James C. Peyton

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-&ggr; 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.

Inhibition of neutrophil migration at the site of infection increases remote organ neutrophil sequestration and injury.

Up-regulation of the leukocyte β2 integrin, CD18, is a key event in neutrophil-endothelial adhesion and neutrophil-mediated organ injury. Inhibition of CD18 with monoclonal antibodies reduces lung and liver neutrophil sequestration in animal models of Gram-negative bacteremia or endotoxemia. However, with a persistent septic challenge, interference with host leukocyte phagocytic defense could adversely affect outcome. To assess the effects of inhibiting CD18 on organ neutrophil responses, bacteremia, and organ injury after fecal peritonitis, mice underwent cecal ligation and puncture (CLP). At the time of CLP and 12 h later, mice received intravenous anti-CD18 antibody or control IgG. At 3, 6, and 18 h after CLP, lung and liver tissue neutrophil content were measured by myeloperoxidase (MPO) assay, peritoneal cells and blood leukocytes were differentially counted, blood was cultured, and serum aspartate aminotransferase was measured. There was a significant reduction in peritoneal neutrophil migration and an increase in blood neutrophils after anti-CD18 treatment compared with results from treatment with the control antibody. In the anti-CD18-treated group, liver MPO was increased fivefold at 6 and 18 h, while lung MPO was increased two-fold at 18 h when compared with the control antibody-treated group. The anti-CD18-treated group also had an increase in bacteria cultured from the blood at 6 and 18 h and an increase in serum aminotransferase at 18 h. Our data demonstrate that peritoneal neutrophil migration in response to an endogenous fecal challenge is CD18-dependent, and that this mechanism forms a vital part of host defense. Inhibition of CD18 increased neutrophil sequestration in the liver and lung and increased liver injury. This study demonstrates a paradoxical increase in organ neutrophil sequestration using a leukocyte anti-adhesion therapy during sepsis and suggests that anti-adhesion therapies targeted towards neutrophil may worsen outcome if given during an ongoing, localized infection.

Neutrophil migration into the peritoneum is P-selectin dependent, but sequestration in lungs is selectin independent during peritonitis.

ABSTRACT Neutrophil (PMN) influx into the peritoneal cavity in response to bacterial peritonitis is an indispensable aspect of host defense. PMNs also are responsible for the remote organ injury observed after major abdominal infection. The aim of this study was to examine the effect of selectin blockade on PMN migration into the peritoneum and on PMN sequestration in the lungs, early in the course of peritonitis. Cecal ligation and puncture (CLP) was performed on P-selectin-deficient (P-def) mice and their genetic controls (C57). Both groups were treated with anti-E-selectin antibody, anti-L-selectin, or isotypic control immunoglobulin G at the time of CLP. 6 h after CLP, mice were sacrificed. Peritoneal PMN migration decreased in P-def mice compared with C57 controls after CLP. Blockade of E- or L-selectin alone in controls did not alter peritoneal PMN influx or circulating PMNs after CLP. In the P-def mice, treatment with anti-E-antibody or anti-L-antibody nearly eliminated PMN influx into the peritoneum. In contrast, circulating PMNs markedly increased after CLP in P-def mice when compared with baseline values. Lung myeloperoxidase increased in all groups of mice following CLP. Blockade of P-selectin with anti-P-selectin antibody elicited a response similar to that observed in the P-def mice. In conclusion, P-selectin mediates PMN influx into the peritoneal cavity, while E- and L-selectins do not appear to play any significant role in the 6 h time period following CLP. Lung PMN sequestration, after CLP, occurred independent of P-, E-, or L-selectin expression. Blockade of P-selectin during peritonitis appears to be potentially deleterious by preventing early PMN influx into the compartment containing the septic focus.

Leukotriene B4 Receptor (BLT-1) Modulates Neutrophil Influx into the Peritoneum but Not the Lung and Liver during Surgically Induced Bacterial Peritonitis in Mice

ABSTRACT Leukotriene B4 (LTB4) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1−/−) mice were used to determine the effects of LTB4 on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB4, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1−/− mice. LTB4 levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1−/− mice. Survival was significantly improved in BLT-1−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB4 modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.

Passive immunization against tumor necrosis factor and interleukin-1 fails to reduce lung neutrophil sequestration in chronic sepsis.

The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are produced within the lung during sepsis, and may induce neutrophil sequestration resulting in neutrophil-mediated lung injury. We hypothesized that, if there is a cause and effect between TNF alpha or IL-1 production and lung neutrophil sequestration during chronic sepsis, TNF alpha mRNA and IL-1 mRNA levels in the lung after cecal ligation and puncture should correlate with the number of sequestered neutrophils as measured by the myeloperoxidase (MPO) content of the lung. To test this hypothesis, Swiss Webster mice were subjected to varying degrees of infectious challenge by single and double-puncture cecal ligation and puncture, or simultaneous antibiotic treatment, and their lungs and blood were harvested at 24 h. Lung TNF alpha and IL-1 beta mRNAs were measured by the reverse-transcription differential polymerase chain reaction, and MPO was measured by colorimetric assay. TNF alpha serum levels showed no correlation with the MPO content of the lung, whereas IL-1 levels were undetectable. Lung TNF alpha mRNA correlated weakly, and IL-1 beta mRNA exhibited a strong correlation with lung MPO (r = .9, p < .01), but administration of anti-TNF alpha- or anti-IL-1-neutralizing antibodies did not prevent a rise in lung MPO. IL-1 beta mRNA in bronchoalveolar macrophages correlated well with whole lung tissue IL-1 beta mRNA levels (r = .91, p < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

STAT4 Is Required for Antibacterial Defense but Enhances Mortality during Polymicrobial Sepsis

ABSTRACT The signal transducer and activator of transcription factor 4 (STAT4) pathway mediates the intracellular effects of interleukin-12 (IL-12), leading to the production of gamma interferon, induction of a T helper type 1 response, and increased natural killer cell cytotoxicity. The purpose of this study was to determine the role of the STAT4 pathway during polymicrobial peritonitis in the cecal ligation and puncture (CLP) model. CLP was performed on STAT4-deficient (STAT4−/−) and wild-type control (BALB/c) mice. At 4 h after CLP, STAT4−/− mice had significantly higher bacterial counts in the peritoneal lavage fluid, liver, and blood. This difference persisted for 18 h in the peritoneal lavage fluid and blood. Neutrophil migration to the site of infection and into remote tissues was unaffected. Despite higher bacterial counts locally and systemically, STAT4−/− mice had a lower mortality rate than BALB/c controls. In contrast, blockade of IL-12 in BALB/c mice was detrimental to host survival. A blunted serum IL-12 response at 18 h after CLP was exhibited in STAT4−/− mice. These results suggest several critical roles for the STAT4 pathway in the resolution of polymicrobial infections. Additionally, the disparate effects observed with IL-12 blockade and STAT4 deficiency on host survival suggest that IL-12 may activate alternate pathways promoting survival.

Alteration of mononuclear cell immune-associated antigen expression, interleukin-1 expression, and antigen presentation during intra-abdominal infection.

Intact peritoneal macrophage (Mø) function is critical to successful localization of intra-abdominal infection. Peritoneal macrophage antigen presentation capacity (APC), interleukin-1 (IL-1) expression, and immune-associated (la) antigen expression and abscess formation were determined following cecal ligation and puncture. APC and IL-1 expression were measured by coculture with a T-helper cell clone and by measuring subsequent proliferation, la expression was determined in blood, peritoneal Mø, and splenocytes using anti-la monoclonal antibody stain and flow cytometric analysis. Significant reductions in both la expression and APC were found 1 and 4 days after CLP with no change in IL-1 expression. Muramyl dipeptide, which enhances Mø phagocytosis, partially abrogated the depression in antigen presentation but did not affect la expression. Peritoneal Mø la expression and APC, but not IL-1 expression, were depressed after experimental peritonitis. The recovery of Mø function by day 14 coincides with clinical recovery and abscess formation, and restoration of early Mø depression may improve outcome.

Heparin binding protein increases survival in murine fecal peritonitis.

