Pathogenesis of murine cytomegalovirus infection: the macrophage as a permissive cell for cytomegalovirus infection, replication and latency.

Macrophages harvested from the peritoneal cavities of mice of several strains were permissive to infection with murine cytomegalovirus (MCMV). Macrophages from six mouse strains released equivalent amounts of plaque-forming virus into the culture fluids and cells from mouse strains scored similarly in numbers of infectious centres. Twenty to 50% of the infected macrophages obtained after thioglycollate activation formed infectious centres. When studied by in situ hybridization, more than 82% of infected macrophages (with or without thioglycollate activation) contained MCMV DNA. Macrophages obtained from latently infected mice were examined for their content of MCMV. Using co-cultivation assays, MCMV was frequently recovered from thioglycollate activated macrophages harvested from latently infected mice but only rarely recovered from non-activated macrophages. MCMV DNA--mouse DNA hybridization assays revealed four to seven virus genome DNA copies per 100 cells. These studies indicate that macrophages harvested from mice susceptible (BALB/cSt) or resistant (C3H) to MCMV infection replicated virus equivalently and that macrophages are a reservoir of MCMV during latent and chronic infections. Activation of macrophages may be one of the important steps leading to the exacerbation of in vivo latent infections.     

用原位杂交技术研究小鼠巨细胞病毒对巨噬细胞的感染

目的 检测巨细胞病毒（CMV）是否能够感染并影响小鼠腹腔巨噬细胞。方法 以小鼠巨细胞病毒（MCMV）攻击小鼠腹腔巨噬细胞后,用地高辛标记的152bp MCMV DNA探针进行原位杂交测定。结合受染细胞形态学改变,巨细胞包涵体检测,以及细胞表达Fc受体表达（Fc花环形成）的变化,观察MCMV对单核－巨噬细胞的作用。结果 原位杂交技术检测表明受MCMV攻击的巨噬细胞的胞浆和核内,有杂交阳性的灰蓝色颗粒沉积,与阳性对照细胞片（小鼠成纤维细胞3T3）相一致;未受MCMV攻击的巨噬细胞片（阴性对照）中,则未见有着色颗粒。同时发现,受染细胞的形态改变,核呈现典型的嗜酸性包涵体;受染细胞表达Fc受体表达呈上调趋势。结论 巨细胞病毒能够感染巨噬细胞并影响其形态和功能。     

Modulation of chemiluminescence in a murine macrophage cell line by neuroendocrine hormones

This study determined the effects of neuropeptides and neuroendocrine hormones at the cellular level of the immune response using a murine macrophage cell line, J774, which exhibits a chemiluminescent oxidative burst acute with stimulation with zymosan. We report that the zymosan-triggered oxidative burst of J774 cells can be modulated by the opioid peptides β-endorphin β-END and dynorphin A (DYN) in a naloxone-reversible fashion. Norepinephrine (NE) also modulated chemiluminescence (CL) emission of J774 cells, with dose-dependent suppression of CL dependent upon co-incubation with γ-interferon (γ-INF). Without γ-INF co-incubation, NE shared with the opioid peptides β-END and DYN the ability to modulate oxidative burst, producing an inverted-U dose response. These data indicate the J774 cells may be useful for explaining some mechanisms through which the neuroendocrine system interacts with the immune system.     

The roles of tumour necrosis factor-alpha, interleukin-1 and interleukin-12 in murine cytomegalovirus infection.

The swine is a useful model for immunobiological studies as it has a highly heterogeneous lymphocyte pool, containing several subsets not easily accessible in humans and rodents. In particular, the CD8-positive (CD8+) cells contain a variety of lymphocyte subsets, such as alpha beta-T cells, gamma delta-T cells, CD4 CD8 double-positive (DP) cells and natural killer (NK) cells. In order to define these subsets further, we have selected four monoclonal antibodies (mAb) with differential reactivity on CD8+ cells. Thus, mAb CD8.1 (PPT20) bound to CD8hi and CD8lo subpopulations in a similar way to the conventional anti-CD8. The mAb CD8.2 (PPT21), though binding to all of the CD8+ cells, reacted preferably with CD8hi. Two other mAb, CD8.3 (PPT22) and CD8.4 (PPT23), were specific for CD8hi alpha beta-T-cell subpopulation. These results, complemented by immunoprecipitation, co-modulation and enzyme-linked immunosorbent assay experiments, suggest that CD8.1 and CD8.2 react putatively with the CD8 alpha-chain and CD8.3 and CD8.4 with the CD8 beta-chain. Tissue distribution studies revealed that CD8+ thymocytes and peripheral CD8hi alpha beta-T cells expressed both putative CD8 alpha- and beta-chains while peripheral CD4+ CD8+ alpha beta-T cells, CD8lo gamma delta-T cells and NK cells expressed only putative CD8 alpha-chain. Functional studies indicated that the CD8hi alpha beta-T and CD8lo gamma delta-T cells were effector cells in the CD3-redirected cytotoxicity.     

Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity

Summary The administration of bovine lactoferrin (LF) with 1 mg/g body weight before the murine cytomegalovirus (MCMV) infection completely protected the BALB/c mice from death due to the infection. In these LF-treated mice, a significant increase in the activity was found in the NK cells but not in the cytolytic T lymphocytes which recognized an MCMV-derived peptide. Moreover, the elimination of the NK cell activity by an injection with anti-asialo GM1 antibody abrogated such augmented resistance, thus supporting the hypothesis that the LF-mediated antiviral effect in vivo is performed through the augmentation of NK cell activity. No such LF-mediated antiviral effect in vivo with the increased NK cell activity was found in athymic nude mice, whereas it was restored completely by the transfer of splenic T cells from LF-treated donors. These findings therefore suggest that T lymphocytes induce both the augmentation of NK cell activity and the resultant antiviral effect in the LF-treated hosts.     

鼠巨细胞病毒感染RAW264.7细胞的体外实验研究

目的 体外观察鼠巨细胞病毒(murine cytomegalovirus,MCMV)对鼠巨噬细胞株RAW264.7的感染特点及其对细胞增殖和凋亡的影响.方法 分别以感染复数(multiplicity of infection,MOI)为1、0.1、0.01的MCMV Smith株感染RAW细胞,于感染后6、12、24、36、48、72、96和120 h收集细胞和培养上清,光镜下观察细胞病变,透射电镜观察感染细胞内病毒颗粒和细胞超微结构变化;免疫细胞化学检测MCMV早期抗原(early antigen,EA)表达以及蚀斑形成实验监测病毒增殖情况;MTT法和流式细胞术分别评估感染细胞增殖与凋亡的改变,以MCMV敏感的鼠胚胎成纤维细胞为阳性对照.结果 MCMV感染后24～48 h可见RAW细胞肿胀和脱落,电镜显示RAW胞浆中存在完整的病毒颗粒且细胞器肿胀明显;病毒感染RAW细胞后6 h(MOI:1和0.1)～12 h(MOI=0.01)可见病毒EA的阳性表达;感染后24 h培养上清中病毒滴度即明显上升,至96～120 h达高峰;RAW细胞增殖在感染后72～120 h受到明显抑制;当MOI=0.1时,感染后72～120 h细胞死亡率明显增高,但凋亡率改变不显著.结论 巨噬细胞株RAW264.7在体外对MCMV易感,较鼠胚胎成纤维细胞更快形成溶细胞性的产毒性感染;病毒可抑制细胞增殖但不影响细胞凋亡,为深入探讨CMV相关免疫学致病机制提供了一个良好的体外细胞模型.     

Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-β₂) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.     

Lectin-mediated enhancement of dengue virus infection in a mouse macrophage cell line Mk1

Summary Treatment of a mouse macrophage cell line Mk1 with pokeweed mitogen (PWM) either before or during but not after virus inoculation resulted in an enhancement of dengue virus (DV) infection. The infection enhancement was primarily due to an increase in the number of DV-infected cells but not to increased virus production in a cell. These results suggested that PWM treatment mediated increased DV binding and/or penetration to Mk1 cells, thereby resulting in the infection enhancement.N-acetylglucosamine (GlucNAc) did not suppress PWM-mediated enhancement of DV infection when added to Mk1 cells after PWM treatment was done, although GlucNAc clearly suppressed the effect of PWM when added simultaneously with PWM. The results implied the possibility that the PWM-mediated increase in viral binding/penetration was not due to a cross-linking by PWM between DV and a cell-surface receptor, but due to another mechanism, presumably exposure of a masked DV receptor(s). The DV receptor, unidentified as yet, involved in the PWM-mediated infection enhancement appeared to have no relation with IgG Fc receptors that are known to be involved in antibody-mediated enhancement of DV infection.     

