IFN-gamma is a potent macrophage activator and induces a number of early and delayed genes. crg-2, the presumed murine homologue of human IP-10, belongs to a family of proinflammatory chemokines and is induced as an immediate early gene in response to IFN-gamma in macrophages. In contrast, class II MHC or Ia genes which are essential for Ag presentation are induced as a delayed response to IFN-gamma. We studied the expression of crg-2 and compared it with Ia in astrocytes and microglia of the central nervous system since, like macrophages, these cells can also produce a number of cytokines, express Ia molecules, and present Ag. We showed that crg-2 mRNA was induced in astrocytes and microglia by IFN-gamma as well as a paramyxovirus, Newcastle disease virus (NDV). Crg-2 protein was detected in the cytoplasm and in the supernatants of IFN-gamma-treated astrocytes and microglia. IFN-gamma and NDV or UV irradiated-NDV (UV-NDV) also induced Ia mRNA in these cells. The kinetics of expression of crg-2 and Ia mRNA were compared in the same systems. While crg-2 mRNA appeared within 2 h and reached a maximum in 6 to 8 h, Ia mRNA was not detected before 8 h. Cycloheximide superinduced crg-2 mRNA induced by IFN-gamma or UV-NDV but it abolished Ia mRNA induction by the same stimuli. The induction of crg-2 in astrocytes and microglia likely contributes to the development of immune-mediated inflammation in response to viruses or in autoimmune diseases of the central nervous system.

Characterization of lymphokine-activated genes in mouse macrophages led to the identification previously of Mumig, an interferon-γ-inducible murine gene that encodes a member of the chemokine family of cytokines. The Mumig cDNA probe was used to screen a cDNA library prepared from cultures of the THP-1 human monocytic cell line that had been treated with interferon-γ. This led to the identification of Humig, a new human member of the chemokine gene family. Humig is induced in THP-1 cells and in peripheral blood mononuclear cells by interferon-γ but not by interferon-α or by lipopolysaccharide. Analysis of mouse and human genomic DNAs suggested that the Mumig and Humig genes are true mouse-human homologues. The Humig mRNA encodes a predicted secreted HuMig protein of 103 residues, Mr 11,725.

To identify genes induced during macrophage activation, a cDNA library was prepared from cultures of the RAW 264.7 mouse macrophage cell line that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the cDNA library was screened by differential plaque hybridization. Eleven cDNA clones, designated CRG-1 through CRG-11, corresponding to mRNA species inducible in RAW 264.7 cells by the spleen cell conditioned medium, were isolated. Inductions were not blocked by cycloheximide. All of the mRNAs were inducible by gamma interferon, and some were also inducible by alpha and beta interferons, by lipopolysaccharide, by phorbol 12-myristate 13-acetate, and by the calcium ionophore A23187. Sequencing of the cDNAs revealed that CRG-1, CRG-3, and CRG-5 are cDNAs of recently identified transcription factors IRF-1, zif/268, and LRF-1 respectively. As previously reported, CRG-2 and CRG-10 (MIG) encode new members of the platelet factor 4 family of cytokines. CRG-6 corresponds to a new member of a family of interferon-inducible genes clustered on mouse chromosome 1, CRG-9 corresponds to a prostaglandin synthase homolog, CRG-8 corresponds to beta 2-microglobulin, and CRG-4 corresponds to metallothionein II. CRG-11 contains sequences of a truncated L1Md repetitive element as well as nonrepetitive sequences. The nonrepetitive sequence of CRG-11 as well as the sequences of CRG-7 are not closely related to published sequences. The CRG genes and proteins are of interest because of their involvement in macrophage activation, because of their roles as mediators of the effects of gamma interferon and other pleiotropic agents, and because of their usefulness as tools for studying the signal pathways through which gamma interferon and other inducers exert their effects on gene and protein expression.

A newly described member of the platelet factor 4 family of cytokine genes, mig, is selectively induced by interferon gamma-(IFN-gamma), and not IFN-alpha, in the mouse macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with IFN-gamma activated mig gene transcription as determined by nuclear run-on assays. mig genomic clones were isolated, and constructs containing genomic fragments that included the mig promoter region and the CAT reporter gene were prepared. In RAW 264.7 cells transfected with these constructs, CAT activity was found to be selectively induced by IFN-gamma. A 278-bp genomic fragment containing 235 nucleotides 5' of the transcription start site was sufficient for IFN-gamma-selective induction of CAT activity. Analysis of 5' deletion mutants localized a region essential for activation by IFN-gamma to within 64 nucleotides extending from -235 to -172. A genomic fragment containing this sequence was capable of conferring IFN-gamma inducibility to constructs with a heterologous promoter.

In order to identify novel mediators synthesized in activated macrophages, a cDNA library was prepared from cultures of the mouse macrophage cell line RAW 264.7 that had been treated with lymphokine-rich conditioned medium from mitogen-stimulated mouse spleen cells. Differential plaque hybridization identified a cDNA, designated m119, that detected a 1.6-kilobase mRNA that accumulated in response to gamma-interferon (IFN-gamma) but not in response to other macrophage activators, including IFN-alpha, IFN-beta, and lipopolysaccharide. The mRNA encoded a predicted protein of Mr 14,461 containing a 21-amino acid signal peptide. The primary structure of the predicted protein indicated that it is a member of a recently described family of cytokines related to platelet factor 4, including Gro/melanoma growth stimulatory activity and neutrophil-activating peptide/interleukin 8. The selective induction of the m119 mRNA by IFN-gamma that the predicted m119 protein mediates a macrophage activity regulated by IFN-gamma. The m119 protein may be a cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory responses. It is proposed that the gene encoding this protein be called mig, for monokine induced by gamma interferon.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.