Alpha-defensin 1 (human neutrophil protein 1) as an antichemotactic agent for human polymorphonuclear leukocytes.

Medium conditioned by tumor necrosis factor alpha (TNF-alpha)-stimulated polymorphonuclear leukocytes (PMN) (CM-TNF) suppresses PMN migration. Therefore, we wished to identify the agent(s) in CM-TNF that mediated antichemotactic activity. CM-TNF was fractionated by high-performance liquid chromatography, and one fraction with antichemotactic activity contained the bactericidal protein human neutrophil protein 1 (HNP-1). We showed that HNP-1 suppresses PMN migration to formyl-methionyl-leucyl-phenylalanine but not to interleukin 8.

Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis.

BACKGROUND: Fas/Fas ligand (FasL) system is one of the major pathways triggering apoptosis that has been shown to play an important role in development and pathogenesis of various diseases including liver and gastrointestinal diseases. Studies indicate that FasL deficiency provides a survival advantage in mice subjected to polymicrobial sepsis. However, the extent to which Fas/FasL contributes to organ injury during sepsis is unclear. Thus, the aim of this study was to determine whether in vivo administration of a Fas-signaling inhibitor during sepsis preserves organ function. METHODS: Male adult C3H/HeN mice were subjected to cecal ligation and puncture (CLP) or sham CLP (sham). Twelve hours after CLP, mice received either Fas-receptor fusion protein (FasFP) (200 microg/kg body weight) or the saline vehicle. Twenty-four hours after the onset of sepsis, cardiac output and organ blood flow were measured with radioactive microspheres. Plasma levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were assessed as indexes of liver damage. Changes in systemic cytokines were measured by enzyme-linked immunosorbent assay. RESULTS. The data indicate that although cardiac output and organ blood flow in the liver, intestine, kidneys, spleen, and heart decreased markedly at 24 hours after CLP, treatment with FasFP maintained the measured hemodynamic parameters and improved hepatic, intestinal, and heart blood flow (P <.05) and partially restored spleen and renal blood flow. Moreover, FasFP treatment markedly attenuated the systemic rise in alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and interleukin 10 (P <.05). CONCLUSIONS: These results not only indicate that there is a role for Fas/FasL-mediated processes in the induction of organ injury but suggest that inhibition of Fas/FasL pathway may represent a novel therapeutic modality for maintaining organ perfusion and preventing liver injury during sepsis.

A 9-year, single-institution, retrospective review of death rate and prognostic factors in adult respiratory distress syndrome.

OBJECTIVE: To evaluate, at a single institution, the adult respiratory distress syndrome (ARDS) death rate in critically ill ventilated surgical/trauma patients and to identify the factors predicting death in these patients. SUMMARY BACKGROUND DATA: The prognostic features affecting mortality at the onset of ARDS have not been clearly defined. Defining rare characteristics would be valuable because it would allow for better stratification of patients in clinical trials and more appropriate utilization of constrained resources in ICU environments. METHODS: A retrospective analysis of 980 ventilated surgical and trauma intensive care unit patients from January 1990 to December 1998 was performed at Rhode Island Hospital. One hundred eleven adult intensive care unit patients with ARDS were identified using the criteria of Lung Injury Score more than 2.50 and the definition from the American-European Consensus Conference. Slightly more than half were trauma patients, 57% were men, and the median age was 59 years. The overall death rate was 52%. Patients were segregated by admission date to the intensive care unit (before or after January 1, 1995). Severity of illness was measured by the Revised Trauma Score for trauma patients and the Acute Physiology and Chronic Health Evaluation III for surgical patients. The Multiple Organ Dysfunction Score was determined on the day of onset of ARDS for all patients. Other recorded variables were age, sex, intensive care unit length of stay, length and mode of ventilation, presence or absence of tracheostomy, ventilation variables of peak and mean airway pressures, lung injury scores, elective versus emergency surgery, and presence or absence of pneumonia. RESULTS: There was a significant decrease in the ARDS death rate from the period 1990 to 1994 to the period 1995 to 1998. The major reason for the decline was a reduction in the posttraumatic ARDS death rate. Lung-protective ventilation strategies were used more frequently in the second period than in the first, and the death rate was significantly decreased in trauma patients in the second period when lung-protective ventilation modes were used. Predictors of death at the onset of ARDS were advanced age, Multiple Organ Dysfunction Score of 8 or more, and Lung Injury Score of 2.76 or more. CONCLUSION: In this single-institution series, the death rate from ARDS declined from 1990 to 1998, primarily in posttraumatic patients, and the decrease is related to the use of lung-protective ventilation strategies. Based on this patient population, the authors developed a statistical model to evaluate important prognostic indicators (advanced age, organ system and pulmonary dysfunction measurements) at the onset of ARDS.

