Factors affecting human cytomegalovirus gene expression in human monocyte cell lines

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.     

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.     

Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture

The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.     

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.     

Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor necrosis factor alpha production

Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-alpha) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-kappaB, leading to TNF-alpha production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-kappaB and colocalization of p65/NF-kappaB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-alpha release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-kappaB but not p38 mitogen-activated protein kinase in induction of TNF-alpha. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-kappaB as a promising target for development of new therapeutic intervention strategies.     

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.     

Human cytomegalovirus productively infects primary differentiated macrophages.

Monocytes are one of the predominant cell types in the peripheral blood that are infected by human cytomegalovirus (HCMV). Although virus can be detected in these cells in vivo, HCMV replication in cultured monocytes has been unsuccessful. In this study, we demonstrate efficient HCMV replication in cultured monocytes. HCMV permissiveness in these cells was dependent on nonadherent cell-induced stimulation of the monocyte, with subsequent morphological differentiation into macrophages. Approximately 40% of the cells infected by virus were detected by immunofluorescent staining with both immediate-early and late antibodies. In addition, viral plaque assays demonstrated significant productive infection of macrophages. These observations are consistent with the suggestion that the monocyte/macrophage serves as a source of viral amplification and dissemination.     

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.     

Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes

A cDNA clone encoding human thymosin-beta 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-beta 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. Nine of 132 nucleotides were different in the coding sequences (93% homology), but the deduced amino acid sequences were identical. No signal peptide was found in the deduced protein sequence. Human thymosin-beta 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-beta 4 mRNA. Expression of the human thymosin-beta 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-beta 4 mRNA varied as a function of differentiation stage. Thymosin-beta 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-beta 4 and the immune response. Thymosin-beta 4 mRNA was more highly expressed in mature granulocytes than in immature blastic cells. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-lambda reduced the levels of thymosin-beta 4 mRNA. Its level decreased after differentiation of THP-1 cells into Ia+ macrophages, but increased after differentiation of HL-60 cells into Ia- macrophages. The pattern of thymosin-beta 4 gene expression suggests that it may play a fundamental role in the host defense mechanism.     

The nef gene of simian immunodeficiency virus SIVmac1A11.

The role of the simian immunodeficiency virus (SIV) nef gene in viral replication was investigated in several tissue culture systems. SIVmac1A11 is a molecularly cloned virus which replicates in both peripheral blood mononuclear cells (PBMC) and macrophages, although no disease is observed in infected rhesus macaques. In this report, we demonstrate that SIVmac1A11 contains a full open reading frame for nef which specifies a 37-kDa protein. To investigate the effects of nef on viral replication, a 70-bp deletion was introduced into the nef gene of SIVmac1A11. Analysis of infected cell extracts by immunoblotting revealed that both SIVmac1A11 and nef deletion virus SIVmac1A11 delta nef produced the same viral proteins, except that Nef was absent in the mutant virus. The deletion mutation did not affect viral replication in PBMC, in monocyte-derived and alveolar macrophages obtained from rhesus macaques, and in human cell lines HUT-78 and CEMx-174. In addition, SIVmac1A11 and SIVmac1A11 delta nef exhibited similar patterns of cytopathologic changes and ultrastructural appearances in infected cells. SIVmac1A11 and SIVmac1A11 delta nef did not infect human tumor macrophage cell line U937, GCT, THP-1, or HL-60 cells, although virus was produced after these cells were transfected with either wild-type or nef mutant viral DNA. Similar levels of virus were recovered from U937 and THP-1 cells transfected with mutant and parental proviral DNAs. In transient expression assays in a T-cell line and a macrophage line, the nef protein of SIVmac1A11 did not significantly suppress or enhance expression of the chloramphenicol acetyltransferase reporter gene linked to the SIVmac long terminal repeat. Thus, abrogation of nef did not affect several in vitro properties of SIVmac1A11, including patterns of viral infection in rhesus PBMC, rhesus macrophages, or human T-cell lines.     

Human immunodeficiency virus infection in cells of myeloid-monocytic lineage

We established persistent infection with a strain of human immunodeficiency virus type 1, HTLV-IIIB, in a promyelomonocytic cell line, ML-1 (CD4 antigen nearly negative and CD4 mRNA negative), and a promonocytic cell line, THP-1 (CD4 antigen positive). Different reaction of giant cell formation was found after co-cultivation of infected and uninfected cells of ML-1, HL-60, THP-1 and U-937 cell lines with uninfected and infected MOLT4 (a T-lymphoma cell line).     

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.