Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin.

Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-alpha and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-alpha production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-alpha. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.

Proteasome-mediated regulation of interleukin-1beta turnover and export in human monocytes.

Interleukin-1beta is a secreted protein that accumulates in the cytosol as an inactive precursor (pIL-1beta) before processing and release of biologically active protein. To understand the impact of this property on IL-1beta production, we examined the intracellular stability of pIL-1beta in lipopolysaccharide (LPS)-stimulated human monocytes. Precursor IL-1beta was degraded with a relatively short half-life of 2.5 h in the promonocytic cell line, THP-1, and in primary monocytes. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) stabilized pIL-1beta levels in THP-1 cells, suggesting that degradation was proteasome-mediated, but this inhibitor was toxic for primary monocytes, causing release of pIL-1beta as well as the cytoplasmic enzyme, lactate dehydrogenase (LDH) into supernatants. In contrast, clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome, caused a dose-dependent stabilization of intracellular pIL-1beta, and this led to a corresponding increase in mIL-1beta and pIL-1beta but not LDH release into culture supernatants. Therefore, by regulating intracellular levels of precursor IL-1beta, the proteasome plays an important and previously unrecognized role in controlling the amount of biologically active IL-1beta that is exported by activated monocytes.

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway.

Upon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identified. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.

Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes.

Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection. LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression of actA and hly during intracellular growth to that during growth in broth cultures. The hly and actA genes were transcriptionally fused to Escherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L. monocytogenes chromosome. A chloramphenicol resistance assay indicated that the hly fusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures. Quantitation of promoter activity on the basis of beta-galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions. However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO. Finally, in comparison to induction in broth cultures, actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.

Protection against murine disseminated candidiasis mediated by a Candida albicans-specific T-cell line.

The role of T lymphocytes in disseminated candidiasis in a mouse model of irradiation-induced immunosuppression was investigated. A continuously cultured Candida albicans-specific T-cell line mediated protection of sublethally irradiated mice from disseminated candidiasis as measured by both the fungal load in the kidneys and mortality. These results are the first to demonstrate directly a role for antigen-specific T cells in the protective immune response against murine disseminated candidiasis.

Methylxanthine-induced inhibition of the antigen- and superantigen-specific activation of T and B lymphocytes.

Methylxanthines have been shown to have a variety of effects on hematopoietic cell activation and function. These compounds inhibit cAMP-specific phosphodiesterase activity resulting in increased levels of intracellular cAMP. In the present study, we examined the effects of two methylxanthines, pentoxifylline (PTX) and caffeine, on the responses of both mouse and human lymphocytes to stimulation with polyclonal T- and B-cell mitogens, antigens, and the microbial superantigen, staphylococcal enterotoxin B (SEB). Both PTX and caffeine significantly inhibited mitogen- and SEB-induced proliferation by murine spleen cells, SEB- and antigen-induced proliferation and lymphokine secretion by murine Th1 and Th2 clones, and the generation of antigen-specific antibody producing murine spleen cells. These compounds also inhibited the proliferative responses of human lymphocytes to phytohemagglutinin, SEB, and tetanus toxoid. Efforts to determine whether these methylxanthine compounds mediated their inhibitory effects through a specific protein kinase pathway revealed a role for cAMP-dependent protein kinase A in methylxanthine-induced immunomodulation. However, it is possible that a protein kinase A-independent pathway may also be involved. These data demonstrate that the methylxanthines, PTX and caffeine, have profound effects on cells of the immune system and may have a potential use as immunotherapeutic agents in the treatment of various inflammatory conditions and autoimmune diseases.

A role for complement receptor-like molecules in iron acquisition by Candida albicans.

Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism.

Altered macrophage antigen-presenting cell function following Friend leukemia virus infection.

To investigate the mechanism by which Friend leukemia virus (FV) causes immunosuppression, the ability of peritoneal macrophages to mediate antigen-specific T-cell activation following FV infection was examined. Decreased IL-2 production was observed when antigen-primed T cells were cultured with antigen-pulsed macrophages from mice infected with FV, compared to T cells cultured with macrophages from control mice. Macrophages from FV-infected mice demonstrated decreased phagocytic and pinocytic activity, suggesting that antigen uptake may be impaired in these cells. In addition, FV-infected mice had decreased numbers of MHC class II positive macrophages compared to uninfected controls, as measured by immunofluorescence. The alterations in antigen uptake and class II expression observed in macrophages from FV-infected mice may be the result of infection of these cells by FV, which was demonstrated by in situ hybridization using a FV-specific probe. The ability of FV to infect and modulate the functions of macrophages may account, at least in part, for the immunosuppression observed in FV-infected mice.

Effect of Friend leukemia virus infection on susceptibility to Candida albicans.

Previous studies have demonstrated that Friend leukemia virus (FLV) induces a profound immunosuppression in susceptible mice. The studies described in this report indicate that mice infected with FLV have an increased susceptibility to subsequent infection with the opportunistic pathogen Candida albicans, as measured by increased numbers of C. albicans CFU in the kidneys of FLV-infected mice relative to uninfected controls. Experiments in which the NB-tropic and N-tropic strains of FLV were used suggest that virus replication or the resulting virus burden may be important in the observed increased susceptibility to C. albicans. Since neutrophils are believed to be important in the response of mice to systemic Candida infections, the effect of FLV infection on neutrophil candidacidal activity was investigated. The percentage of neutrophils present in unfractionated Proteose Peptone-elicited peritoneal exudates of mice infected with FLV for 14 days was significantly lower than in uninfected control mice or mice infected with FLV for 6 or 10 days. When neutrophils from FLV-infected and control mice were purified, adjusted to equal concentrations, and tested for in vitro candidacidal activity, neutrophils from mice infected with FLV for 14 days were deficient in their ability to kill C. albicans relative to normal controls and mice infected with FLV for 6 or 10 days. Addition of normal mouse serum increased killing in all groups but did not restore candidacidal activity of neutrophils from mice infected with FLV for 14 days to levels of control neutrophils or neutrophils from mice infected for 6 or 10 days with the virus. These results suggest a defect in neutrophil function, at the later stages of FLV infection, involving in vitro candidacidal activity. In addition, neutrophils from FLV-infected mice may be deficient in in vivo chemotactic activity. These defects in neutrophil function could account, at least in part, for the observed increased susceptibility of FLV-infected mice to C. albicans.