Diagnosis of acute cholecystitis: sensitivity of sonography, cholescintigraphy, and combined sonography-cholescintigraphy.

BACKGROUND: Radiographic diagnosis of acute cholecystitis can be established using ultrasonography (US), cholecystoscintigraphy (HIDA), or both. Although both modalities have been effective in diagnosing acute cholecystitis (AC), physicians from the emergency department and admitting surgeons continue to request both tests in an attempt to increase the diagnostic accuracy of AC. This article reports the institutional experience of a large tertiary care health care facility, with respect to the sensitivity of US, HIDA, and combined US and HIDA. STUDY DESIGN: We conducted a retrospective review of 132 patients diagnosed with AC who underwent laparoscopic cholecystectomy during the same hospitalization. Patients were stratified into three groups: Group 1 (Gp1, n = 50) included patients who underwent US alone, group 2 (Gp2, n = 28) included patients who underwent HIDA scan alone, and group 3 (Gp3, n = 54) included patients who underwent both US and HIDA. RESULTS: The three groups did not differ with respect to age, liver chemistry, time to operation, and hospital length of stay. The sensitivity of US, HIDA, and combined US/HIDA as diagnostic modalities for acute cholecystitis was referenced to histopathologic confirmation. Sensitivity was 24 of 50 (48%), 24 of 28 (86%), and 49 of 54 (90%) for US, HIDA, and the combination of US/HIDA, respectively. CONCLUSIONS: HIDA scan is a more sensitive test than US in diagnosing patients with AC. Based on the results of this study, we recommend that HIDA scan should be used as the first diagnostic modality in patients with suspected acute cholecystitis; US should be used to confirm the presence of gallbladder stones rather than to diagnose AC.

Prolonged lactate clearance is associated with increased mortality in the surgical intensive care unit.

BACKGROUND: Failure of arterial serum lactate to achieve normal levels has been associated with an increased mortality among medical and trauma patients. At our institution the ability of the patient to normalize arterial serum lactate has been utilized as an end point of resuscitation. In this study, we examine the correlation between length of time to lactate normalization and mortality. METHODS: The charts of 95 consecutive surgical intensive care unit (SICU) patients requiring hemodynamic monitoring or therapy were reviewed retrospectively. Hemodynamic, demographic, and laboratory data were recorded. Patients were stratified by lactate normalization time, and a subgroup analysis of survivors and nonsurvivors was performed by univariate and multivariate analysis. RESULTS: Patients not achieving a normal lactate level sustained a 100% hospital mortality rate. Those clearing between 48 and 96 hours sustained a 42.5% mortality rate. Patients normalizing in 24 to 48 hours had a 13.3% mortality rate, and those clearing in less than 24 hours had a mortality rate of 3.9%. Subgroup analysis by survival revealed differences in time to lactate clearance, initial blood pressure, and initial lactate on univariate analysis. On multivariate analysis only time of lactate clearance was found to differ. CONCLUSIONS: Prolongation of lactate clearance is associated with increasing mortality. Failure of a patient to normalize lactate is associated with 100% mortality. Measurement of arterial serum lactate is a simple and effective predictor of outcome and end point of therapy.

TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production.

