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.

Tularemia is a zoonotic infectious disease caused by the facultative intracellular bacterium Francisella tularensis. The initial clinical presentation and dominant features of this condition reflect the mode of transmission and the route of infection. The ulceroglandular form, most frequently acquired from bites of arthropods, is characterized by an initial papular skin lesion that can progress to an eschar or ulcer with regional lymphadenopathy. Inhalation of aerosolized bacteria or secondary hematogenous dissemination of infection to the lungs results in pneumonic tularemia characterized by fever, a broad range in the severity of respiratory symptoms, and a high mortality rate. In the so-called "typhoidal" form of tularemia, patients present with similar systemic symptoms without a clear focus of primary infection. They may show sepsis and rhabdomyolysis with multiorgan system failure, including renal impairment. Following pulmonary and gastrointestinal infection, systemic dissemination of the pathogen leads to the colonization of the liver, where organisms infect both macrophages and hepatocytes (4, 6). At autopsy, the livers of infected human beings often show areas of focal coagulation necrosis throughout the parenchyma of the organ (18).The infection of mice with the live vaccine strain (LVS) of F. tularensis subsp. holarctica is a commonly used animal model and is associated with the formation of multifocal hepatic microgranluomas containing CD11b ϩ macrophages and Gr-1 ϩ

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Human milk lymphocytes (ML) can be partially purified and propagated in vitro as a means of assessing their immunological function. When exposed to a variety of stimuli known to activate T lymphocytes, ML respond in a unique manner that indicates a selected population of immunocompetent cells. ML are hyporesponsive to to nonspecific mitogens and respond in a reduced manner to histocompatibility antigens on allogeneic cells. In most cases, they are completely unresponsive to C. albicans although blood lymphocytes from the same patients respond to the antigen. The Kl capsular antigen of E. coli induces significant proliferation in lymphocytes obtained from milk, but fails to stimulate blood lymphocytes. This dichotomy of reactivity does not appear to result from suppressive factors or cells in milk or insufficient adherent cell function. Rather it appears to reflect the accumulation of particular lymphocyte clones in the breast and the local nature of mammary tissue immunity at the T-lymphocyte level.

Pseudomonas aeruginosa alkaline protease and elastase are thought to contribute to bacterial invasiveness, tissue damage, and immune suppression in animals and patients infected with the bacterium. This study examined the ability of the two proteases to inactivate a number of cytokines that mediate immune and inflammatory responses. Human recombinant gamma interferon (rIFN-y) and human recombinant tumor necrosis factor alpha were inactivated by both proteases. Murine rIFN-,y was relatively resistant to alkaline protease but was inactivated by elastase, and human recombinant interleukin-la and recombinant interleukin-lo were resistant to the effects of both proteases. Western immunoblots suggested that cytokine inactivation by these proteases, where it occurred, required only limited proteolysis of the polypeptides. The ability of different P. aeruginosa strains to inactivate IFN--y appeared to require the production of both proteases for optimum activity. These results indicate that in vitro cytokine inactivation by Pseudomonas proteases is selective, requires only limited proteolysis, and in certain instances reflects the cooperative effects of both proteases.Pseudomonas aeruginosa is an important pulmonary pathogen in conditions like cystic fibrosis (15,42). It has been suggested that the ability of P. aeruginosa to establish itself in the respiratory tract may be promoted by its suppressive effects on pulmonary immune responses (2,16,40). The mechanisms of this immunosuppression are not entirely understood, but proteolytic enzymes secreted by the bacterium have been shown to degrade surface receptors on hematogenous cells (36, 43) and inactivate interleukin-2 (IL-2) and gamma interferon (IFN--y) (16,17,41).In the case of human IFN--y, cytokine inactivation was caused by either Pseudomonas alkaline protease (AP) (17) or elastase (E) (16). Significant reductions in antiviral and immunomodulatory activities were associated with limited proteolysis of IFN--y. Of particular interest were the synergistic effects on IFN--y seen when both purified proteases were added to reaction mixtures (16). These results would predict that Pseudomonas strains that produce both enzymes should be particularly immunosuppressive, a property that may aid the bacterium in establishing initial colonization by significantly altering immune and inflammatory responses in infections like those seen in cystic fibrosis.This study was undertaken to address three questions relating to the effects of Pseudomonas protease on cytokines. First, what are the specificities of these proteases relative to the inactivation of cytokines that might be involved in Pseudomonas infections? Second, is limited proteolysis of the type seen with human IFN--y sufficient for the inactivation of other cytokines? Third, is the ability to inactivate cytokines a common property of Pseudomonas strains, and how does this property relate to their production of the two proteases? MATERIALS AND METHODSHuman subjects. The studies reported here were approved by the Human Subj...