Induction of IFN-gamma in macrophages by lipopolysaccharide.

In this paper we report that macrophages can be stimulated to express detectable levels of IFN-gamma-specific mRNA. Macrophages from lipopolysaccharide (LPS)-responsive, C3H/OuJ mice are induced by LPS to increase steady-state levels of IFN-gamma-specific mRNA, while those from LPS-hyporesponsive C3H/HeJ mice are not. This interstrain variation is apparently the result of LPS-specific signal differences since macrophages derived from both Lpsn and Lpsd mouse strains are able to produce comparable levels of IFN-gamma-specific mRNA following stimulation with polyinosinic-polycytidylic acid. The identity of the cell type responsible for this IFN-gamma message appears to be the macrophage as IFN-gamma-specific mRNA was also detectable following T and natural killer cell depletion, in the LPS-stimulated RAW 264.7 cell line, and in a homogeneous population of mature macrophages propagated in vitro by stimulation of bone marrow progenitors with recombinant colony stimulating factor-1. Immunofluorescent staining of fixed and permeabilized LPS-stimulated macrophages confirmed the presence of immunoreactive IFN-gamma protein. The possible significance of IFN-gamma production by macrophages is discussed in the context of normal macrophage differentiation as well as the inflammatory immune response.

Selective alteration of the turnover of interferon beta mRNA in peritoneal macrophages from LPS-hyporesponsive mice and its role in the defective expression of spontaneous interferon.

Basal levels of interferon (IFN)-beta mRNA transcription were detected in both freshly explanted LPS-responsive (Lpsn) and LPS-hyporesponsive (Lpsd) peritoneal macrophages (PM). In vitro cultivation of PM resulted in a time-dependent reduction in the level of IFN-beta mRNA, which was far more rapid in Lpsd than in Lpsn PM. Treatment of Lpsn PM with cycloheximide (CHX) resulted in a marked accumulation of IFN-beta mRNA, which was not associated with an increase in IFN-beta gene transcription. However, treatment of Lpsd PM with CHX did not induce accumulation of IFN-beta mRNA. CHX induced the accumulation of IFN-alpha-4 mRNA in both Lpsn and Lpsd PM, CHX enhanced the accumulation of two cytoplasmic factors interacting with AU-rich sequences within the 3' untranslated region of IFN-beta mRNA. We conclude that Lpsd PM exhibit an impaired capacity to stabilize IFN-beta mRNA that may account for their low expression of IFN-beta.

Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor.

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59.

Mouse hepatitis virus-A59 (MHV-A59), a murine coronavirus, can utilize as a cellular receptor MHVR, a murine glycoprotein in the biliary glycoprotein (BGP) subfamily of the carcinoembryonic antigen (CEA) family in the immunoglobulin superfamily (G.S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991). Several different BGP isoforms are expressed in tissues of different mouse strains, and we have explored which of these glycoproteins can serve as functional receptors for MHV-A59. cDNA cloning, RNA-mediated polymerase chain reaction analysis, and Western immunoblotting with a monoclonal antibody, CC1, specific for the N-terminal domain of MHVR showed that the inbred mouse strains BALB/c, C3H, and C57BL/6 expressed transcripts and proteins of the MHVR isoform and/or its splice variants but not the mmCGM2 isoform. In contrast, adult SJL/J mice, which are resistant to infection with MHV-A59, express transcripts and proteins only of the mmCGM2-related isoforms, not MHVR. These data are compatible with the hypothesis that the MHVR and mmCGM2 glycoproteins may be encoded by different alleles of the same gene. We studied binding of anti-MHVR antibodies or MHV-A59 virions to proteins encoded by transcripts of MHVR and mmCGM2 and two splice variants of MHVR, one containing two immunoglobulin-like domains [MHVR(2d)] and the other with four domains as in MHVR but with a longer cytoplasmic domain [MHVR(4d)L]. We found that the three isoforms tested could serve as functional receptors for MHV-A59, although only isoforms that include the N-terminal domain of MHVR were recognized by monoclonal antibody CC1 in immunoblots or by MHV-A59 virions in virus overlay protein blot assays. Thus, in addition to MHVR, both the two-domain isoforms, mmCGM2 and MHVR(2d), and the MHVR(4d)L isoform served as functional virus receptors for MHV-A59. This is the first report of multiple related glycoprotein isoforms that can serve as functional receptors for a single enveloped virus.

