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Polymorphonuclear neutrophils (PMNs) and macrophages are recognized to serve as the first line of defense against bacterial contamination during the perioperative period. Chemoattractants produced by macrophages cause PMN accumulation at the inflammatory site. Proinflammatory cytokines released by macrophages increase adhesion molecule expression on the surface of endothelial cells. P- and E-selectins produce leukocyte rolling, whereas beta 2-integrin-ICAM-1 interaction causes firm adhesion of leukocytes to the endothelium, followed by migration to the interstitium. Thus appropriate activation of the inflammatory cascade leads to leukocyte migration to the infectious focus. In particular, during the first several hours after the onset of bacterial contamination, massive exudation of PMNs is generally observed. PMNs and macrophages phagocytose the bacteria via opsonization and kill them with oxygen products or proteolytic enzymes. Malnutrition and the lack of enteral nutrition are assumed to impair PMN-macrophage-dependent host defense by derangement of adhesion molecule expression and the cytokine milieu. Recently, sticky PMNs before surgery have been reported to increase infectious morbidity after surgery. In addition, augmented alpha 4-integrin-VCAM-1 interaction has been demonstrated during sepsis, which is possibly a mechanism for increased PMN adhesion and resultant organ injury. Further study is needed to clarify the mechanisms of the disturbed function of PMNs and macrophages under various conditions.

Appropriate polymorphonuclear neutrophil (PMN) recruitment is essential for host defense against infection. We investigated the significance of the preoperative PMN adhesion-migration process, as assessed by the flow chamber method, on postoperative infectious complications. Thirty-one consecutive patients with gastrointestinal malignancies, 21 colorectal and 10 gastric, who were undergoing elective surgery were enrolled. PMNs, isolated preoperatively from each patient's venous blood, were perfused onto a tumor necrosis factor alpha-stimulated human umbilical vein endothelial cell (HUVEC) monolayer through the flow chamber. We evaluated the adherent PMN number, the migrated PMN number, and the stuck PMN number by directly inspecting PMN interactions with a HUVEC monolayer under continuous shear flow simulating postcapillary venules. The expression of adhesion molecules on circulating PMNs was also measured. Patients were grouped into an infectious and a noninfectious group according to the occurrence of postoperative infectious complications defined by the Centers for Disease Control criteria. Eleven patients developed postoperative infectious complications. Although the number of preoperative in vitro adherent PMNs in patients with postoperative infection was significantly higher than in those without postoperative infection (P = 0.01), migrated PMN number was similar in both groups. Stuck PMN number tended to be higher in the infectious group than in the noninfectious group. The migrated PMN number showed a significant positive correlation with the adherent PMN number in the noninfectious group but not in the infectious group. Preoperative CD31 expression on circulating PMNs was significantly lower in the infectious group than in the noninfectious group. Preoperative in vitro derangement of the PMN adhesion-migration process is closely associated with postoperative infectious complications.

Dietary restriction impairs polymorphonuclear neutrophil (PMN) recruitment into the local inflammatory site, resulting in susceptibility to infection. Probiotics enhance host immunity via conditioning host intestinal microflora. Oral administration of Bifidobacterium longum culture condensate (BCC) in a diet-restricted murine peritonitis model may enhance PMN recruitment into the inflammatory site. Male ICR mice (n = 40) were assigned in equal numbers to control or BCC groups and subjected to 75% restricted food intake for 7 d. During dietary restriction, controls received only standard mouse chow, whereas the BCC group received standard mouse chow containing 1% BCC. Mice were killed before (0 h) or after (2 or 4 h) intraperitoneal glycogen injection. Peritoneal lavage fluid and exudative cells were recovered by peritoneal lavage. Peritoneal exudative cell number was counted. Tumor necrosis factor-alpha, interleukin-6, macrophage inflammatory protein-2, and interleukin-10 concentrations in peritoneal lavage fluid were determined by enzyme-linked immunosorbent assay. CD11b, CD18, CD31, and CD62L expressions on circulating PMNs were measured by flow cytometry. Oral BCC administration upregulated PMN recruitment into the peritoneal cavity and increased peritoneal fluid cytokine concentrations as well as CD18 and CD62L expressions on circulating PMNs during glycogen-induced peritonitis. Oral BCC administration in a diet-restricted murine peritonitis model augmented PMN recruitment into the inflammatory site by upregulating cytokine concentrations in the local inflammatory site and adhesion molecule expression on circulating PMNs. Oral BCC administration may be a favorable modality for improving dietary restriction-induced host immunosuppression.

Antibiotic therapy is an essential treatment for gram-negative bacterial infections. Antibiotic-induced endotoxin release and subsequent production of inflammatory cytokines reportedly depend on the type of antibiotic action. This study examined the effects of various beta-lactam antibiotics on cell death of human polymorphonuclear neutrophils (PMNs) cocultured with Escherichia coli (E. coli) in vitro. E. coli morphology after antibiotic treatment was determined. PMNs and E. coli were cocultured with antibiotics for 0, 4, or 12 h. Levels of endotoxin and cytokines (TNF-alpha, IL-1beta, and IL-6) in the supernatants were measured. The filtrates of antibiotic-treated E. coli supernatants were cocultured with PMNs for 0, 4, or 12 h. In all experiments, ampicillin (ABPC), cefazolin sodium (CEZ), cefoperazone sodium (CPZ), latamoxef sodium (LMOX), imipenem (IPM), and polymyxin B sulfate (PLB) were used at 30 microg/mL. PMNs were isolated from healthy volunteers. PMN cell death was assessed by flow cytometry and light microscopy. ABPC, CEZ, CPZ, and LMOX, which induce bacterial filament formation with lysis, caused PMN necrosis when cocultured with E. coli. In contrast, IPM, which induces bacterial spheroplast formation with lysis, caused PMN apoptosis. Levels of endotoxin, TNF-alpha and IL-6 in the supernatants with IPM and PLB were significantly lower than in those with other beta-lactam antibiotics. The filtrates of IPM- and PLB-treated E. coli supernatants induced PMN apoptosis, whereas those treated with other beta-lactam antibiotics increased PMN necrosis. Beta-lactam antibiotics have different impacts on the types of PMN cell death after E. coli killing. Underlying mechanisms and the clinical relevance of IPM-induced PMN apoptosis in severe gram-negative infection warrant further investigation.

Neutrophil adhesion to the pulmonary endothelium is prerequisite to neutrophil transmigration and activation, both of which may lead to lung injury. A simple method to evaluate neutrophil adherence in the lung would be useful for developing new strategies for neutrophil-mediated lung injury. The purpose was to establish a simple method to evaluate neutrophil adhesion in the lung using ex vivo fluorescence microscopy. Rats were anesthetized, and the right jugular veins were catheterized. Neutrophils were isolated from another set of rats and labeled with 5,(6)-carboxyfluorescein diacetate. Animals were killed 120 s after a 1 x 10(6) labeled neutrophil injection. The pulmonary labeled neutrophil number was counted under a fluorescence microscope. In the first experiment, rats were given 0, 20, 200, or 2000 microg/kg lipopolysaccharide (LPS) i.p. At 4 h after challenge, the pulmonary labeled neutrophil number was determined. Kinetic studies were also performed at 0, 1, 4, and 8 h after 200 microg/kg LPS. Finally, anti-ICAM-1 Ab was injected i.v. before LPS 200 microg/kg, and the labeled neutrophil number in the lung was determined at 4 h. The number of pulmonary labeled neutrophils was higher after LPS 200 or 2000 microg/kg than after the other doses. The pulmonary labeled neutrophil number was increased at 4 h compared with the other time points. ICAM-1 blocking normalized the pulmonary labeled neutrophil number in the LPS group. In conclusion, our method seems to reflect ICAM-1-mediated neutrophil adherence to the endothelium in the present setting. This simple technique may be useful for evaluating neutrophil adhesion.

