An updated model of cell-mediated immunity--listeriosis: clinical and research aspects.

Listeria monocytogenes causes sepsis and meningitis in immunocompromised hosts and a devastating maternal/fetal infection in pregnant women. In recent years a more benign gastroenteritis in normal hosts has been described. Listeria has been increasingly identified as a food-borne pathogen, and large-scale contamination of processed foods with resulting outbreaks has occurred in recent years, possibly as a result of consolidation of the food industry. Experimental listeriosis in mice has proven to be an extraordinarily useful model for analyzing cell-mediated immune host defenses. Contrary to original concepts, we found that neutrophils, not macrophages, are the prime effectors during early infection. CD8+ T cells are then responsible for lysing infected hepatocytes through perforin-related (early primary and secondary infection) or Fas-L/Fas mechanism (late primary). Of interest, non-classical MHC class Ib restricted recognition mechanisms exist early, whereas MHC class Ia mechanisms can be detected throughout infection.

Innate defenses in the liver during Listeria infection.

The majority of pathogens entering the bloodstream are cleared by the liver. Listeria monocytogenes, an important natural pathogen of humans, is a useful tool for examining protective immune responses during systemic infections of mice. Innate immunity contributes to blood clearance and eventual sterilization of the liver subsequent to Listeria infections. Effector mechanisms expressed in the liver early after infections are orchestrated by complex interactions between resident populations, i.e. hepatocytes and Kupffer cells, with infiltrating monocytes, neutrophils, and natural killer cells. These interactions include cell to cell contact through adhesion molecules, as well as communication through secretion of cytokines and chemokines. The liver environment, as the interface between blood-borne pathogens and innate host defenses, is reviewed here.

Neutrophil-Kupffer-cell interaction in host defenses to systemic infections.

Most relevant textbooks characterize phagocytosis by Kupffer cells as the principal mechanism for clearing bacterial pathogens from the bloodstream and eliminating them from the liver. Here, Stephen Gregory and Edward Wing discuss recent evidence indicating that the actual mechanism is far more complicated, dependent upon the complex interaction of Kupffer cells with neutrophils that immigrate into the liver following infection.

Fas (CD95)-dependent cell-mediated immunity to Listeria monocytogenes.

Two distinct and complementary pathways, one mediated by perforin and the other dependent upon CD95 (Fas), effect cell-mediated cytotoxicity. We examined the relative roles of these pathways in host defenses against the intracellular bacterial pathogen Listeria monocytogenes by using murine listeriosis as a model system. Mice which lacked both perforin and Fas (P0L0) were generated, and their responses to primary and secondary listeriosis were compared to those of wild-type (WT), Fas-deficient (L0), and perforin knockout (P0) mice. Relative to WT mice during primary listeriosis, P0 mice exhibited a reduced capacity to clear the infection from their spleens but not their livers whereas L0 mice had elevated bacterial titers in their livers and a modestly increased titer in their spleens. In contrast, bacterial titers in P0L0 mice were increased approximately 50- to 560-fold in their spleens and 230- to 1, 000-fold in their livers; eventual clearance of listeriae from both organs was significantly delayed. Furthermore, the resistance of P0L0 mice to secondary listeriosis was significantly reduced in their spleens and livers compared to that of WT, P0, or L0 mice. In vitro experiments indicated that immune cytotoxic T lymphocytes (CTL) lysed L. monocytogenes-infected hepatocytes primarily via a Fas-dependent, perforin-independent mechanism. The absence of Fas severely abrogated the lysis of infected hepatocytes by immune CD8(+) CTL. Taken together, these results provide the first evidence for Fas-dependent CTL-mediated lysis of L. monocytogenes-infected hepatocytes and demonstrate complementary roles for Fas and perforin in host defenses against an intracellular bacterial pathogen.

IL-6 produced by Kupffer cells induces STAT protein activation in hepatocytes early during the course of systemic listerial infections.

Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.

Listeria monocytogenes-infected hepatocytes are targets of major histocompatibility complex class Ib-restricted antilisterial cytotoxic T lymphocytes.

Subclinical infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes results in the development of protective antilisterial immunity. L. monocytogenes can infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyse Listeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whether L. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1(b) mRNA and protein. We further showed that Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derived Listeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish that Listeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restricted Listeria-specific CTL.

Internalin B promotes the replication of Listeria monocytogenes in mouse hepatocytes.

