Mark A. Mercer-Jones

Regulation of early peritoneal neutrophil migration by macrophage inflammatory protein-2 and mast cells in experimental peritonitis.

Neutrophil (PMN) migration into the peritoneal cavity in response to fecal peritonitis is an important mechanism of host defense against bacterial invasion. We show that the murine C‐X‐C (PMN‐specific) chemokine, macrophage inflammatory protein‐2 (MIP‐2), on intraperitoneal injection in mice, causes PMN migration into the peritoneum. MIP‐2 mRNA and protein were expressed by peritoneal leukocytes after cecal ligation and puncture (CLP) in mice and neutralization of MIP‐2 reduced peritoneal PMN migration. A prerequisite for neutrophil‐endothelial adhesion and subsequent migration from the circulation is selectin‐mediated rolling. Pretreatment of mice with an anti‐P‐selectin antibody before intraperitoneal injection of MIP‐2 significantly reduced peritoneal PMN migration. However, there are no reports that a C‐X‐C chemokine can up‐regulate endothelial selectins. We postulated that MIP‐2, when injected intraperitoneally, interacts with a cell that is known to release factors that up‐regulate endothelial selectins. A likely candidate is the mast cell, which contains histamine and tumor necrosis factor α (TNF‐α), and both of these factors induce selectins. Intraperitoneally injected MIP‐2 caused an early significant increase in peritoneal TNF‐α, whereas histamine levels were unaffected. In a subsequent experiment, mast cell‐deficient mice and their normal controls were then injected intraperitoneally with MIP‐2 or underwent CLP. Significantly fewer PMNs migrated into the peritoneal cavity in the mast cell‐deficient mice after MIP‐2 injection or CLP. Thus, our findings indicate that mast cells and MIP‐2 are necessary for PMN migration into the peritoneum in response to intra‐abdominal infection, and that MIP‐2 appears to facilitate this through an increase in TNF‐α release. J. Leukoc. Biol. 65: 249–255; 1999.

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.

THE PULMONARY INFLAMMATORY RESPONSE TO EXPERIMENTAL FECAL PERITONITIS : RELATIVE ROLES OF TUMOR NECROSIS FACTOR-ALPHA AND ENDOTOXIN

The roles of endotoxin (LPS) and tumor necrosis factor-α (TNF-α) in the causation of organ injury during sepsis are unclear. To study LPS and TNF-α in the genesis of lung inflammation after cecal ligation and puncture (CLP), we used endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive mice (C3H/HeOuJ). We examined lung neutrophil sequestration, interleukin 1 (IL-1)β mRNA expression, IL-1β protein expression, and injury. We also determined the expression of two C-X-C chemokine mRNAs, macrophage inflammatory protein-2 (MIP-2) and KC, in the lung to determine whether in vivo, endotoxin, or TNF-α are significant modulators of MIP-2 and KC mRNA expression. After CLP, increased neutrophils sequestrated in the lungs of both strains of mice and coincided with an increase in expression of IL-1β, MIP-2 and KC mRNAs, and IL-1β protein. Lung and serum TNF-α were significantly increased in the C3H/HeOuJ strain but not in the C3H/HeJ strain. Histologic studies of the lung revealed similar injury in both strains. Our results suggest that bacterial factors other than endotoxin cause lung neutrophil sequestration and injury after CLP and further, that TNF-α production is not a prerequisite. Our findings also suggest a potential role for local pulmonary chemokine production in the control of neutrophil sequestration after CLP.

NEUTROPHIL SEQUESTRATION IN LIVER AND LUNG IS DIFFERENTIALLY REGULATED BY C-X-C CHEMOKINES DURING EXPERIMENTAL PERITONITIS

C-X-C chemokines play an important role in the migration and activation of neutrophils (PMNs) during an inflammatory event. We measured mRNA and protein expression of the murine C-X-C chemokines macrophage inflammatory protein-2 (MIP-2) and KC in the lungs, liver, blood, and peritoneal cavity of Swiss Webster mice after cecal ligation and puncture (CLP). Neutralizing antibodies to MIP-2 and KC were also used to determine the biological effects of these chemokines on neutrophil sequestration and organ injury in the CLP model. The data showed that early after CLP, MIP-2 mRNA and protein were expressed predominantly by the lung, whereas KC mRNA and protein were expressed by the liver. Inhibition of MIP-2 reduced both lung neutrophil sequestration and peritoneal neutrophil migration. Inhibition of KC had no effect on overall neutrophil sequestration in liver but reduced injury as measured by serum transaminases. An early survival benefit was found with anti-KC treatment, although overall survival was not different. Our study showed a differential expression by organs of C-X-C chemokines during sepsis and suggested that such chemokine effects are tissue-specific.

SEPSIS AND SEPTIC COMPLICATIONS IN THE SURGICAL PATIENT: WHO IS AT RISK?

The estimation of patients who are at risk for infection, sepsis, and organ dysfunction/failure is crucial not only for inclusion in treatment algorithms but also for entry into appropriate clinical trials of prophylaxis and therapy. Patients on the surgical service who have sustained major trauma or who have undergone transplantation are clearly at the greatest risk. Other immunosuppressed patients at risk for sepsis include those receiving myelosuppressive chemotherapy, those with overwhelming malignancy, and those who suffer from cirrhosis, diabetes mellitus, and severe malnutrition. We have focused on the trauma patient, in whom infection and organ failure are the leading causes of late death, major morbidity, and prolonged hospital stay. Over a 10 yr period, we have surveyed a number of host defense parameters that pertain to an adequate immune response and found a suppressed response shortly after injury in many. All were anergic to a standard skin test panel, and the duration of anergy varied with the clinical course of infection. Immunoglobulin levels were low after major injury as well as specific antibodies to both Gram-positive and Gram-negative organisms. The ability of serum from the trauma patient to opsonize heat-killed bacteria was markedly depressed 24 h after injury in those patients who subsequently died of infection. Class II major histocompatibility antigen expression on peripheral blood monocytes correlated closely with clinical outcome and led to the development of an Outcome Predictive Score. This score can identify patients within hours of hospitalization who are at risk of subsequently developing overt clinical infection and sepsis. Intervention then can be applied to such at-risk populations prior to the onset of sepsis and to evaluate the efficacy of prophylaxis. Patients in whom prophylaxis fails could be eligible for trials of therapeutic intervention as well.

Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60‐min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate‐labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright‐red fluorescent PMNs. Our experiments with two different bacteria, various PMN‐to‐bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

CD14 expression in injured patients correlates with outcome.

OBJECTIVE The authors determined the correlation between monocyte CD14 expression and outcome in severely injured patients. SUMMARY BACKGROUND DATA Human leukocyte antigen-DR (HLA-DR) expression CD14 positive monocytes correlates with the development of major infection and subsequent death in severely injured patients. Recent studies show that CD14 is not only a marker for mature monocytes, but also is an important endotoxin/lipopolysaccharide receptor. METHODS Flow cytometry data obtained by dual staining techniques (CD14 and HLA-DR) of monocytes in 213 severely injured patients were analyzed over a 30-day period. Outcome criteria included survival and the development of both major and minor infections. RESULTS The percentage of cells expressing CD14 (%CD14) correlated with clinical outcome, reaching significance (p < 0.05) between noninfected survivors (n = 74) and nonsurvivors (n = 21) at days 3, 7, 11, 17, 24, and 30. At days 3, 7, and 17, the %CD14 also was different between noninfected and infected survivors. After 7 days, differences were only seen between survivors and nonsurvivors (p < 0.05). The mean fluorescence intensity (MC CD14) in monocytes of all patients was significantly reduced at day 3 compared with day 1 and remained low for 30 days (p < 0.05). The nonsurvivor group had consistently low MC CD14 values, which were significant at day 5 (p < 0.05). CONCLUSIONS In addition to HLA-DR expression, CD14 expression on monocytes is an indicator of clinical outcome after injury and could represent a more precise target for treatment.

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.

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.