Linda Eskra

Abstract Interferon (IF) inducers and IF-containing preparations of leukocyte origin have recently been reported to augment natural killer (NK) cell activity. Our studies were undertaken to determine whether highly purified human fibroblast interferon (HFIF), induced by polyinosinic: polycytidylic acid (poly I:C), would augment human NK cell activity against an NK-sensitive leukemia cell line, K562, and, more importantly, to determine whether HFIF would activate normal lymphocytes to lyse human leukemia cells that have not been adapted to tissue culture or established as cell lines. Pretreatment of peripheral blood mononuclear cells for 1 to 16 hr with purified HFIF (150 to 300 units/ml) caused 2.5- to 5-fold increases in cytotoxicity against the K562 cells. Both native and HFIF-augmented cytotoxicities were mediated by FcR+ lymphocytes (lymphocytes expressing receptors for the Fc portion of IgG). Removal of FcR+ lymphocytes, but not monocytes, prevented HFIF from augmenting cytotoxicity, suggesting that HFIF activates preexisting FcR+ NK cells and that monocytes are not required for this effect. Our findings that NK cell activity is augmented by poly I:C-induced fibroblast IF, which lacks lymphokines and viral products present in virus-induced leukocyte IF preparations previously used, together with the fact that our IF preparation was more highly purified than any previously used for such studies, strongly suggest that the IF protein itself augments NK activity. HFIF-treated cells from some individuals lysed many patients' leukemia cells; however, the HFIF-treated cells were not cytotoxic for autologous or allogeneic normal lymphocytes or concanavalin A blasts. FcR+ cells were required for the HFIF-induced cytotoxicity against the patients' leukemia cells. These results thus demonstrate that not only leukemia cell lines, but also human leukemia cells that have not been adapted to tissue culture or established as cell lines, can be lysed by IF-activated human NK cells.

Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection. Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals. Using microarray technology, the response of macrophages 4 h following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages. Of the >6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed. First, an increase in the transcription of a number of proinflammatory cytokines and chemokines, such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-1alpha, and members of the SCY family of proteins, that may constitute a general host recruitment of antibacterial defenses was evident. Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection. Second, transcription of receptors and cytokines associated with antigen presentation, e.g., major histocompatibility complex class II and IL-12p40, were not evident at this 4-h period of infection. Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking. Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways. In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage, permitting intracellular survival of the bacterium. These results provide insights into the pathogenic strategies used by Brucella for long-term survival within a hostile environment.

Purified polyribonucleotide-induced human fibroblast interferon (HFIF) was tested for its effects on proliferative and cytotoxic human T cell responses to alloantigens. The addition of HFIF (100 to 400 IFU/ml) to mixed leukocyte cultures decreased alloantigen-induced lymphocyte proliferative responses as determined by both recovery of responding cells and by 3H-thymidine incorporation into responding cells. However, HFIF, but not the mock interferon preparation, increased the cytotoxic response of T cells to allogeneic cells by 4- to 5-fold when expressed in terms of lytic units. Although fibroblast and leukocyte interferons have different physicochemical and biologic properties, the results reported here are in concert with previous findings concerning the effects of virus-induced leukocyte interferon on human T cell functions.

The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for Brucella. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B. abortus genes. Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively produced gfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K(+) transport gene), and site-specific recombination (xerD gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5' phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of B. abortus genes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.