Hypothesis: Patients with malnutrition are susceptible to infection. Polymorphonuclear neutrophils (PMNs) are the major effector of the nonspecific immune response in host resistance to infection. Dietary restriction may impair PMN-mediated immunity in the peritoneal cavity by reducing PMN exudation, adhesion molecule expression on PMNs, and chemokine production., Design: Randomized study of murine glycogen-induced peritonitis with dietary restriction., Setting: University research laboratory., Materials: Male C57BL/6J mice., Interventions: Mice (N = 204) were assigned to ad libitum, moderate, and severe diet-restricted groups receiving mouse chow ad libitum (132 g/kg, 66 g/kg, and 33 g/kg daily for 7 days, respectively). After dietary restriction with or without 1 day of refeeding, mice were administered glycogen intraperitoneally to induce cell exudation., Main Outcome Measures: CD11b, CD18, and CD62L expressions on circulating PMNs, phagocytosis, and reactive oxygen intermediate production by exudative PMNs were measured after glycogen installation. The levels of PMN-specific chemokine, macrophage inflammatory protein 2 (MIP-2), in peritoneal lavage fluid were also measured. These parameters were measured after glycogen installation in the refeeding experiment., Results: Seven days of dietary restriction decreased CD11b/CD18 expression on circulating PMNs, MIP-2 levels in peritoneal lavage fluid, and subsequent PMN exudation into the peritoneal cavity early in peritonitis. Both CD11b and CD18 expression on circulating PMNs and MIP-2 levels correlated significantly with numbers of exudative PMNs. Seven days of dietary restriction also impaired phagocytosis, while up-regulating reactive oxygen intermediate production by exudative PMNs. Only 1 day of ad libitum refeeding normalized CD11b/CD18 expression with PMN exudation into the peritoneal cavity., Conclusions: Short-term dietary restriction impairs PMN exudation into local inflammatory sites in murine peritonitis by reducing CD11b/CD18 expression and MIP-2 production. Even brief nutritional replenishment in diet-restricted patients may improve host defense via restoring these PMN functions and chemokine production at local inflammatory sites.

Background: The fate of exudative polymorphonuclear neutrophils (PMNs) at the local site after surgery is not well understood. We evaluated the fate and functions of exudative PMNs at the local site in patients who were undergoing major surgery. We also investigated the relation between PMN apoptosis and cytokine levels at the local site during the postoperative period., Methods: Exudative PMNs were isolated from 11 patients during the postoperative period. Apoptosis, reactive oxygen intermediates (ROI) production, CD16, and tumor necrosis factor receptor expression of the PMNs were determined by flow cytometry. Cytokine levels in the drainage fluid were measured., Results: Exudative PMN apoptosis was markedly inhibited on postoperative day 1 and then increased in a time-dependent manner. IL-6 and granulocyte macrophage colony-stimulating factor were significant factors to inhibit exudative PMN apoptosis; tumor necrosis factor-alpha and IL-10 were the factors to increase apoptosis. ROI production and CD16 expression of exudative PMNs were augmented when PMN apoptosis was inhibited in the early postoperative period., Conclusions: Exudative PMN apoptosis was inhibited after surgery; PMN function was augmented after surgery. Cytokines at the local site may modulate exudative PMN apoptosis. Exudative PMN apoptosis reflected the inflammatory response after surgery. Understanding the mechanisms of PMN apoptosis and its pathophysiologic significance at local inflammatory sites in vivo may help in the design of more rational treatments.