ObjectiveTo test the effectiveness of recombinant heparin-binding protein (HBP), a neutrophil-derived multifunctional protein with monocytic-specific properties, in fecal peritonitis and polymicrobial sepsis. DesignProspective, controlled animal trial. SettingAnimal research laboratory. SubjectsSwiss Webster mice challenged with cecal ligation and puncture (CLP) and treated with recombinant HBP and 60 mg/kg cefoxitin twice a day. InterventionsHBP was administered to mice at different concentrations and different intervals before and after CLP. Rat albumin (1%) was administered to control animals. Measurements and Main ResultsMortality Rate: Survival was increased in mice pretreated intraperitoneally 24 hrs before CLP with 10 &mgr;g or 100 &mgr;g of HBP without cefoxitin (p = .01, Cox-Mantel log-rank test). Compared with control animals, survival was increased significantly (from 5% to 47%, p = .014) in mice that received cefoxitin and 50 &mgr;g ip HBP immediately after CLP, followed by continuous administration of HBP (12 &mgr;g/24 hrs). Intravenous administration of HBP (0.1, 1, and 10 &mgr;g) at the time of CLP showed an opposite dose effect; low doses (0.1 &mgr;g) prolonged early survival, whereas high dose (10 &mgr;g) shortened survival (p = .036). Compared with control animals, overall survival was not different. Chemotaxis: Cytospin preparations from peritoneal exudate cells (PECs) 48 hrs after administration of 10 &mgr;g and 100 &mgr;g ip HBP demonstrated a 1.7-fold increase in the total number of macrophages compared with carrier control (p < .05). Phagocytosis: A flow cytometric in vitro assay demonstrated that administration of 10 &mgr;g ip HBP alone did not enhance phagocytosis of fluorescent Escherichia coli in PECs. However, 24-hr pretreatment with 10 &mgr;g of HBP followed by CLP increased phagocytosis in PECs 1.8-fold compared with the control CLP group (p = .04). Receptor expression: CD16/CD32w expression in PECs did not change after HBP or CLP. CD11b and CD18 expression in PECs was increased significantly after CLP compared with PECs from non-CLP-challenged animals (p < .05). Pretreatment with 10 &mgr;g of HBP did not further enhance CD11b/CD18 expression in PECs. ConclusionsRecombinant HBP increases survival in murine fecal peritonitis. The mechanisms by which HBP reduces septic death are not fully understood, but they include monocyte chemotaxis and increased phagocytosis of E. coli by PECs. Our data suggest that the inflammatory response induced by CLP is important for the effect of HBP to enhance phagocytosis.

Vaccinia virus complement control protein increases early bacterial clearance during experimental peritonitis.

BACKGROUNDnComplement is one of the first immunological pathways activated in peritonitis. It functions to initiate and augment the innate immune response. Complement activation has also been shown to contribute to multiple organ failure after sepsis. Vaccinia virus complement control protein (VCP) is an immunomodulatory protein encoded by vaccinia virus and binds complement components C3b and C4b of the complement cascade to inhibit both the classical and alternative pathways of complement activation. This study investigates the effect of complement inhibition by recombinant (r) VCP on bacterial clearance after cecal ligation and puncture (CLP).nnnMETHODSnSwiss Webster mice were intravenously given either 20 mg/kg rVCP in 0.2 mL of normal saline, or 0.2 mL of normal saline alone, at the time of CLP. After 4 and 18 h, samples of peritoneal washout, blood, liver, and lung were collected for bacteriology, myeloperoxidase (MPO) assay for neutrophil accumulation, differential cell counts, and interleukin (IL)12 ELISA. Statistical analysis was by Mann-Whitney U test for bacteriology, and analysis of variance (ANOVA) for MPO and IL-12 concentrations.nnnRESULTSnAerobic and anaerobic bacterial levels were significantly lower at 4 h after treatment with rVCP (p < 0.05) in peritoneal lavage, blood, and liver compared with controls. There were no differences in bacterial levels at 18 h. There were no differences in myeloperoxidase concentrations or in the differential cell counts between the groups at either 4 or 18 h after CLP. IL-12 concentrations in serum or peritoneal washout were also not different.nnnCONCLUSIONSnrVCP enhances early bacterial clearance in mice after CLP, although not through neutrophil recruitment, as MPO concentrations and cell counts were not different. rVCP may, however, increase neutrophil function potentially by prevention of accumulation of complement factors that inhibit leukocytes. Further studies will be needed to elucidate this pathway.

Leukocyte—Endothelial Cell Interactions: Review of Adhesion Molecules and Their Role in Organ Injury

The causes of multiple organ injury, which are likely multifactorial, include microcirculatory vasoconstriction, local organ inflammatory mediator production, endothelial activation, and inappropriate polymorphonuclear neutrophil (PMN) sequestration and activation. This review focuses on leukocyteendothelial interactions involving neutrophils and leading to tissue injury.