Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Detecçäo do vírus da hepatite murina utilizando-se a reaçäo em cadeia pela polimerase / Detection of mouse hepatitis virus in mouse colonies using the nested polymerase chain reaction

Desenvolveu-se a técnica de transcriçäo reversa - reaçäo em cadeia pela polimerase para detectar o vírus da hepatite do camundongo em tecido hepático. Para se eliminar possíveis falhas na reaçäo de amplificaçäo RT-PCR usou-se um segundo ciclo de amplificaçäo com primers internos para confirmar a especificidade e aumentar a sensibilidade do teste. Após a optimizaçäo do protocolo, descreve-se a detecçäo da amplificaçäo específica da seqüência-alvo em camundongos de 18 das 20 colônias examinadas

Antiviral activity of interferon gamma in vivo during mouse hepatitis virus infection

A/J mice became resistant to experimental MHV3 infection after immunization with UV-inactivated MHV3 (0 percent mortality, 0/10). Depletion of interferon (IFN) gamma-producing CD4+ T lymphocytes with monoclonal antibodies to CD4+ led to susceptibility to virus infection (60 percent of mortality, 6/10). The resistance to MHV3 infection of CD4+ T lymphocyte-depleted-A/J mice was restored by treatment with 1000 U of IFN gamma on days -1, 0, 1, 2, 3 and 4 (10 percent of mortality, 1/10). The low virus titers observed in resistant mice (controls or CD4+ depleted plus IFN gamma treated) were cleared 6 days after infection and the virus titers observed among susceptible mice (CD4+ depleted) increased gradually and peaked on day 6, when the animals died. Previous data, taken together with the direct evidence presented in this paper, provide strong evidence supporting the concept of an in vivo antiviral role of IFN gamma through a central action on the mechanisms of resistance to MHV3 infection

Reduction of the accumulation of viral mRNA and of cellular rRNA synthesis in MHV3-infected macrophages of resistant mice

1. After MHV3 infection, only macrophages from resistant A/J mice partially restricted virus growth compared to those from susceptible BALB/c mice (2 logs of difference in virus titer). 2. Cellular ribosomal ribonucleic acid (rRNA) synthesis by MHV3-infected macrophages was decreased only in A/J mouse macrophages as indicated by accumulation of the 28S rRNA fraction. 3. The accumulation of viral messenger ribonucleic acids (mRNAs) in MHV3-infected macrophages was also reduced in A/J mouse macrophages compared to BALB/c mice. 4. In pulse-chase experiments of viral protein synthesis, the appearance, glycosylation and cleavage of glycoprotein S, as well as the metabolism of nucleoprotein N were delayed in A/J mouse macrophages. 5. These data show that MHV3 infection of A/J mouse macrophages induced an imbalanced accumulation of the 28S fraction of rRNA. Furthermore the synthesis of mRNAs correlated with viral protein synthesis in both A/J and BALB/c macrophages, but was delayed in A/J mice. 6. These results suggest that the partial restriction of MHV3 replication in macrophages of resistant A/J mice may take place during or before the mRNA synthesis, although it is correlated with the appearance, glycosylation, cleavage and metabolism of viral proteins

Mouse hepatitis virus 3 and interferon gamma binding to extracted macrophage proteins correlate with virus growth in A/J and BALB/c mice

1. After immunization, adult A/J mice are resistant and BALB/c mice are susceptible to MHV3 infection. After IFN gamma activation, only macrophages originating from A/J mice were able to partially restrict MHV3 growth. 2. When the binding of MHV3 and interferon (IFN) gamma to solubilized cytoplasmic and membrane macrophage proteins of mice was determined by ELISA, there was more binding of MHV3 to proteins extracted from BALB/c macrophages than to proteins extracted from A/J macrophages. When the proteins were obtained from IFN gamma-activated macrophages, decreased MHV3 binding was observed only in proteins originating from A/J macrophages. 3. ELISA showed a comparable binding of IFN gamma to A/J or BALB/c macrophage proteins. When the proteins were obtained from IFN gamma-activated macrophages, only IFN gamma-binding to A/J macrophage proteins was increased. 4. The results indicate a different expression and IFN gamma modulation of MHV3 receptors in macrophages from A/J and BALB/c mice, which directly correlated with their acquired resistance or susceptibility to MHV3 infection

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response