Background: The effects of malnutrition on polymorphonuclear neutrophil (PMN) exudation are not well understood. The purpose of this study was to examine the effects of short-term dietary restriction on adhesion molecule expression on circulating PMNs and PMN exudation into the inflamed site in a glycogen-induced peritonitis model., Methods: Twelve mice were randomly assigned to one of two groups. The ad libitum and diet-restricted groups received mouse chow ad libitum (estimated consumption: 132 g/kg per day) and 33 g/kg per day, respectively, for 7 days. Then, 2 mL of a 1% glycogen solution was intraperitoneally administered to all mice. After 4 hours, the animals were killed. Whole blood was drawn by cardiac puncture. Peritoneal exudative cells were harvested by lavaging the peritoneal cavity. Expressions of CD11b, CD18, and CD62L were measured by flow cytometry., Results: Dietary restriction did not affect the numbers of circulating leukocytes, PMNs, or monocytes. However, CD11b and CD18 expressions on circulating PMNs were significantly lower in the diet-restricted than in the ad libitum group. In contrast, CD62L expression on circulating PMNs was not affected by dietary restriction. The number of exudative PMNs was significantly lower in the diet-restricted group than in the ad libitum group. The expressions of CD11b, CD18 and CD62L on exudative PMNs were unaffected by dietary restriction. There was a significant positive correlation between exudative PMN numbers and CD18 expression on circulating PMNs., Conclusions: Severe dietary restriction in our murine model decreased beta2 integrin expression on circulating PMNs and inhibited PMN exudation into inflamed sites in the early phase of inflammation. These events may increase susceptibility to bacterial infection. Nutritional replenishment may improve host defense in part by enhancing PMN adhesion molecule expression.

The energy substrate for neutrophils has been believed to be glucose. However, a recent investigation has demonstrated that neutrophils use glutamine (Gln) as well as glucose. Nevertheless, little is known about the effects of Gln on neutrophil function. Thus, this study was designed to investigate the effects of Gln on phagocytosis and reactive oxygen intermediate (ROI) production by neutrophils from postoperative patients in vitro. Eleven patients who had undergone major gastrointestinal surgery were randomly selected. Peripheral blood was drawn before surgery and on postoperative days (PODs) 1, 3, and 7. The blood was washed with medium to remove plasma. Washed whole blood was incubated in RPMI 1640 medium containing neither Gln nor glucose for 24 h at 37 degrees C. The medium was supplemented with Gln at a concentration of 0, 500, 1000, or 2000 microM. Whole blood was then assessed for phagocytosis by flow cytometry using fluorescent beads. ROI production by phagocytes was measured by flow cytometry using dihydrorhodamine 123. In each assay, the neutrophil population was gated and analyzed. Serum amino acids were also measured. Postoperative serum Gln level decreased significantly until POD 7. Phagocytosis by neutrophils on PODs 3 and 7 was significantly greater at 2000 microM Gln than at other Gln concentrations. Neutrophil ROI production was significantly greater at 2000 microM Gln than at 0 microM Gln at each time point. In conclusion, supplemental Gln enhances both phagocytosis and ROI production by neutrophils from postoperative patients in vitro.

The energy source for neutrophils (PMNs) has long been believed to be glucose. However, it has been shown recently that PMNs use glutamine as well as glucose. Nevertheless, the comparative effects of glucose and glutamine on PMN function remain to be clarified. This study investigated the relative effects of glucose and glutamine on reactive oxygen intermediate (ROI) production by PMNs. In experiment 1, PMNs (1 x 10(6)/mL) isolated from healthy volunteers were incubated in RPMI 1640 medium containing neither glucose nor glutamine for 4, 12, 18, and 24 h at 37 degrees C. The medium was supplemented with 0 or 200 mg/dL (0 or 11 mM, respectively) glucose and glutamine (0, 0.5, 1, or 2 mM). PMN cell death was assessed on the basis of hypodiploid DNA by flow cytometry using propidium iodide DNA staining. ROI production by PMNs was determined by flow cytometry using dihydrorhodamine 123. In experiment 2, isolated PMNs were cultured in RPMI 1640 medium containing neither glucose nor glutamine. The medium was supplemented with glucose (0 or 11 mM) and a competitive inhibitor of glycolysis, 2-deoxy-D-glucose (2-DG; 0 or 20 mM). Each medium was supplemented with glutamine (0, 0.5, 1, or 2 mM) and incubated for 12 h at 37 degrees C. Then, ROI production by PMNs was measured. PMN cell death was not affected by glucose or glutamine in this experiment. In contrast, ROI production by PMNs was greater at 11 mM glucose than at 0 mM glucose at all incubation times studied. At 11 mM glucose, supplemental glutamine enhanced PMN ROI production after 18 and 24 h culture. In contrast, at 0 mM glucose, glutamine augmented ROI production by PMNs after 12 h as well as with 18 and 24 h incubations. PMN ROI production after 12 h culture was significantly greater at 11 mM glucose without 2-DG than at both 11 and 0 mM glucose with addition of 2-DG. In addition, supplemental glutamine enhanced ROI production by PMNs when 2-DG was added at 11 and 0 mM glucose. Glucose is essential for PMN ROI production. Under conditions of glucose depletion in vitro, glutamine is of importance in ROI production by PMNs, whereas the enhancing effect of glutamine on PMN ROI production is minor compared to that of glucose.