Hantaan virus infection of human endothelial cells.

The primary pathophysiologic finding of the viral disease known as Korean hemorrhagic fever, the etiological agent of which is Hantaan virus (HTV), is vascular instability. To investigate whether HTV was able to infect cells derived from human vascular tissue and alter their behavior, we infected in vitro primary adult human endothelial cells from saphenous veins (HSVEC). We were able to detect the presence of viral antigens in infected cells both by immunofluorescence and by Western blot (immunoblot) analysis as early as day 1 postinfection. HSVEC infected with HTV produce infectious virus during the first 3 days of infection but, at later times (days 4 to 8), show decreasing yields of virus. This contrasts with the HTV growth pattern observed for the permissive simian CV-7 cell line, which generates infectious virus up to day 12 after infection. Further investigation showed that the late decrease in viral production in HSVEC is the result of the induction of beta interferon and can be reversed by the addition of anti-beta interferon serum to the culture medium. At no time during the course of infection of HSVEC with HTV was any obvious cytopathic effect observed. When tests for changes in mRNA levels of other cytokines and endothelial cell gene products following HTV infection of HSVEC were done by reverse transcription and polymerase chain reaction methods, no significant changes were observed in the levels of interleukin 1, interleukin 6, or von Willebrand factor mRNA. We hypothesize that, while HTV can replicate in human vascular endothelial cells, the mechanism of microvascular damage seen with Korean hemorrhagic fever is not likely to be a direct effect of virus replication but may conceivably be the consequence of an immune-mediated endothelial injury triggered by viral infection.

Binding of the coronavirus mouse hepatitis virus A59 to its receptor expressed from a recombinant vaccinia virus depends on posttranslational processing of the receptor glycoprotein.

Recently, we showed that a murine member of the carcinoembryonic antigen family of glycoproteins serves as a cellular receptor (MHVR) for the coronavirus mouse hepatitis virus A59 (MHV-A59) (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; R. K. Williams, G.-S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). To examine the role of posttranscriptional modification of MHVR on virus-receptor interactions, a vaccinia virus-based expression system was employed. Expression from the vaccinia virus recombinant (Vac-MHVR) in BHK-21 cells resulted in high levels of MHVR glycoprotein on the cell surface and made these cells susceptible to MHV-A59 infection. Nonglycosylated core MHVR proteins were made in Vac-MHVR-infected BHK-21 cells in the presence of tunicamycin by in vitro translation of MHVR mRNA in a rabbit reticulocyte cell-free system in the absence of microsomal membranes and by expression of an N-terminal deletion clone of MHVR lacking its signal peptide. These three nonglycosylated MHVR proteins were recognized by polyclonal antibody against affinity-purified receptor but did not bind antireceptor monoclonal antibody (MAb) CC1 or MHV-A59 virions. Partial glycosylation of MHVR, either expressed in Vac-MHVR-infected cells treated with monensin or synthesized by in vitro translation with microsomal membranes, restored both the MAb CC1- and the virus-binding activities of the MHVR glycoprotein. Deletion of 26 amino acids at the carboxyl terminus of MHVR resulted in a secreted protein which was able to bind MAb CC1 and MHV-A59. These results suggest that either a carbohydrate moiety is an element of the MHVR-binding site(s) for virus and MAb CC1 or a posttranslational membrane-associated process is required for functional conformation of the receptor glycoprotein.

Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV.

The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59.

Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody.

Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.

Effects of aspirin, dipyridamole, and dibutyryl cyclic adenosine monophosphate on platelet-derived growth factor A chain mRNA levels in human saphenous vein endothelial cells and smooth muscle cells.