Expression of murine N-MYC by insertion of retrovirus sequences in a murine macrophage cell line (RAW264).

RAW264 cells, reported to be originated from Abelson-virus-induced B lymphomas, are widely used as a murine monocyte cell line. We found that RAW264 show enhanced expression of murine N-MYC. Murine cDNA clones associated with N-MYC were separated from (lambda)gt11 cDNA library constructed by using mRNA from the macrophage cell line, RAW264 cells. Sequencing analysis of the longest cDNA clone N-MYCL showed that the length of the coding region was 18 bases shorter than that of the predicted full length N-MYC cDNA, and the 3' untranslated region had the 5' long terminal repeat (LTR) sequence of the Moloney-like proviral sequence, suggesting the expression of N-MYC by insertion of the proviral sequence. This suggests that expression of N-MYC plays a role in the establishment of macrophage cell line RAW264. Integration of LTR and overexpression of the N-MYC gene might have existed in the parental lymphoma cells, playing a role in the development of lymphoma or in the establishment of macrophage cell line.     

Persistent infection with human cytomegalovirus in a lymphoblastoid cell line.

Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus (HCMV). The infected culture has persistently yielded HCMV with titers ranging from 2 × 10⁴ to 3 × 10⁵ PFU/ml over a period of 1 year. Infectious center and fluorescent antigen assays and electron microscopic examination indicated that 1–10% of the cells were infected. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells rather than to a defective or temperature-sensitive mutant of HCMV. The treatment of persistently infected cultures with anti-HCMV serum resulted in curing the virus infection. Cured cells in culture grew at the same rate as normal uninfected cells and became resistant to HCMV infection and relatively resistant to HSV infection.     

A soluble immunosuppressive activity induced by murine cytomegalovirus infection of mouse embryo fibroblasts

Summary Murine cytomegalovirus infection of mouse embryo fibroblast cultures resulted in the production of a soluble activity which inhibited spontaneous and mitogen induced proliferation of fresh spleen cultures. The production of this soluble immunosuppressive activity by mouse fibroblasts was not inhibited by indomethacin nor could its activity be mimicked by alpha, beta or gamma interferons.     

A murine macrophage cell line, immortalized by v-raf and v-myc oncogenes, exhibits normal macrophage functions

In vitro immortalized cell lines with the morphology and phenotype of mature macrophages (M phi) have been generated by infecting freshly isolated bone marrow cells from C3H/HeJ mice with a recombinant retrovirus carrying v-raf and v-myc oncogenes. All of the clones obtained had M phi-like phenotypes, and one such clone, GG2EE, has been compared to normal M phi to ascertain the effects of immortalization on the expression of the biological functions of the lines. GG2EE cells expressed cytotoxic activity against L5178Y, P815 or RL male 1 target cells in response to stimulation with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes; in contrast, they failed to kill YAC-1 target cells. GG2EE cells did not constitutively express I-A or I-E antigens; nevertheless, I region-coded antigens could be induced by IFN-gamma treatment. GG2EE cells produced interleukin 1 upon stimulation with a T cell-derived lymphokine; they were weakly phagocytic, yet became highly phagocytic following IFN-gamma treatment. Since c-fos mRNA is augmented in peritoneal exudate M phi by protein kinase C activators but not by IFN-gamma, we evaluated the effects of calcium ionophore, phorbol myristate acetate, L-alpha-1-oleoyl-2-acetoyl-sn-3 glycerol (OAG) and IFN-gamma on the levels of c-fos mRNA in GG2EE cells. We found that calcium ionophore, PMA and OAG stimulation enhanced the expression of c-fos mRNA, but IFN-gamma treatment did not. The kinetics of c-fos induction in GG2EE cells were also comparable to those observed in peritoneal exudate M phi. Overall, the GG2EE cell line has the same biological properties as normal tissue M phi. Because it is capable of both constitutive and inducible M phi-like functions, this cell line provides a valuable tool for studying the molecular mechanisms controlling induction and/or expression of biological activities in M phi. It is striking that a cell line immortalized in vitro by two oncogenes, v-raf and v-myc, behaves, according to the criteria mentioned above, like a normal M phi population.     

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2'-7'-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation.     

