Michael J. Parmely

Francisella tularensis Induces Extensive Caspase-3 Activation and Apoptotic Cell Death in the Tissues of Infected Mice

ABSTRACT Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80+ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.

Programmed cell death and the pathogenesis of tissue injury induced by type A Francisella tularensis.

Francisella tularensis is a highly virulent bacterial species that causes various forms of tularemia in humans. The urgency in understanding the pathogenesis of these diseases has stimulated unprecedented interest in this bacterial species over the past few years. Recent findings underscore a number of important distinctions between the Francisella ssp. and emphasize the importance of using type A F. tularensis strains when characterizing pathophysiological responses that are relevant to the lethal forms of human disease. This review focuses on the mediators of cell death induction in infected tissues and the implications of these processes on the pathophysiological changes observed in various host species.

NK Cells and Gamma Interferon Coordinate the Formation and Function of Hepatic Granulomas in Mice Infected with the Francisella tularensis Live Vaccine Strain

ABSTRACT Host innate immune responses to many intracellular pathogens include the formation of inflammatory granulomas that are thought to provide a physical barrier between the microbe and host. Because two common features of infections with the live vaccine strain (LVS) of Francisella tularensis within the mouse liver are the formation of granulomas and the production of gamma interferon (IFN-γ), we have asked what role IFN-γ plays in hepatic granuloma formation and function. Francisella antigens were predominantly localized within granulomas of the livers of mice infected with F. tularensis LVS 4 days postinfection. Hepatic granulomas also contained large numbers of dying cells, some of which coexpressed the F4/80 macrophage antigen and activated caspase-3. IFN-γ-deficient mice did not form normal numbers of hepatic granulomas and showed widely disseminated Francisella antigens within the liver. The incidence of cell death within hepatic granulomas also decreased significantly in the absence of IFN-γ. Inducible NO synthase (iNOS) expression was restricted to the granulomas of wild-type mice but was not seen for IFN-γ-deficient mice. Cell death within granulomas was also significantly decreased for iNOS-deficient mice. The predominant IFN-γ-expressing cells in the liver were NK cells. Depleting NK cells resulted in the expression of bacterial antigens and iNOS outside the granulomas and the appearance of extensive hepatic focal necrosis. These findings indicate that IFN-γ and hepatic NK cells that are activated during F. tularensis LVS infections regulate hepatic granuloma formation, the spatial containment of infection, the expression of iNOS, and the induction of cell death within the liver.

Toll-like receptor 2 controls the gamma interferon response to Francisella tularensis by mouse liver lymphocytes

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-γ-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon‐γ (IFN‐γ)‐induced priming of macrophages (mfs) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal mφs from Lewis (LEW/N) rats for 18 h with recombinant rat IFN‐γ (rRaIFN‐γ) in the presence of either adenosine (100 μM) or MDL 201,112 (10 μM) resulted in a fourfold decrease in superoxide anion (02 ‐) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN‐γ treatment. Peritoneal mφs from LEW/N rats stimulated with LPS/rRaIFN‐γ were observed to secrete immunoreactive and bioactive TNF‐α over 18 h in vitro and this cytokine could be dose‐dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of 02 ‐ release mediated by MDL 201,112. Furthermore, incubation of LEW/N mφs for 18 h in vitro with rRaIFN‐γ resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 μM) or adenosine (100 μM). MDL 201,112 and adenosine were also effective in decreasing mφ opsonized zymosan‐stimulated 02 ‐ levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down‐regulation of heat‐killed M. tuberculosis‐activated LEW/N mφ MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat mf activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases. J. Leukoc. Biol. 56: 133–144; 1994.

Coactivating Signals for the Hepatic Lymphocyte Gamma Interferon Response to Francisella tularensis

ABSTRACT The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-γ), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-γ when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

CELLULAR DETERMINANTS OF MAMMARY CELL-MEDIATED IMMUNITY IN THE RAT: KINETICS OF LYMPHOCYTE SUBSET ACCUMULATION IN THE RAT MAMMARY GLAND DURING PREGNANCY AND LACTATION*

The present study was undertaken to determine the pattern of T- and B-lymphocyte accumulation in the rat mammary gland during gestation, lactation, and weaning. Adoptive transfer experiments were then used to identify the likely source and extent of immigrating cells during this period. Our results indicate that the mammary gland accumulates levels of cIgA+, W3/13+, and W3/25+ cells that are approximately one third their densities in the small intestine. Cell migration studies suggest that the input of immigrating B-cells is not sufficient to maintain these levels in the gland, and we have suggested that substantial clonal expansion must occur among cIgA+ B-cells within the mammary tissue itself. The migratory behavior of T-cells, their pattern of appearance throughout gestation and lactation, and the predominance of the T-helper cell subset suggest that T-cells regulate B-cell development within the mammary gland.