Aspirin and dipyridamole have frequently failed to control intimal hyperplasia in vascular grafts in animal and clinical trials. These trials were based on the concept that the smooth muscle mitogen, platelet-derived growth factor (PDGF) released from platelets, was a major cause of intimal hyperplasia. Both endothelial and smooth muscle cells (ECs and SMCs), however, can also release PDGF-like SMC mitogens that might cause intimal hyperplasia. We therefore tested whether aspirin and dipyridamole alone or together can affect PDGF-A chain mRNA levels in cultured human saphenous vein ECs and SMCs. Cultures were exposed for 72 hours to 3 x 10(-5) mol/L aspirin and/or 5 x 10(-6) mol/L dipyridamole. Cellular RNA was then extracted, and PDGF-A chain mRNA signal levels were measured by a reverse transcription/polymerase chain reaction method. mRNA for glyceraldehyde-3-phosphate dehydrogenase was used as a constitutively expressed control RNA species. Signal strength on Southern blots of amplified polymerase chain reaction products was measured by densitometry. Neither aspirin nor dipyridamole alone or together reduced the ratio (PDGF-A chain signal/glyceraldehyde-3-phosphate dehydrogenase signal) below that of control cultures. PDGF-A chain expression was not a constitutive artifact of culture because dibutyryl cyclic AMP (5 x 10(-4) mol/L) reduced PDGF-A chain signal from a control index of 1.0 to 0.5 +/- 0.1 (mean +/- SE) (n = 3; p less than 0.05) in EC cultures and to 0.2 (mean) (n = 2) in SMC cultures. These data may explain why aspirin and dipyridamole fail to reduce intimal hyperplasia in some animal and clinical trials despite effective inhibition of platelet aggregation.

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells.

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of toxic viral-associated double-stranded RNA (dsRNA) in influenza-infected lung.

While many of the molecular events in viral replication are well studied, the molecular mechanisms by which viral infections trigger such constitutional symptoms as fever and 'malaise' are unknown. The hypothesis that these viral constitutional symptoms can be triggered by the toxic action of dsRNA associated with viral replication was investigated. Total lung RNA from mice acutely infected with PR8 influenza virus, but not from sham-infected mice, was shown to induce fever and altered sleep (excess slow-wave sleep, enhanced amplitudes of electroencephalographic slow waves, and reduced rapid eye movement sleep) when injected into the rabbit brain. Viral-associated dsRNA was shown to be responsible for the rabbit responses by differential nuclease digestion. Influenza viral dsRNA was directly demonstrated in the active lung RNA preparations by reverse transcriptase-polymerase chain reaction techniques. The time course of the responses paralleled those seen in the same model inoculated with nanogram quantities of the synthetic dsRNA polyriboinosinic-polyribocytidylic acid and suggested that they were mediated by induced cytokines. A model for the role of viral-associated dsRNA in eliciting both local cytotoxicity and viral constitutional symptoms is presented.

2',5'-Oligoadenylate synthetase gene expression in revertants of ras-transformed NIH3T3 fibroblasts.

Persistent revertant (PR) cells of Ha-ras-transformed NIH3T3 fibroblasts, isolated after prolonged treatment with interferon (IFN), have been previously described. PR cells remain nontumorigenic even after IFN withdrawal. To investigate the mechanisms responsible for the stable phenotypic reversion, we have now examined the potential involvement of an endogenous IFN and the 2',5'-oligoadenylate (2-5A) synthetase pathway. Northern blot analysis revealed an increased level of 2-5A synthetase transcripts in PR cells compared to parental Ha-ras-transformed cultures. Although inducible on treatment with exogenous IFN alpha/beta, this mRNA was not detectable in untreated NIH3T3 cells. 2-5A synthetase expression following IFN treatment was also significantly higher in PRs than in the normal or ras-transformed NIH3T3. The increased levels of synthetase mRNA correlated with a similarly elevated enzymatic activity in cell extracts from PR cells. This increased expression was biologically functional, since the revertant cells were more resistant to the cytolytic action of mengovirus than normal or ras-transformed NIH3T3 fibroblasts. Another class of IFN-induced genes, H-2 class I antigens, showed enhanced expression in PRs. Antibodies directed against mouse IFN alpha/beta did not reduce the constitutive expression of 2-5A synthetase in PR cells. Furthermore, conditioned medium from PR cultures or cocultivation with PRs failed to induce the enzyme message in NIH3T3 cells. Finally, there was no detectable elevation in the mRNA specific for IFN beta in the PR cultures, as determined using a sensitive polymerase chain reaction amplification protocol. These results show that the Ha-ras revertants constitutively produce a functional 2-5A synthetase, which appears to be independent of the production of an endogenous interferon alpha or beta.