The uptake of Listeria monocytogenes by a variety of cell types in vitro is facilitated by the protein products of the inlAB (internalin) operon expressed by the organism. In the case of mouse hepatocytes, the extent to which inlAB expression influenced the uptake of Listeria in vitro was markedly dependent upon the ratio of bacteria to cells. At a ratio of 100:1, greater than 40-fold fewer transposon-induced inl4B mutant listeriae entered hepatocytes compared to the isogenic wild-type control; the difference was only fourfold, however, in cultures inoculated at a 1:1 ratio. Similarly, the uptake of in-frame inlB or inlAB deletion mutants differed only fourfold from the uptake of wild-type or inlA mutant Listeria at a 1:1 multiplicity of infection. Mutations affecting inlB or inlAB, on the other hand, resulted in a marked decrease in the capacity of Listeria to proliferate within mouse hepatocytes in vivo and in vitro. Electron micrographs of Listeria-infected hepatocytes demonstrated the impaired capacity of inlB mutants to escape from endocytic vacuoles and to enter the cytoplasm where proliferation occurs. These findings indicate that the protein product of inlB exerts a significant effect on the intracellular replication of Listeria.

Immune CD8+ T lymphocytes lyse Listeria monocytogenes-infected hepatocytes by a classical MHC class I-restricted mechanism.

Hepatocytes constitute the principal site of listerial replication in the livers of mice infected i.v. CD8+ T lymphocytes play a predominant role in the host defenses to Listeria monocytogenes. In vitro experiments by others undertaken to delineate the functions of CD8+ T lymphocytes have focused primarily on their interaction with Listeria-infected macrophages. Such experiments do not address directly the role of CD8+ T lymphocytes in eliminating the bulk of Listeria replicating within the liver. Here, we report that immune CD8+ T cells at an E:T cell ratio > or = 10:1 lysed Listeria-infected hepatocytes as judged by the following two criteria. Aspartate aminotransferase activity in the culture supernatants, indicative of hepatocyte damage, increased significantly. Conversely, infected hepatocytes cocultured with immune CD8+ T cells exhibited a marked reduction in viable intracellular Listeria assessed by CFUs. Neither immune CD4+ T cells nor nonimmune CD8+ T cells caused a similar increase in aspartate aminotransferase activity released or a decrease in intracellular bacteria. Immune CD8+ T cell-mediated lysis of infected hepatocytes was restricted by classical MHC class I (H-2Kb) molecules and was inhibited by the presence of either brefeldin A or mAb specific for CD8. These results suggest that the predominant role of CD8+ T lymphocytes in host resistance to listerial infections of the liver may be due to their capacity to lyse infected hepatocytes.

Lymphokine-activated killer cells lyse Listeria-infected hepatocytes and produce elevated quantities of interferon-gamma.

The bulk of Listeria monocytogenes injected intravenously into mice is taken up in the liver, where hepatocytes serve as the principal site of intracellular replication. NK cells have been implicated in host defenses to a variety of intracellular pathogens. To explore the role of NK cells in resistance to listerial infections of the liver, lymphokine-activated natural killer (LAK) cells were cocultured with Listeria-infected hepatocytes. The aspartate aminotransferase activity in the medium (evidence of cytotoxicity and hepatocyte damage) was elevated significantly in these cultures. Conversely, the viability of intracellular Listeria organisms was reduced. Increased quantities of interferon-gamma (IFN-gamma) were also detected. IFN-gamma production by LAK cells was modulated by interleukin (IL)-2 and IL-12. These findings suggest that the response of LAK cells to infected hepatocytes may play a critical role in host defenses to Listeria organisms taken up in the liver.

Expression of the inlAB operon by Listeria monocytogenes is not required for entry into hepatic cells in vivo.

Listeria monocytogenes injected intravenously into mice is taken up in the liver, where hepatocytes serve as the principal site of intracellular replication. The factors effecting entry of L. monocytogenes into hepatic cells remain to be determined. Others have shown that the protein products of the inlAB (internalin) operon are required for maximum entry of L. monocytogenes into a number of cell lines in vitro. Likewise, we report here that expression of the inlAB operon was required for maximum uptake of L. monocytogenes by primary cultures of mouse hepatocytes. Uptake of an inlAB mutant strain of L. monocytogenes was approximately 10-fold less than that of the isogenic wild-type control. In contrast, inlAB expression was not a factor in (i) clearance of L. monocytogenes injected intravenously into mice and taken up in the liver, (ii) the distribution of L. monocytogenes among hepatocytes and nonparenchymal cells in the liver, or (iii) internalization of L. monocytogenes by hepatic cells in vivo. These latter findings suggest that infection of hepatic cells by L. monocytogenes in vivo does not require the protein products of the inlAB operon.

Bacteria in the bloodstream are trapped in the liver and killed by immigrating neutrophils.

The critical role of the liver in the resolution of systemic bacterial infections is well documented. In the case of Listeria monocytogenes, approximately 60% of bacteria inoculated i.v. into mice are recovered in the liver at 10 min after infection. Here we report that the Listeria recovered at 10 min were distributed equally among the hepatocyte and nonparenchymal liver cell populations. The majority (>/= 75%) of these organisms were bound extracellularly as judged by their sensitivity to gentamicin. In contrast, >/= 93% of Listeria recovered in the liver at 6 h were located within hepatocytes. The listerial burden of the liver decreased 0.5 to 1.0 log, between 10 min and 6 h after infection. This decrease correlated with a sevenfold increase in the percentage of neutrophils that constituted the nonparenchymal cell population. Mice rendered neutrophil deficient by pretreatment with anti-granulocyte (RB6-8C5) mAb exhibited a significant increase (>300%) rather than a decrease in liver Listeria and a marked increase in hepatocyte damage. Similarly, neutrophil-deficient mice exhibited a reduced capacity to eliminate Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus that were cleared by the liver and bound extracellularly to hepatocytes and nonparenchymal cells. These findings document the crucial role of immigrating neutrophils in nonspecific host defenses to systemic bacterial infections expressed within the liver.

Factors associated with the development of candidemia and candidemia-related death among liver transplant recipients.

OBJECTIVE: The authors' objective was to identify factors associated with candidemia and candidemia-related death among adult liver transplant recipients. SUMMARY BACKGROUND DATA: Invasive candidiasis is the most common severe fungal infection occurring after liver transplantation and is associated with high morbidity and mortality rates. Although candidemia is not always found during invasive candidiasis, it has been considered as an indicator of invasive candidiasis in immunocompromised patients. METHODS: A time-matched case-control study of 26 patients with candidemia, which was defined as the isolation of Candida from at least one blood culture, and 52 control patients without candidemia was reported. Two control patients were matched with each case patient regarding time of transplantation and duration of follow-up. RESULTS: Between December 1985 and December 1992, candidemia developed in 1.4% of adult liver transplant recipients a median of 25 days after transplantation (range, 2-1690 days). The overall mortality rate among patients with candidemia was 81%, and 71% of these deaths were related to candidemia. Conditional logistic regression analysis was used to identify factors associated with candidemia, which were 1) hyperglycemia treated with insulin up to 2 weeks before candidemia (odds ratio [OR], 16.15; p = 0.002), and 2) exposure to more than three different intravenous antibiotics before development of candidemia (OR, 11.15; p = 0.005). The variables predictive of death related to candidemia were abdominal surgery performed up to 1 week before candidemia (relative risk [RR], 7.25; p = 0.02), high white blood cell count (RR, 1.10; p = 0.01), lower platelet count (RR, 0.99; p = 0.02), and elevated AST with candidemia (RR, 1.001; p = 0.01). CONCLUSIONS: Hyperglycemia that requires insulin and exposure to more than three antibiotics are the factors associated with the development of candidemia in liver transplant recipients. When candidemia develops shortly after abdominal surgery and in patients with elevated AST, high white blood cell count, or low platelet count, it is associated with a high mortality rate.

Hepatocytes can serve as accessory cells in the response of immune T lymphocytes to heat-killed Listeria monocytogenes.

Previous findings in our laboratory indicated that the bulk of Listeria monocytogenes injected intravenously into mice and recovered in the liver is taken up and replicates within hepatocytes. Other investigators have shown that hepatocytes can display costimulatory adhesion molecules, express major histocompatibility complex class I and II molecules, and secrete a number of cytokines, including interleukin-1 (IL-1), IL-6, and IL-8. These data suggest that hepatocytes may serve as accessory cells in the immune response to L. monocytogenes. The accessory function and capacity of hepatocytes to present listerial antigens, however, have never been explored. We undertook a series of experiments to examine the response of Listeria-immune T lymphocytes to murine hepatocytes preincubated with heat-killed listeriae (HKL). Electron micrographs showing the organism within membrane-limiting vacuoles demonstrated the capacity of hepatocytes to internalize HKL. T cells cocultured with hepatocytes pulsed with HKL exhibited a 5- to 10-fold increase in [methyl-3H]thymidine incorporation relative to T cells cultured with either hepatocytes or HKL alone. Similarly, gamma interferon production by immune T cells was elevated significantly in cultures that contained both hepatocytes and HKL. The optimal response of T cells required lysosomal processing of HKL by hepatocytes and contact between the two cell populations. Furthermore, maximum T-cell proliferation and gamma interferon production were dependent upon the presence of CD4+ T lymphocytes and the expression of Ia antigens. Taken together, these findings demonstrate that hepatocytes pulsed with HKL can stimulate the antigen-specific response of immune T lymphocytes. These results suggest that hepatocytes can serve as accessory cells in host defenses to listerial infections of the liver.

Arginine analogues suppress antigen-specific and -nonspecific T lymphocyte proliferation.

Using analogues of arginine to inhibit nitric oxide (NO.) production, investigators have demonstrated the intermediary role of NO. in a variety of physiological events including the antimicrobial activity exhibited by macrophages in vitro. In an effort to establish the effector function of NO. in the antimicrobial activity expressed by macrophages in vivo, several groups report treating infected animals with relatively high concentrations of these same analogues. In the present study, we found that the arginine analogues NG-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester, aminoguanidine, and L-canavanine at concentrations > or = 10 mM significantly inhibited both the antigen-specific and -nonspecific proliferation of T lymphocytes in culture. These findings indicate that in vivo experiments demonstrating the suppressive effect of arginine analogues on host defenses are subject to alternative interpretations that do not directly involve the microbicidal activity of macrophages.

Rapamycin but not FK506 inhibits the proliferation of mononuclear phagocytes induced by colony-stimulating factors.

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cell-conditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population.

Macrophage colony-stimulating factor and the enhanced migration of monocytes are essential in primary but not secondary host defenses to Listeria organisms.

Nonimmune mice infected with Listeria monocytogenes exhibited elevated expression of macrophage colony-stimulating factor (M-CSF) mRNA and the enhanced migration of Mac-1 antigen-positive bone marrow-derived mononuclear phagocytes (BMMP) to their livers. Treatment with monoclonal anti-M-CSF antibody diminished the traffic of BMMP and promoted the replication of listeriae. Immune animals infected with listeriae expressed significantly lower levels of M-CSF mRNA than did nonimmune animals. Moreover, listerial infections did not elicit the migration of BMMP to the livers of immune mice, nor did anti-M-CSF affect the capacity of immune animals to respond to infection. Adoptive immunization experiments suggest that T lymphocytes can mediate protective immunity to listeriae in the absence of M-CSF and migrating BMMP. These findings indicate that M-CSF and the enhanced migration of BMMP are critical factors in primary but not secondary host defenses to listerial infections.

IFN-gamma inhibits the replication of Listeria monocytogenes in hepatocytes.

We previously reported that the bulk of Listeria monocytogenes injected intravenously into mice is taken up in the liver and replicates within the parenchymal cells (hepatocytes). Although IFN-gamma is known to play an important role in host defenses to listerial infections of the liver, the mechanism(s) that underlies this role remains to be fully delineated. In the initial experiments presented here, we demonstrated the elevated expression of IFN-gamma message in the livers of mice during primary listerial infections. Subsequent experiments showed that the listerial burden of the hepatocyte population was increased significantly in mice administered monoclonal anti-IFN-gamma. Conversely, the administration of murine rIFN-gamma resulted in a marked (2 log10) decrease in the number of hepatocyte-associated Listeria. In vitro, IFN-gamma stimulated the listericidal activity of purified hepatocytes. Infected hepatocytes incubated in the presence of > 0.1 U/ml murine rIFN-gamma exhibited a significant reduction in intracellular Listeria. The elevated antilisterial activity of IFN-gamma-treated hepatocytes in culture was abrogated by the presence of compounds that scavenged or inhibited the production of reactive oxygen intermediates. Taken together, these findings suggest that activation of the oxygen-dependent, antimicrobial activity of hepatocytes may constitute a principal effector function of IFN-gamma in host defenses to listerial infections of the liver.