Dysregulated neutrophil (polymorphonuclear PMN) apoptosis is thought to contribute to the onset of adult respiratory distress syndrome (ARDS) in critically ill patients. Tumor necrosis factor-alpha (TNFalpha), which is present in elevated levels in the bronchoalveolar lavage fluid in patients with ARDS, is thought to play a central role in regulating PMN function in the lungs. Studies have shown that short-term culture with TNFalpha increases apoptosis yet extended culture with TNFalpha suppresses apoptosis. However, it is unclear whether this latter effect of TNFalpha is directly or indirectly mediated through production of anti-apoptotic cytokines such as interleukin (IL)-8. To investigate the role of IL-8 in TNFalpha-induced apoptosis PMN were exposed to TNFalpha (100 ng/mL) in the presence or absence of antibodies to IL-8, and the extent of apoptosis was assessed. An enzyme-linked immunoassay was used to measure levels of the anti-apoptotic cytokine IL-8, induced by TNFalpha-stimulation. Because TNFalpha may mediate its effect through various cell-signaling pathways, we next assessed the effect of kinase inhibition on the ability of TNFalpha to effect apoptosis and IL-8 production. Treatment with TNFalpha had a biphasic effect: at 4-8 h, apoptosis was increased but was markedly suppressed at 24 h (P < 0.05). PMN cultured for 24 h with TNFalpha also showed markedly increased levels of IL-8. Neutralization of IL-8 inhibited the ability of TNFalpha to suppress apoptosis (P < 0.05). Incubation of TNFalpha + p38-mitogen-activated protein kinase (MAPK) inhibitor SB202190 increased apoptosis (P < 0.01) and decreased IL-8 production to PMN control. To a lesser extent, incubation of TNFalpha with inhibitors to NF-kappaB (SN50) and PI3K (LY294002) also increased apoptosis and decreased IL-8 production (P < 0.05). These data illustrate a novel mechanism by which TNFalpha can indirectly elicit an anti-apoptotic effect via p38-MAPK induced release of the anti-apoptotic chemokine IL-8. The exploitation of such a pathway represents a potential target for regulation of PMN-mediated acute lung injury.

Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity.

BACKGROUND: Hypoxia has been shown to delay the onset of neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis. With the use of antisense oligonucleotides, we have previously demonstrated that Mcl-1 is necessary for this effect. We wanted to further characterize the expression of Mcl-1 and examine signaling pathways required for the delay in apoptosis that is mediated by hypoxia. METHODS: For kinase signaling inhibition, PMNs were incubated for 12 hours with the following inhibitors: PD98059 Mitogen Activated Protein Kinase Kinase (MEK), SB202190 (p38 mitogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphologic features or were lysed for Western blot analysis. RESULTS: Western blot analyses, immunofluorescent staining, and quantification showed an upregulation of Mcl-1 expression after 12 hours of incubation in response to hypoxia. When inhibitors of either MEK or p38 MAPK were incubated with PMNs during hypoxia, apoptosis increased to similar levels as normoxia. We further wanted to determine whether signaling through p38 MAPK or MEK led to increased Mcl-1 expression. Western blot analysis confirmed that the inhibition of p38 MAPK led to a significant decrease in Mcl-1 expression. CONCLUSIONS: We have documented a novel mechanism by which hypoxia can modify PMN apoptosis in the wound site by the activation of p38 MAPK signaling, thereby inducing the anti-apoptotic protein Mcl-1.

The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis.

The regulation of polymorphonuclear leukocyte (PMN) apoptosis can influence the duration of the inflammatory response. We have previously shown that PMN apoptosis is delayed by matrix adhesion and hypoxia; however, the mechanisms responsible for this delay are not well understood. Mcl-1, an antiapoptotic Bcl-2 family member, is present in neutrophils; therefore, we sought to characterize its localization and function as it relates to PMN apoptosis. We found that Mcl-1 localized to the nucleus and cytoplasm and that expression levels decreased as PMN were aged in culture. Reducing available Mcl-1 through the use of antisense oligonucleotides demonstrated that Mcl-1 is necessary to delay apoptosis during normal PMN aging and hypoxia but is not required for suppression of apoptosis by laminin adhesion. Our results demonstrate a distinct expression pattern of Mcl-1 and that Mcl-1 is crucial for the delay of apoptosis initiated by certain antiapoptotic factors.

Neutrophils regulate their own apoptosis via preservation of CXC receptors.

BACKGROUND: Dysregulated neutrophil (PMN) apoptosis is thought to contribute to an exaggerated inflammatory response in diseases such as acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS). The CXC chemokines, interleukin-8 (IL-8) and growth-related oncogene alpha (Gro-alpha), contribute to the inflammatory response and suppress PMN apoptosis. We hypothesized that PMN generation of CXC chemokines is an autocrine/paracrine mechanism for amplification of the PMN inflammatory response via suppression of apoptosis. METHODS: Freshly isolated human PMNs from healthy donors were incubated with IL-8 or Gro-alpha (100 ng/ml) for 0-12 h, and apoptosis was analyzed at 24 h. De novo synthesis of IL-8 or Gro-alpha was measured using an ELISA. To determine if receptors were available to bind these newly synthesized ligands (125)I radiolabeled monoclonal antibodies specific for each receptor (CXCRI, CXCRII) were used to determine PMN receptor density. Comparison was by one-way ANOVA. RESULTS: Significant suppression of apoptosis was seen at 24 h with only 4 h exposure to IL-8 or Gro-alpha (n = 5, P < 0.05). PMNs cultured with IL-8 for 4 h produced 31 +/- 4.3 ng/ml IL-8 by 24 h; PMNs cultured with Gro-alpha produced 19.7 +/- 4.0 ng/ml Gro-alpha (n = 6, P < 0. 05). Neither chemokine induced significant production of the other chemokine. The addition of either ligand promoted upregulation of CXCR1 (n = 4, P < 0.05) at 24 h. However, CXCR2 was downregulated by Gro-alpha and IL-8 to 71 +/- 7.5 and 79 +/- 6.3% of control, respectively (P < 0.05). CONCLUSION: IL-8 and Gro-alpha, which suppress apoptosis, stimulate their own production after short-term incubation with PMNs. PMNs maintain the ability to respond to these chemokines through expression of the CXC receptors which suggests that PMNs are active participants in the suppression of apoptosis at inflammatory sites. CXCRI remains upregulated after prolonged stimulation and may be an important target for mediating neutrophil responses to IL-8.

CXC chemokine suppression of polymorphonuclear leukocytes apoptosis and preservation of function is oxidative stress independent.

Interleukin 8 (IL-8) and growth-related oncogene alpha (Gro-alpha) delay neutrophil apoptosis, which is thought to be important for the resolution of inflammation. We hypothesized that (IL-8) and Gro-alpha interfere with extracellular death receptor signaling or intracellular caspase activation to suppress neutrophil apoptosis. In addition, we sought to determine if prolonged neutrophil half-life was associated with preservation of function. Polymorphonuclear leukocytes (PMN) were cultured with IL-8 or Gro-alpha (0-100 ng/mL) in normoxia or hypoxia, and the extent of apoptosis was assessed by histology and TdT-mediated dUTP nick end labeling (TUNEL). Subsequently, to determine the role of apoptotic-associated receptors, PMN were cultured with IL-8 and neutralizing monoclonal antibody to Fas (CD95), TNFR55, and TNFR75. To establish the effect of IL-8 or Gro-alpha on pro-apoptotic caspase activity, the cleavage of specific colorimetric substrates was assessed. Functional changes in PMN included the capacity to produce superoxide anion and phagocytosis of Escherichia coli. At the 100 ng/mL dose, the addition of IL-8 and Gro-alpha maximally suppressed PMN apoptosis from 54% (untreated) to 5% and 6%, respectively. The addition of neutralizing antibodies to Fas, TNFR55, or R75 caused no change in IL-8 suppression of apoptosis. Caspase 3 activity was markedly suppressed at 24 h by the inclusion of either IL-8 and Gro-alpha. IL-8 and Gro-alpha-stimulated PMN released more superoxide anion and had an increased phagocytic index vs. control PMN. IL-8 and Gro-alpha suppress neutrophil apoptosis to a similar level that is not influenced by oxygen tension at high doses. The effect of IL-8 and Gro-alpha does not depend on activation of the Fas, TNFR55, or R75 receptor pathways but involves suppression of caspase 3 activity. IL-8 or Gro-alpha extends the functional half-life of neutrophils and may explain their role in disease states such as acute respiratory distress syndrome.

Effects of fluconazole administration in critically ill patients: analysis of bacterial and fungal resistance.