Gamma Interferon Prevents the Inhibitory Effects of Oxidative Stress on Host Responses to Escherichia coli Infection

ABSTRACT Oxidative stress occurs in animals challenged with bacterial endotoxin and can affect the expression of important host inflammatory genes. However, much less is known about the effects of oxidative stress on responses to gram-negative bacteria. The current study compared the effects of redox imbalance on hepatic responses of mice to Escherichia coli bacteria versus purified endotoxic lipopolysaccharide (LPS). Oxidative stress induced by glutathione depletion virtually eliminated hepatic tumor necrosis factor alpha responses to both E. coli and LPS. Inducible NO synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) expression was also markedly inhibited by glutathione depletion in LPS-challenged mice, but was unaffected in E. coli-infected animals. Three findings suggested that gamma interferon (IFN-γ) production explained the differences between LPS and bacterial challenge. Glutathione depletion completely inhibited the IFN-γ response to LPS, but only partially inhibited IFN-γ production in infected mice. Exogenous IFN-γ restored iNOS and ICAM-1 responses to LPS in stressed mice. Conversely, IFN-γ-deficient, glutathione-depleted mice showed a marked decrease in iNOS and ICAM-1 expression when challenged with E. coli. These findings indicate that both the nature of the microbial challenge and the production of IFN-γ can be important in determining the effects of redox imbalance during gram-negative bacterial infections.

Inhibition of nuclear factor-κB activation in mouse macrophages and the RAW 264.7 cell line by a synthetic adenyl carbocyclic nucleoside

Adenyl carbocyclic nucleosides have potent anti-inflammatory effects on a number of cell types. Notable in this regard is their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) by mouse macrophages that have been activated with bacterial lipopolysaccharide (LPS). Because the transcriptional activation of the mouse TNF-alpha gene is highly dependent on kappaB enhancers, the present study determined whether the synthetic carbocyclic nucleoside 9-[(1S,3R)-cis-cyclopentan-3-ol]adenine (cPA) inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in these cells. Stimulation of either mouse peritoneal macrophages or RAW 264. 7 macrophage-like cells with LPS led to the appearance of four distinct kappaB-binding nucleoprotein complexes detected by gel mobility shift assays. Cells treated with 100 microM cPA showed significantly reduced levels of NF-kappaB activation as evidenced by measurements of nucleoprotein kappaB-binding activity and diminished kappaB-dependent transcriptional activation. However, both the LPS-induced degradation of the cytoplasmic NF-kappaB inhibitor IkappaBalpha and the nuclear translocation of the NF-kappaB p50, p65, and c-Rel peptides were unaffected by treatment of the cells with the nucleoside. These findings suggest that certain adenyl carbocyclic nucleosides inhibit the activation of NF-kappaB/Rel complexes by a novel mechanism that results in an inhibition of their DNA-binding activities, without blocking their dissociation from IkappaBalpha or their nuclear translocation.

Murine interferon-γ enhances resistance to infection with strains of pseudomonas aeruginosa in mice

This study focused on the role of recombinant murine interferon-gamma (rMuIFN-gamma) in modulating resistance to infections produced by Pseudomonas aeruginosa in mice. CBA/C mice were treated with rMuIFN-gamma, exposed to thermal injury, and challenged with P. aeruginosa organisms. Survival of infected, burned mice was determined over a period of 14 days and compared with control animals not receiving rMuIFN-gamma. Two clinical isolates of P. aeruginosa that differed originally in the ability to produce metalloproteases, were used to infect the mice. When the mice were pretreated with rMuIFN-gamma before the infection, no statistically significant differences in survival were observed with either of the two strains of bacteria compared to controls. However, when mice were pretreated and injected therapeutically with rMuIFN-gamma (continued receipt of rMuIFN-gamma after infection), there was a significant protective effect in animals infected with one of the strains of P. aeruginosa. We conclude that rMuIFN-gamma may be useful in treating infections with certain strains of P. aeruginosa in mice.