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The interleukin-3 family of cytokines, which play an important role in the development of myeloid lineages, transduce signals through the JAK-STAT pathway. Previous studies demonstrate that this process entails the activation of four distinct isoforms of STAT5, where two shorter isoforms are activated in a distinct population of cells. We now demonstrate that the shorter isoforms represent carboxy-terminal truncations. Moreover, these truncations are not generated by RNA processing, but by a specific proteolytic activity. Consistent with the notion that truncated STAT5 isoforms transduce distinct signals, they fail to promote the activation of several known interleukin-3 target genes. These studies suggest that the activity of a specific protease may play a critical role in defining the biological responses transduced by STAT5.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

We have isolated DNA clones containing the interferon (IFN)-inducible guanylate-binding protein-1-encoding gene (GBP1) from a human genomic library. Two overlapping phage clones contained the entire GBP1 gene. The 2880 bp corresponding to the GBP1 cDNA were subdivided into eleven exons which were interrupted by a total of 9500 bp of intron DNA. All exon/intron junctions contained consensus splice donor and acceptor sequences. Using hybrid rodent cell lines containing human chromosomes, GBP1 was mapped to human chromosome 1. In addition to GBP1, we detected and partially characterized a novel gene, GBP3, with a structure related to GBP1 and a high degree of sequence homology to both GBP1 and GBP2. Our data suggest that the GBP family of genes contains members other than the previously characterized GBP1 and GBP2.

The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE.