Stephen H. Gregory

Listeria monocytogenes: Clinical and Experimental Update

Listeria monocytogenes, a small gram-positive bacillus, causes sepsis and meningitis in immunocompromised patients and a devastating maternal/fetal infection in pregnant women. Recent outbreaks demonstrated that L. monocytogenes can cause gastroenteritis in otherwise healthy individuals and more severe invasive disease in immunocompromised patients. Centralized processing in the food industry may be the cause of these large-scale listeriosis outbreaks. The mouse model of listeriosis, which was developed in the 1960s, has been extraordinarily useful for studying T cell-mediated immunity. Contrary to the original concept that macrophages are the principal effector cells in listeriosis, we found that immigrating neutrophils play the predominant role in early liver defenses. At later time points, CD8(+) T cells lyse infected hepatocytes by both perforin- and Fas-L/Fas--dependent mechanisms. Of interest, nonclassical major histocompatibility complex (MHC) class Ib--restricted cytolytic activity is expressed early during primary infection, whereas MHC class Ia--restricted activity is predominant through late primary and secondary infections.

Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections

Most bacteria that enter the bloodstream are taken up and eliminated within the liver. The specific mechanisms that underlie the role of the liver in the resolution of systemic bacterial infections remain to be determined. The vast majority of studies undertaken to date have focused on the function of resident tissue macrophages (Kupffer cells) that line the liver sinusoids. Indeed, it is often reported that Kupffer cells ingest and kill the bulk of organisms taken up by the liver. Recent studies indicate, however, that phagocytosis by Kupffer cells is not the principal mechanism by which organisms are eliminated. Rather, elimination depends on the complex interaction of Kupffer cells and bactericidal neutrophils that immigrate rapidly to the liver in response to infection. We discuss the critical role of neutrophil‐Kupffer cell interaction in innate host defenses and, conceivably, the development and expression of adaptive immunity in the liver.

Effector function of hepatocytes and Kupffer cells in the resolution of systemic bacterial infections

It has been suggested that mononuclear phagocytes serve as the principal site of replication for a number of intracellular pathogens including Listeria monocytogenes. To determine the role of the tissue macrophages (Kupffer cells) in the proliferation of Listeria taken up in the liver, the hepatic cell populations were purified and the associated bacteria were quantified at periodic intervals postinfection. Here we report that the bulk of Listeria injected intravenously into nonimmune mice replicated within hepatocytes rather than Kupffer cells. Whereas a 200‐fold increase in the number of hepatocyte‐associated Listeria occurred during the first 3 days following infection, a relatively small (less than 2‐fold) increase in number of Kupffer cell‐associated Listeria was observed. The Listeria injected intravenously into immune animals, on the other hand, were eliminated rapidly from the hepatocyte as well as the Kupffer cell population. The latter findings suggest that uptake and elimination of pathogenic organisms by “nonprofessional phagocytes” in the liver (i.e., hepatocytes) may be an important effector mechanism in host defenses.

Complementary Adhesion Molecules Promote Neutrophil- Kupffer Cell Interaction and the Elimination of Bacteria Taken Up by the Liver

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to ∼15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.

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.

Differential Effects of Macrophage Inflammatory Chemokine-2 and Keratinocyte-derived Chemokine on Hemorrhage-induced Neutrophil Priming for Lung Inflammation: Assessment by Adoptive Cells Transfer in Mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 ± 5 mmHg and were then administered anti-MIP-2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringers lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; &mgr;U/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (↑MPO, ↑% esterase+ cells in tissue) into the lung and local tissue inflammation (↑IL-6/MIP-2, ↓IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.