Contribution of tumor necrosis factor α and interleukin-1 α on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in a murine macrophage cell line,RAW264.7

The production of several inflammatory cytokines, such as murine macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor (TNF), and interleukin (IL)-1, was investigated in response to respiratory syncytial virus (RSV) infection in a murine macrophage cell line, RAW264.7, with special reference to mutual relation of their productions. The kinetics of MIP-2 production showed a trend for a biphasic pattern, that is, MIP-2 levels became detectable from 2 h postinfection (p.i.) and increased markedly until 8 h p.i. Thereafter, this level fell to the same level until 16 h p.i. and then increased again. TNF α was also detectable at 2 h p.i. and then increased sharply until 8 h p.i., when the peak level attained. Compared with the levels of MIP-2 and TNF α, that of IL-1 α/β, especially IL-1 β, was lower (ng versus pg/ml order). The presence of anti-TNF α or anti-IL-1 α antibody did not influence the early phase of MIP-2 production but significantly inhibited the late phase, suggesting that MIP-2 is induced by the combined effects of RSV infection via direct induction and indirectly after initial induction of TNF α and IL-1 α productions. Although RSV-infected RAW264.7 cells had no alteration inviability compared with mock-infected control, these data demonstrate that RSV is a potent inducer of inflammatory cytokines by direct induction and indirectly via the initial production of other cytokines. J. Med. Virol. 53:145–149, 1997. © 1997 Wiley-Liss, Inc.     

Abortive infection of human diploid cells by murine cytomegalovirus

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 mug/ml) or puromycin (20 mug/ml), but 5'-fluoro-2'-deoxyuridine at 1 x 10(-4)m did not prevent CPE or the development of intranuclear inclusions.     

Characterization of macrophage subsets regulating murine natural killer cell activity

We examined the relationship of I-A expression by normal murine macrophages to their immunoregulatory role on natural killer cell activity. Macrophages were isolated on the basis of plastic adherence; characterized on the basis of conventional markers such as phagocytic ability, cytoplasmic non-specific esterase activity, surface MAC-1 and F4/80 antigen expression; and then used for functional studies relative to their expression of surface I-A. Two functional macrophage subsets were identified: NK-stimulatory and NK-suppressive subsets. The former function was associated with splenic macrophages, which were predominantly I-A+ as identified with a radioautographic immunolabeling technique; the latter function with peritoneal macrophages which were predominantly I-A-. Loss of macrophage I-A expression in vitro was delayed in the presence of indomethacin and enhanced in the presence of PGE2, indicating that PGE2 down-regulates I-A expression on macrophages. The NK stimulatory function of I-A+ macrophages was attributable to a soluble mediator, identified as IFN-gamma, since the stimulatory ability was abrogated with an anti-IFN-gamma antibody. I-A expression appears to be important for the stimulatory function, since some interference with this function was noted in the presence of anti-I-A antibody. The NK-suppressor function of I-A- macrophages was attributable to the soluble mediator PGE2, since this function was abrogated with indomethacin or anti-PGE2 antibody. These results are relevant to the understanding of normal in vivo immunoregulation by macrophages.     

Enhancement of murine alveolar macrophage functions by orally administered beta-glucan.

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF1 mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for 10 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H2O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyer's patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine.     

Interaction of Bartonella henselae with the murine macrophage cell line J774: infection and proinflammatory response.

Bartonella henselae is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with B. henselae display typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of B. henselae with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-gamma) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis N(G)-monomethyl L-arginine. These findings suggest that IFN-gamma-mediated activation of macrophages may be important for the clearing of B. henselae infection and that anti-B. henselae microbicidal activity of IFN-gamma-activated macrophages is mediated to a large extent by NO production.     

Enhancement of hamster alveolar macrophage phagocytic activity by lymphokines

Modulation of phagocytic activity of resident hamster pulmonary alveolar macrophages was accomplished by incubation of the cells in lymphokines prepared by stimulation of hamster splenocytes with concanavalin A or alloantigens in mixed lymphocyte cultures. Alveolar macrophages preincubated in either of these lymphokine preparations possessed significantly greater ability to ingest IgG or IgM plus complement-coated sheep erythrocytes, via their Fc or complement receptors, respectively, than macrophages exposed to control preparations. Ingestion of yeast particles also was enhanced with macrophages incubated in supernatants from cultures of stimulated splenocytes. Supernatant fluids from either mitogen- or alloantigen-stimulated splenocytes possessed migration inhibitory activity with characteristics similar to MIF from other animals; the phagocytosis-enhancing activity shared some of these characteristics.