This study was designed to investigate how live Escherichia coli influence the fate of polymorphonuclear neutrophils (PMNs) in vitro. PMNs from 10 healthy volunteers were cocultured with or without live E. coli at different ratios. Heat-killed E. coli (Hk) were also added to PMNs at a ratio of 1:10. The PMNs were then analyzed by flow cytometry for cell death, reactive oxygen intermediates (ROI) production, and CD16 expression. Morphologic features were also assessed. PMN apoptosis was confirmed by DNA gel electrophoresis. Low doses of E. coli (PMN:E. coli ratios of 1:0.01 and 1:0.1) inhibited PMN apoptosis. In contrast, a high dose of E. coli (PMN:E. coli ratio of 1:10) increased PMN necrosis. ROI production was significantly greater at PMN:E. coli ratios of 1:10 and 1:10 (Hk) than at ratios of 1:0.01 and 1:0.1, or in PMNs cultured alone after a 15 or 30 minute coculture. CD16 expressions were significantly lower in PMNs cocultured with E. coli than in those cultured alone after a 4 or 12-h coculture. Tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6 levels in cell-free supernatants were also measured. The mean percentages of apoptosis at PMN:E. coli ratios of 1:0.01 and 1:10 (Hk), and in PMNs cultured alone after a 12-h coculture showed significant inverse correlations with these cytokine levels in cell-free supernatants at 12 h. Our results demonstrate that low doses of live E. coli inhibits predominantly PMN apoptosis, whereas a high dose of E. coli increases necrosis. Augmented PMN bactericidal function, via inhibition of PMN cell death, may be beneficial for host defense against bacterial infection and/or sepsis.

Hypothesis: The levels of intestinal interleukin 10 and interleukin 4, inhibitors of intercellular adhesion molecule-1 (ICAM-1) expression, decline with total parenteral nutrition (TPN). These cytokine changes induced by lack of enteral nutrition may increase ICAM-1 expression, resulting in polymorphonuclear neutrophil accumulation in intestine., Design: Prospective randomized experimental trials., Setting: Laboratory., Materials: Male mice., Interventions: Sixty-three mice were randomized to chow, intravenous TPN, or intragastric TPN., Main Outcome Measures: Experiment 1: After diet manipulation, iodine 125-labeled anti-ICAM-1 antibody and iodine 131-labeled nonbinding antibody were injected to quantify ICAM-1 expression on endothelial cells in the lung, liver, kidney, and small intestine. Measurement of myeloperoxidase was used to quantify polymorphonuclear neutrophil accumulation in the organs. Experiment 2: Intestine was harvested for both ICAM-1 and myeloperoxidase levels after chow refeeding of mice in the intravenous TPN group., Results: In experiment 1, uninjured mice fed intravenous TPN showed significantly increased intestinal ICAM-1 expression and polymorphonuclear neutrophil accumulation with no significant changes in the lung, liver, or kidney. No changes occurred in mice fed chow or intragastric TPN. In experiment 2, reinstitution of enteral feeding returned intestinal ICAM-1 and myeloperoxidase levels to normal., Conclusion: Gut changes associated with lack of enteral feeding induce endothelial changes and an immunologic response, which may influence subsequent responses to injury.