Invariant Natural Killer T Cells Suppress the Neutrophil Inflammatory Response in a Mouse Model of Cholestatic Liver Damage

BACKGROUND & AIMS NK1.1(+) TCRalphabeta(int) CD1-restricted T (NKT) cells are a unique subset of T lymphocytes that are believed to have an immunoregulatory role in a wide range of diseases. Most mouse NKT cells express a T-cell receptor that contains an invariant Valpha14Jalpha18 chain and recognizes antigenic glycolipids presented in association with major histocompatibility complex class Ib (CD1d) molecules. These invariant NKT (iNKT) cells have been implicated in cholestatic liver injury. METHODS We examined the role of iNKT cells in liver injury associated with biliary obstruction in mice with ligations of the common bile duct. RESULTS The number of activated iNKT cells increased markedly in the livers of mice following bile duct ligation. Plasma alanine aminotransferase levels, an indicator of liver injury, were significantly higher in iNKT cell-deficient (Jalpha18(-/-)) mice compared with wild-type mice following bile duct ligation. Photo image analysis of histologic sections confirmed that more damage was present in the livers of Jalpha18(-/-) mice; liver damage correlated with increases in keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) production as well as neutrophil sequestration. Liver injury was significantly reduced in Jalpha18(-/-) mice treated with anti-KC and anti-MIP-2 or rendered neutrophil deficient before bile duct ligation. Similarly, Jalpha18(-/-) mice that were injected with iNKT cells before bile duct ligation exhibited significant decreases in neutrophil accumulation and liver damage. CONCLUSIONS These data document the role of iNKT cells in suppressing the neutrophil proinflammatory response and neutrophil-dependent cholestatic liver damage.

The Role of Hepatic Invariant NKT Cells in Systemic/Local Inflammation and Mortality during Polymicrobial Septic Shock

NKT cells have been described as innate regulatory cells because of their rapid response to conserved glycolipids presented on CD1d via their invariant TCR. However, little is known about the contribution of the hepatic NKT cell to the development of a local and/or systemic immune response to acute septic challenge (cecal ligation and puncture (CLP)). We found not only that mice deficient in invariant NKT cells (Jα18−/−) had a marked attenuation in CLP-induced mortality, but also exhibited an oblation of the systemic inflammatory response (with little effect on splenic/peritoneal immune responsiveness). Flow cytometric data indicated that following CLP, there was a marked decline in the percentage of CD3+α-galactosylceramide CD1d tetramer+ cells in the mouse C57BL/6J and BALB/c liver nonparenchymal cell population. This was associated with the marked activation of these cells (increased expression of CD69 and CD25) as well as a rise in the frequency of NKT cells positive for both Th1 and Th2 intracellular cytokines. In this respect, when mice were pretreated in vivo with anti-CD1d-blocking Ab, we observed not only that this inhibited the systemic rise of IL-6 and IL-10 levels in septic mice and improved overall septic survival, but that the CLP-induced changes in liver macrophage IL-6 and IL-10 expressions were inversely effected by this treatment. Together, these findings suggest that the activation of hepatic invariant NKT cells plays a critical role in regulating the innate immune/systemic inflammatory response and survival in a model of acute septic shock.

Dendritic cells, regulatory T cells and the pathogenesis of chronic hepatitis C

Hepatitis C virus (HCV) is a small, enveloped RNA virus and a major cause of chronic liver disease. Resolution of primary HCV infections depends upon the vigorous responses of CD4+ and CD8+ T cells to multiple viral epitopes. Although such broad-based responses are readily detected early during the course of infection regardless of clinical outcome, they are not maintained in individuals who develop chronic disease. Ostensibly, a variety of factors contribute to the diminished T cell responses observed in chronic, HCV-infected patients including impaired dendritic cell function and the induction of CD4+FoxP3+ regulatory T cells. Overwhelming evidence suggests that the complex interaction of dendritic cells and regulatory T cells plays a critical role in the pathogenesis of chronic hepatitis C.

Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers

BACKGROUND Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. METHODS Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl(3)) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. RESULTS GdCl(3) treatment for either 24 or 48 hours before decompression reduced the numbers of ED2(+) Kupffer cells and ED1(+) inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl(3) pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl(3) treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. CONCLUSION We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation.