS, ARA-LAM upregulated CD14 surface expression after restimulation. Recurrent MAN-LAM treatment either downmodulated or did not induce any change in CD14 expression. The role of MAN-LAM regulated cytokine secretion as well as implications regarding M. tuberculosis infection will be discussed.     

Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.