Background: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis. The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method., Study Design: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli. Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused. Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Lung myeloperoxidase (MPO) activity was also determined. Experiment 2: Rats (n = 23) were given 10(9) E. coli ip. Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused. Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1. Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E. coli ip challenge., Results: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers. Lung MPO also positively correlated with E. coli number and labeled neutrophil number in the lungs. Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h. The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h. No remarkable change was observed in the hepatic labeled neutrophil number. There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number. Hemodynamic and blood gas data reflected a hyperdynamic state., Conclusions: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model. Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs. Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis., (Copyright 1999 Academic Press.)

Background: Growth hormone (GH) regulates the immune and metabolic systems; however, the effects of GH on the functions and cell death of polymorphonuclear neutrophils (PMNs) are not well understood. Therefore, this study was designed to investigate the effects of GH on PMN apoptosis, reactive oxygen intermediates (ROI) production, CD16, and Fas expression. We also investigated the effects of GH on the functions of other circulating leukocytes (ie, monocytes and lymphocytes)., Methods: Venous blood was collected from healthy volunteers. Whole blood was washed and pretreated with GH (0 or 100 ng/mL) for 3 hours and then cultured for 0, 4, or 12 hours. PMNs in washed whole blood were analyzed by flow cytometry for cell death, phorbol myristate acetate-stimulated ROI production, CD16, and Fas expression at each time point. Morphologic features also were assessed. PMN apoptosis was confirmed by chromatin staining and DNA gel electrophoresis., Results: GH inhibited PMN apoptosis at 12 hours of culture. GH enhanced ROI production by PMNs and monocytes throughout the 12-hour culture but had no effects on CD16 expression on PMNs. Furthermore, GH decreased Fas expression on PMNs at 4 hours of culture. However, there were no effects of GH on apoptosis of monocytes or lymphocytes for the duration of this experiment., Conclusions: GH pretreatment down-regulates Fas expression on PMNs, inhibits apoptosis, and up-regulates ROI production. GH pretreatment also increases monocyte ROI production. Although activated PMNs have potentially harmful aspects, our results suggest that GH may improve host defense, mainly through enhancement of the PMN functional life span.

Effects of growth hormone (GH) and insulin-like growth factor (IGF)-I on bactericidal capacity of human polymorphonuclear neutrophils (PMNs) were investigated. Venous blood was collected from healthy volunteers. In Experiment 1, PMNs were isolated, incubated with GH or IGF-I, and cocultured with Escherichia coli. E. coli-killing capacity, viability, and CD11b and CD16 expressions of PMNs were then assessed. Both GH and IGF-I enhanced E. coli killing by PMNs. GH preserved PMN viability during E. coli killing, whereas IGF-I enhanced PMN CD11b expression before coculture with E. coli. In Experiment 2, whole blood was washed and incubated with GH or IGF-I. PMNs in washed whole blood were then analyzed for phorbol myristate acetate (PMA)-stimulated CD11b, CD35, and CD16 expressions and production of reactive oxygen intermediates (ROI), as well as phagocytosis with/without anti-CD11b antibody. IGF-I enhanced PMN expressions of CD11b and CD35, but not CD16, stimulated with PMA. Both hormones enhanced phagocytosis, which was abrogated by anti-CD11b antibody, and intracellular ROI production by PMNs. These results indicate that both GH and IGF-I augment human PMN bactericidal capacity, via increased phagocytosis and intracellular ROI production. Preservation of PMN viability by GH and enhanced complement receptor expression by IGF-I may also be associated with augmented PMN bactericidal capacity. Although PMN activation has potentially harmful aspects, these results encourage additional studies to confirm the clinical relevance of exogenous GH or IGF-I for the prevention or management of septic complications in perioperative or critically ill patients especially with low circulating GH and/or IGF-I levels.

