A natural immune modulator attenuates stress hormone and catecholamine concentrations in polymicrobial peritonitis.

BACKGROUND: Activated hexose correlated compound (AHCC), derived from shiitake mushrooms, increases resistance to infection in immunocompromised hosts with positive effects on dendritic cells, natural killer cell function and interleukin 12 production. It may also be attenuating the systemic inflammatory response by regulating the secretion of cortisol and norepinephrine (NE). METHODS: Female Swiss-Weber mice were pretreated with AHCC (Amino Up Chemical Co., Sapporo, Japan) or water by gavage for 10 days before undergoing cecal ligation and puncture (CLP). Peritoneal exudate cells and blood samples were harvested at 4 hours and 24 hours following CLP. Plasma and peritoneal concentrations of cortisol and NE were obtained using enzyme-linked immunosorbent assay. Peritoneal bacteria were quantified by colony counts after 4 hours and 24 hours. Significance was denoted by a p < 0.05. RESULTS: Plasma and peritoneal cortisol concentrations were increased 4 hours after CLP compared with normal controls, with no difference between the pretreated groups. Concentrations of cortisol decreased from 4 hours to 24 hours after CLP with AHCC (plasma, p = 0.009; peritoneal, p < 0.001), and peritoneal cortisol at 24 hours was lower with AHCC as compared with water (p = 0.028). There was no change in plasma or peritoneal NE concentrations at 4 hours. At 24 hours, higher concentrations of NE were detected in both plasma and peritoneal fluid, with lower plasma concentrations in those gavaged with AHCC (p = 0.015). There was no significant difference in peritoneal bacteria counts. CONCLUSION: Enhanced immune function observed with AHCC could be caused by attenuated concentrations of stress hormones and catecholamines.

Endogenous IL-10 leads to impaired bacterial clearance and reduced survival in a murine model of chronic peritonitis.

We previously observed insufficient neutrophil accumulation and a lack of TNF-alpha response at the site of infection until bacteria numbers >10(5) colony forming units in our model of chronic murine peritonitis, suggesting a defective host response after bacterial challenge with Klebsiella pneumoniae (Klebsiella). The aim of this study was to determine a potentially immunosuppressive effect of IL-10 in this model of chronic peritonitis. Balb/c animals were injected with 10(3) colony forming units Klebsiella intraperitoneally. Gentamicin (5 mg/kg/day BID) was given subcutaneously (s.c.) for two days and then withdrawn. Animals were treated with anti-IL-10 antibody or IgG isotype control (s.c.) before or after Klebsiella administration. Survival was determined over 14 days. Similarly treated animals were harvested after 48 h to obtain liver tissue, peritoneal fluid and blood. Bacteria and neutrophil counts were determined. TNF-alpha and IL-10 were measured by ELISA. Anti-IL-10 antibody significantly increased survival and bacterial clearance in the observed compartments. Anti-IL-10 administration did not lead to an increase in TNF-alpha concentrations or neutrophil accumulation at the site of infection at lower levels of Klebsiella. We conclude that endogenous IL-10 is detrimental for survival and bacterial clearance in this model of chronic peritonitis.

Mortality in murine peritonitis correlates with increased Escherichia coli adherence to the intestinal mucosa.

During peritonitis, bacterial adherence is the initial step in a series of events that include mucosal infection, bacterial translocation, organ dysfunction, and death. Adherent Escherichia coli levels increase in response to stress. This study was designed to assess the adherence of E. coli to the cecal mucosa after cecal ligation and puncture (CLP) of increasing severity and to determine whether a relationship exists between adherence of bacteria and mortality. Sham surgery, sterile peritonitis (thioglycollate administration), lethal CLP (18-gauge double-puncture), and nonlethal CLP (23-gauge single-puncture) were performed on Swiss Webster mice and compared with normal mice or before CLP (time 0). Specimens of bowel tissue were harvested, and serial log dilutions of homogenized specimens or bowel contents were plated and cultured on media selective for determination of individual bacterial species. Low levels of E. coli and Proteus mirabilis adhered to the mucosa of unmanipulated controls; however, adherence of both species increased significantly by 18 hours after both lethal and nonlethal CLP. After 18 hours, adherent E. coli levels increased by greater than 5 x 10(6)-fold compared to unmanipulated controls, whereas P. mirabilis levels decreased. After nonlethal CLP, adherent P. mirabilis increased 3 x 10(6)-fold compared to unmanipulated animals, whereas E. coli levels did not increase after 24 hours. Sterile peritonitis had little effect on bacterial adherence. Higher levels of adherent E. coli in the cecum correlate with the increased mortality observed after lethal CLP. Higher levels of adherent P. mirabilis appear to prevent the overgrowth of adherent E. coli following nonlethal CLP. Our data indicate that E. coli plays a key role in mortality from polymicrobial peritonitis and that Proteus may be antagonistic to E. coli in murine peritonitis.

A novel model of pneumonia from intraperitoneal injection of bacteria.