Regulation of HIV replication in infected monocytes by IFN-alpha. Mechanisms for viral restriction.

In a survey of 15 different virus isolates, no IFN-alpha or IFN-beta activity was detected in culture fluids of HIV-infected T cells or monocytes. Exogenous rIFN-alpha added to T lymphoblast or monocyte cultures induced restriction in replication of the amphotropic HIV that infect both cell types. With IFN-treated HIV-infected T cells, levels of reverse transcriptase (RT) activity in culture fluids were half those in control cultures, but the frequency of infected cells or the levels of p24 Ag released in culture fluids were unchanged. In contrast to the modest effect of IFN on HIV-infected T cells, IFN-induced antiviral activity in monocytes was quite dramatic. Monocytes treated with IFN at the time of virus challenge showed no evidence of HIV infection: no p24 Ag or RT activity, no viral mRNA, and no proviral DNA. In this system, IFN interrupts one or more early event(s) in the virus replication cycle before formation of proviral DNA. Monocyte cultures infected with HIV 7 days before IFN treatment showed a gradual decrease in levels of p24 Ag and RT activity to baseline by 3 wk. HIV-induced cytopathic changes were markedly reduced, and the frequency of productively infected cells was less than or equal to 1% of total cells. Virus particles released 24 h after IFN treatment were 100- to 1000-fold less infectious than equal numbers of control virions. But, monocytes treated with IFN 7 days after HIV infection were not free of the retroviral pathogen: levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.

Induction of interferon-beta and 2',5'-oligoadenylate synthetase mRNAs by interleukin 6 during differentiation of murine myeloid cells.

Interleukin 6 (IL 6) induces differentiation of murine myelomonocytic leukemia (M1) cells into mature macrophages. This process is monitored by the sequential appearance of surface markers, induction of intracellular enzymes, and changes in morphology as the cells progress from blast cells to mature macrophages. Differentiation is also associated with growth arrest and accumulation of the differentiating cells in the G0/G1 phase of the cell cycle. Interferon-beta (IFN-beta) is known to be involved in the growth arrest of M1 cells by inducing 2'5'-oligoadenylate synthetase (2',5'-AS). We therefore analyzed whether IL 6 has the potential to trigger the full differentiation program directly or whether its effect on M1 cells is mediated through IFN-beta or through the activation of genes that are typically induced by IFN-beta. We first tested whether IL 6 could induce IFN-beta mRNA. Using a reverse transcription/polymerase chain reaction procedure, we found that IFN-beta mRNA was induced by IL 6. By Northern analysis we determined that IL 6 also caused a significant increase in 2',5'-AS gene expression. IL 6, however, induced the expression of two mRNA species (1.7 and 2.4 kb), whereas IFN-beta mainly induced the expression of the 1.7-kb species. Enhancement of 2',5'-AS gene expression by IL 6 was observed even when protein synthesis was inhibited by cycloheximide. Furthermore, IL 6-induced growth arrest of M1 cells was not inhibited by anti-IFN-beta antibodies. Thus induction of 2',5'-AS gene expression is a primary response to IL-6 and not secondary to the induction of IFN-beta.