Background: As nitric oxide (NO) is an antiadhesive molecule, exogenous NO may modulate neutrophil adhesion in organs. This study was designed to examine the effects of the NO donor SNAP (S-nitroso-acetyl penicillamine) on neutrophil adhesion at the inflammatory site and in remote organs, in peritonitis using a fluorescent microscopic method., Materials and Methods: In experiment 1, rats (n = 12) were given saline or 10 micrograms/kg of SNAP intravenously followed by continuous infusion of saline, or of 2, 20, or 200 micrograms/kg/h SNAP until sacrifice. Ten minutes after injection of saline or SNAP, 10(7) Escherichia coli were injected into the peritoneal cavity. Five hours after challenge, 10(6) fluorescein-labeled neutrophils were infused. Peritoneal samples, lungs, liver, and kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. In experiment 2, rats (n = 25) were treated with saline or 10 micrograms/kg of SNAP intravenously and infused with saline or 20 micrograms/kg/h SNAP; E. coli was injected as in experiment 1. Before or 5 h after challenge, hemodynamic data were obtained. Then, labeled neutrophils were infused for counting of neutrophil numbers in organs. Arterial blood gas data and the circulating neutrophil number were also determined., Results: Experiment 1. Twenty and 200 micrograms/kg/h SNAP infusions tended to reduce labeled neutrophil numbers in lungs, while all three SNAP doses decreased the peritoneal labeled neutrophil numbers., Results: Experiment 2. Five hours after bacterial injection, SNAP infusion simultaneously decreased both pulmonary and peritoneal labeled neutrophil numbers. SNAP had no effect on hemodynamic and blood gas data, or on circulating neutrophil numbers., Conclusion: NO donors may be useful for preventing neutrophil-associated lung injury, but should be used with caution in light of the possible adverse effects on host defense in the peritoneal cavity.

Neutrophils play an important role in host defense by phagocytosing and destroying invading bacteria. A recent investigation revealed that glutamine (Gln) augmented the in vitro bactericidal activity of neutrophils from burn patients. However, it is unclear whether Gln enhances the function of neutrophils in postoperative patients. This study was designed to investigate the effect of Gln on the in vitro Escherichia coli-killing activity of neutrophils from postoperative patients. Nine randomly selected patients were included in this study. On the morning of the first postoperative day, blood was drawn and neutrophils were isolated. Eight healthy volunteers served as controls. E. coli was opsonized with pooled normal serum. Neutrophils (5 x 10(6)), together with opsonized E. coli (5 x 10(5)), were incubated for 2 h at 37 degrees C in Hanks' balanced salt solution supplemented with 0, 100, 500, or 1000 nmol/mL of Gln. The bactericidal function of neutrophils was determined by counting the number of viable bacteria. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-8, and granulocyte elastase levels in the cell culture supernatant were measured. Plasma C-reactive protein (CRP), cortisol, and amino acids were also analyzed. The plasma concentration of Gln was significantly lower in the postoperative patients than in the controls. Following culture with patient neutrophils, the number of viable E. coli decreased by 26% as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL (P < 0.01). We defined the Gln 1000/Gln 500 ratio of the number of viable bacteria as the number of viable E. coli at an in vitro Gln concentration of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL. A positive correlation was thus demonstrated between the plasma Gln level and the Gln 1000/Gln 500 ratio of the number of viable bacteria in the patients (r = 0.69, P = 0.04). This finding indicated that as plasma Gln fell, there was an enhancement of neutrophil E. coli-killing activity by neutrophils in in vitro tests when the Gln concentration was increased from 500 to 1000 nmol/mL. Gln supplementation caused no appreciable changes in TNF-alpha, IL-1 beta, IL-8, or granulocyte elastase levels in cell culture supernatants. A negative correlation was recognized between the patient plasma Gln level and the Gln 1000/Gln 500 ratio of the cell culture supernatant IL-8 level (r = -0.73, P = 0.025). In conclusion, Gln supplementation enhanced the in vitro bactericidal function of neutrophils from postoperative patients.

Nitric oxide (NO) is a regulator of leukocyte adhesion in the microcirculation. This study was designed to examine the effects of a NO synthase inhibitor on neutrophil adhesion in the peritoneum, lung, liver, and kidney in a rat peritonitis model using a fluorescence microscopic method. Sprague-Dawley rats were given normal saline (control) or N omega-nitro-L-arginine methyl ester (L-NAME) at dosages of 10 mg/kg (N10) or 100 mg/kg (N100) (n = 66) intraperitoneally. One hour after pretreatment fluorescein-labeled neutrophils were infused without bacterial challenge (0 hr). Other rats received an injection of 10(7) Escherichia coli into the peritoneal cavity 1 hr after pretreatment. Labeled neutrophils were infused 1 and 5 hr after bacterial challenge. Just 2 min after neutrophil injection, blood samples were obtained and the animals were killed. Five peritoneal samples (omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Combined plasma nitrite/nitrate levels were determined. In another set of rats (n = 36), an arterial catheter was inserted after L-NAME treatment and bacterial challenge. At 0, 1, 5, and 12 hr after challenge, blood pressure, heart rate, and arterial blood gas data were measured. One hour after E. coli challenge, the number of neutrophils in the peritoneum was significantly lower in both L-NAME-treated groups than in the control group. In contrast, the number of labeled neutrophils in the lungs was significantly higher in the N100 group than in the control group. Neutrophil accumulation in the lungs and peritoneum at 0 and 5 hr and in the liver and kidney at 0, 1, and 5 hr did not differ among groups, nor did combined plasma nitrite/nitrate levels. L-NAME treatment had no influence on either hemodynamic or blood gas data. In conclusion, administration of L-NAME increases neutrophil adhesion in the lung, while decreasing that in the peritoneum. NO plays an important role in neutrophil adhesion at the inflammatory site, as well as in remote organs, during peritonitis. NO inhibition may be detrimental, due to neutrophil sequestration, in this peritonitis model.

Background: Peritonitis remains a major infectious problem. Neutrophil influx into the peritoneal cavity is one of the most important host defense mechanisms. However, no studies have focused on the site of neutrophil exudation. This study examined the primary anatomic site of neutrophil exudation in bacterial peritonitis., Study Design: Fifty-five rats were injected intraperitoneally with saline solution (control group) or 10(7) Escherichia coli (peritonitis group). In experiment 1, 1 x 10(6) fluorescein-labeled neutrophils were infused 3 hours after the challenge. Then, peritoneal-lavaged fluids and peritoneal tissues (the greater omentum, mesentery, parietal peritoneum, colon, and ileum) were obtained. Subpopulations of peritoneal exudative cells and numbers of labeled neutrophils in tissues were counted. In experiment 2, labeled neutrophils were infused at 10 minutes and at 1 and 5 hours after the challenge. Peritoneal tissues were also harvested. The number of labeled neutrophils in each tissue was determined., Results: In experiment 1, numbers of labeled peritoneal neutrophils and exudative neutrophils were higher in the peritonitis group than in the control group. Numbers of exudative neutrophils showed a positive correlation with numbers of labeled peritoneal neutrophil. In experiment 2, at 1 and 5 hours after the challenge, the number of labeled neutrophils was higher in the peritonitis group than in the control group. The number of neutrophils in the omentum was higher than the number in other peritoneal tissues., Conclusions: Our fluorescence microscopic method is useful for detecting neutrophil adhesion. Neutrophil exudation into the peritoneal cavity was most marked in the omentum. The greater omentum may play an important role in host defense as a source of exudative neutrophils.