BACKGROUND: Pneumonia remains a major clinical problem in the surgical patient. Experimental modeling by intratracheal injection of bacteria is not consistently reproducible. In an attempt to produce peritonitis by Klebsiella, we found evidence of pneumonia on autopsy and further developed this approach as a new experimental model. METHODS: Male Swiss Webster mice were given intraperitoneal (IP) injections of Klebsiella pneumoniae serotype 2 in different doses and this was compared with similar doses given intravenously (IV). A dose dependent survival curve was generated. Subsequently, 10(3) colony forming units (CFU) of bacteria were used in further experiments. Blood, peritoneal fluid and lung tissue were collected at time points up to 72 hours after injection and were cultured for levels of bacteria. Lung weights and myeloperoxidase levels were also measured. RESULTS: Intraperitoneal administration of Klebsiella was uniformly lethal with as few as 10(2) bacteria. Lung weight increased after IP Klebsiella, and all animals became bacteremic within 24 hours correlating with high bacterial levels in the lung. Conversely, most animals (72%) survived IV injection of bacteria, and were able to clear bacteria from the blood and lung. CONCLUSIONS: We found that this model produced no clinically apparent peritonitis after 48 hours, but uniformly resulted in histopathologic changes of pneumonia by 24 hours. Survival time was related to initial dose of Klebsiella and there was a linear correlation between bacterial levels in the blood and lung. This model is reproducible, simple to perform, and the severity is easy to manipulate.

Natural killer cell activation primes macrophages to clear bacterial infection.

Natural killer (NK) cells are major cytokine producers during bacterial sepsis, but their precise role is undefined. This study investigates the effect of NK cell depletion with and without prior activation on macrophage function and bacterial clearance during cecal ligation and puncture. Two different NK cell-depleting antibodies were used: anti-asialo-GM1 (GM1), a nonactivating antibody, and anti-NK1.1 (NK1.1), an NK cell-activating antibody. C57BL/6 mice were NK depleted with either GM1 or NK1.1 by intraperitoneal injection 7 and 3 days before experimentation. Control animals received isotype immunoglobulin G. Depletion was confirmed by flow cytometry. Bacterial levels in peritoneal washout, blood, and liver were determined 4 hours after cecal ligation and puncture. Macrophage activation was measured by phagocytosis ability and by production of nitric oxide and interleukin-6. Depletion with GM1 resulted in significantly higher bacterial levels at 4 hours, whereas depletion with NK1.1 had the opposite effect of significantly decreasing bacterial levels. Macrophage phagocytosis ability was significantly increased in mice depleted with NK1.1 compared with those mice depleted with GM1. We conclude that activation of NK cells improves bacterial clearance by priming macrophages to help clear a subsequent bacterial challenge. Macrophages are less able to clear bacteria when NK cells are depleted without activation. NK cells are therefore important in bacterial clearance through interactions with macrophages.

Natural killer cells participate in bacterial clearance during septic peritonitis through interactions with macrophages.

Natural killer (NK) cells have a well-established role in host defense against viral infections and malignancies. However, their function in bacterial infection and sepsis is poorly defined. We hypothesized that NK cells, as a major producer of interferon-gamma during sepsis, would be important in host defense against bacterial infections. Cecal ligation and puncture (CLP) was performed on Swiss Webster mice depleted of NK cells by pretreatment with anti-asialo GM1 and control mice given immunoglobulin G (IgG) antibody. NK cell-depleted mice had significantly higher anaerobic bacterial counts in the liver and peritoneal lavage fluid, as well as higher aerobic counts in the liver and blood 4 h after CLP. Macrophage phagocytosis, nitric oxide production, and interleukin (IL)-6 levels at 4 h were also decreased in mice depleted of NK cells compared with controls. Greater neutrophil influx into the peritoneum, indicated by higher myeloperoxidase levels, was also seen in NK cell-depleted mice. At 8 and 18 h after CLP, bacterial counts were similar between groups, and overall survival rates were not significantly different. Peritoneal IL-12 levels significantly increased by 18 h in normal mice, but not in NK cell-depleted animals. Our data suggest that NK cells participate in the early local and systemic eradication of bacteria and regulation of IL-12 during polymicrobial sepsis. These effects are likely due to their interactions with macrophages.

Genetic background determines susceptibility during murine septic peritonitis.

BACKGROUND: The tolerance of mouse strains to cecal ligation and puncture (CLP), a clinically relevant model of sepsis, can vary greatly. We compared the immune response and bacterial eradication during CLP in two mouse strains with different susceptibilities to the lethal effects in an effort to understand alterations in tolerance. MATERIALS AND METHODS: CLP of increasing severity was performed on Swiss Webster mice. Interleukin (IL)-12 levels, bacterial counts, and myeloperoxidase were determined. We then compared the same parameters in Swiss Webster and in BALB/c mice and determined survival for both mouse strains after CLP. RESULTS: Bacterial counts locally and systemically as well as serum IL-12 correlated with the severity of CLP in Swiss Webster mice. Lung myeloperoxidase increased with increasing severity CLP, while peritoneal myeloperoxidase decreased. Following CLP, one-half of the Swiss Webster mice survived versus none of the BALB/c mice. Despite worsened survival, BALB/c mice had lower bacterial counts and similar IL-12 levels compared to Swiss Webster mice. Myeloperoxidase and IL-6 levels were similar between experimental groups. CONCLUSIONS: Swiss Webster and BALB/c mice have significantly different susceptibilities to the lethal effects of CLP, and this difference may be related to IL-12 responsiveness.