Tumor necrosis factor-alpha mediates the early pathology in Salmonella infection of the gastrointestinal tract.

Salmonella infection of the intestinal tract results in damage to the gut epithelium. While it is generally believed that bacteria and/or bacterial products account for this pathology, the role of host factors has not been explored. Using a ligated intestinal loop model, we investigated whether tumor necrosis factor-alpha (TNF-alpha) could contribute to the tissue pathology associated with Salmonella infection. Intestinal segments infected with Salmonella typhimurium had high levels of fluid secretion as early as 6 h post-bacterial infection. At this time point, low levels of TNF activity were also present in the fluid obtained from infected segments. At 20 h post-infection, high levels of TNF activity were present in fluids obtained from infected intestinal segments and was characterized as TNF-alpha by neutralization experiments using rabbit antisera to TNF-alpha. TNF-alpha production was further verified by Northern blot analysis using RNA obtained from cells eluted from the infected intestinal segments. In contrast, no TNF activity was found in fluid obtained from intestinal segments challenged with cholera toxin, which induces fluid secretion with little to no inflammatory response. Double labeling by in situ hybridization and immunocytochemistry revealed that macrophages in the lamina propria were producing the TNF-alpha mRNA. To investigate what role TNF-alpha might play in Salmonella-induced inflammation, intestinal segments were injected with recombinant mouse TNF-alpha (rTNF-alpha) or mice were pretreated with antibody to TNF-alpha or a control antibody prior to Salmonella infection. The histological profile of intestinal segments injected with rTNF-alpha appeared identical to segments infected with S. typhimurium. Further, pathology was completely eliminated in infected mice pretreated with antibody to TNF-alpha. These results document the production of TNF-alpha in the intestinal tract following S. typhimurium infection and show that the early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms.

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations.

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.

Interferon production by Shigella flexneri-infected fibroblasts depends upon intracellular bacterial metabolism.

The role of bacterial invasion and subsequent intracellular metabolism or replication, or both, in the induction of interferon (IFN) production in primary cultures of murine embryo fibroblasts (MEFs) was examined. IFN production appeared to be dependent upon bacterial invasion. MEFs that were challenged with Shigella flexneri cultured at 30 degrees C to inhibit the temperature-dependent virulence gene expression that is essential for invasion failed to produce IFN. Furthermore, inhibition of S. flexneri invasion by pretreatment of MEFs with cytochalasin B resulted in a reduction in IFN production. Intracellular bacterial residence alone, however, was not sufficient for the induction of IFN production since an avirulent isogenic variant of S. flexneri which invades but fails to grow intracellularly did not induce IFN production. In fact, the blocking of bacterial RNA synthesis immediately after cellular uptake of S. flexneri by rifampin inhibited IFN production by MEFs. Transfer of the invasion-encoding plasmid to a noninvasive Escherichia coli strain conferred upon the bacteria the ability to invade MEFs and induce IFN production. These results suggest that the induction of IFN production in S. flexneri-infected fibroblasts requires bacterial invasion and intracellular bacterial metabolism or replication, or both.

The induction of interferon production in fibroblasts by invasive bacteria: a comparison of Salmonella and Shigella species.

As the role of interferon (IFN) in host defense against facultative intracellular bacterial infections continues to expand, it has become increasingly important to understand what cell types can produce IFN following infection and/or interaction with these invasive bacteria. We have demonstrated previously that Shigella flexneri was able to induce high levels of IFN in primary cultures of human and murine fibroblasts following bacterial invasion. In this study, we examined the ability of Salmonella typhimurium to induce IFN production in different cell lines. S. typhimurium-infected primary cell cultures of mouse embryo-fibroblasts (MEF) were shown to produce high levels of IFN following bacterial challenge. In contrast to Shigella, Salmonella required a much lower multiplicity of infection for optimal IFN induction. Examination at the RNA level of IFN production by MEF following challenge with either bacteria revealed that the IFN produced was a mixture of IFN alpha and IFN beta (IFN alpha/beta), with IFN beta 1 as the predominant species. As previously demonstrated for Shigella, bacterial invasion of cells appeared to be required for the induction of IFN production by S. typhimurium. Salmonella rendered non-invasive by UV-treatment failed to induce IFN production in MEF. Furthermore, Salmonella LPS, when tested over a wide range of concentrations, was unable to induce IFN production in these cells. In contrast to MEF, human and murine continuous cell lines did not produce IFN following Salmonella challenge. These results taken together suggest that IFN may be a common factor involved in Salmonella and Shigella infections. Furthermore, IFN may play an important role in the front line host defense against these types of infections.

Bacterial invasion of fibroblasts induces interferon production.

Although bacteria and/or bacterial products have been shown to induce interferon (IFN) production in cells of the immune system, the ability of non-lymphoid cells to produce IFN in response to bacterial products and/or bacterial invasion is currently unknown. In this study we examined murine and human fibroblasts for their ability to produce IFN in response to challenge with the invasive bacteria Shigella flexneri and enteroinvasive Escherichia coli. Both human and murine primary cell cultures produced high levels (500 to 1000 U/ml) of IFN-beta within 12 hr after Shigella invasion. The production of IFN appeared to be dependent upon bacterial invasion because no IFN was produced by cells pretreated with a non-invasive isogenic variant of S. flexneri. Furthermore, UV treated invasive S. flexneri, which lose the ability to invade cells, failed to induce IFN production in fibroblasts after bacterial challenge. In contrast to primary cells, most human and murine continuous cell lines did not produce IFN in response to Shigella invasion. The above results demonstrate that bacterial invasion of primary cultures of fibroblasts can induce IFN production. Taken together with previous findings, these results suggest that IFN may play an important front line host defense against invasive bacterial infections.

Natural and recombinant interferons inhibit epithelial cell invasion by Shigella spp.

The effect of natural and recombinant interferons (IFNs) on the abilities of Shigella flexneri, S. sonnei, and Salmonella typhimurium to invade different human and murine cells was examined. Pretreatment of cell monolayers with natural and recombinant IFNs reduced the number of Shigella-infected cells in a dose-dependent manner. Establishment of an anti-invasive cellular state was time dependent, requiring 10 h for 50% inhibition of bacterial invasion. The inhibitory effect of IFN was species specific, with human or murine IFN effective against homologous but not heterologous cells. Gamma IFN was slightly more potent than alpha IFN at inhibiting bacterial invasion. Inhibition of Shigella invasion was dependent on the challenge dose of bacteria. Little inhibition of invasion was seen when cells were pretreated with low concentrations of IFN and challenged with high multiplicities of infection of Shigella sp. In contrast to Shigella invasion, the maximum inhibitory effect of IFN on Salmonella invasion of cells was observed at low levels (5 to 50 U) of IFN. These results suggest that Shigella and Salmonella invasions occur at unique sites on eucaryotic cells or by different penetration mechanisms. More importantly, these data suggest that IFN may play a significant role in host defense against Shigella and Salmonella infections.