HYPOTHESIS: The administration of fluconazole in intensive care unit (ICU) patients leads to the emergence of bacterial and fungal resistance. DESIGN: Retrospective analysis of 2 patient cohorts: (1) critically ill patients treated in surgical, trauma, and medical ICUs between June 1997 and January 1999 who did and did not receive fluconazole; and (2) ICU patients with fungal infections and sensitivity testing results from June 1994 to December 1998. SETTING: University-affiliated tertiary care hospital. PATIENTS: The first cohort included 99 ICU patients with documented microorganism culture(s) who were treated with (n = 50) or without (n = 49) fluconazole; the second cohort included 38 patients with Candida species infection, identification, and antifungal susceptibility testing. RESULTS: Mortality (40% vs 20%; P = .03) and hospital length of stay (33.8 vs 25.6 days; P = .04) were higher in the patients treated with fluconazole compared with patients not treated with fluconazole. The ICU length of stay was also higher in patients treated with fluconazole (23.7 vs 15.1 days; P = .009). An increase in bacterial resistance occurred in patients after fluconazole treatment as opposed to bacterial resistance of patients who were treated for bacterial microorganism(s) without fluconazole (16% vs 4%; P = .049). Comparison of patient populations with Candida species identification before and after December 1997 showed an increase in Candida species resistance to fluconazole (11% vs 36%; P = .16), respectively. Fungal strains were dominated by a combination of Candida albicans and Candida glabrata in both populations (60% [before 1998] vs 82% [after 1998]), with an emergence of Candida non-albicans species tolerant to fluconazole. The amount of fluconazole administered and the number of patients receiving fluconazole treatment in the ICUs has also increased when comparing both periods. CONCLUSIONS: Comparison of critically ill patient populations with and without fluconazole treatment found increased mortality and longer hospital and ICU lengths of stay in the fluconazole-treated group. This group also had higher bacterial pathogen resistance to antibiotics after fluconazole administration compared with bacterial resistance of patients without fluconazole treatment. Our results warrant concern regarding worsening bacterial infections, increased mortality, and an increase in Candida resistance to fluconazole from increased use in ICU patients, with a shift in yeast infection that is more difficult to treat.

IL-1beta stimulation induces paracrine regulation of PMN function and apoptosis.

Polymorphonuclear leukocytes (PMN) play a crucial role in the primary immunological defense against infectious agents. PMN activation and function is influenced in a paracrine manner by cytokines and bacterial products. While cell-cell communication has been demonstrated between PMN and other cell types, little data is available addressing PMN-PMN communication. Therefore, the aim of this study was to determine whether PMN were able to affect PMN function in vitro in a cell-contact independent manner, and whether IL-1beta influenced this effect. Conditioned medias (CM) were prepared by incubating PMN in HBSS +/- IL-1beta for 1-4 h. Incubation of fresh PMN in these conditioned medias had little or no effect on the expression of cell surface FcgammaR expression or oxidative metabolism. However, incubation of PMN in CM-IL1beta, but not control CM, increased phagocytotic activity and suppressed apoptosis. Additionally, CM-IL1beta, but not control CM, slowed the changes in Mac-1 and CR1 cell surface expression that occurred in HBSS within 2 h of incubation. Finally, control CM down-regulated the cell surface expression of PSGL-1; an effect that was not observed with CM-IL1beta. In conclusion, we demonstrate that PMN are able to communicate with and influence the immunological function of other PMN independent of cell-cell contact, and that this influence is regulated by cytokines such as IL-1beta. The major impact of this paracrine regulation is to down-regulate PMN apoptosis with the potential for an upregulated inflammatory response.

Role of granulocyte-macrophage colony-stimulating factor and its receptor in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis.

HYPOTHESIS: That granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor modulate the suppression of apoptosis (Ao) of normal neutrophils incubated in the plasma of patients with postraumatic acute respiratory distress syndrome (ARDS). DESIGN: Experimental study using cultured human neutrophils. SETTING: University hospital, level I trauma center. PARTICIPANTS: Plasma was obtained from 14 patients with early, fulminant posttraumatic ARDS (mean Injury Severity Score, 22). All samples were drawn within 24 hours after injury. Plasma was also taken from up to 21 healthy control subjects. These volunteers were also used as sources of polymorphonuclear leukocytes (PMNs). MAIN OUTCOME MEASURES: (1) Effect of early, fulminant ARDS and normal plasma on spontaneous Ao and GM-CSF receptor expression in PMNs in vitro. (2) Effect of ligation of either GM-CSF or its receptor with a neutralizing monoclonal antibody (mAb) on PMN Ao in ARDS and normal plasma. (3) Correlation of extracellular GM-CSF concentration with rate of PMN Ao. (4) Levels of GM-CSF in ARDS and normal plasma and in culture supernatant of normal PMNs incubated in early, fulminant ARDS and normal plasma. RESULTS: Plasma from patients with ARDS enhanced PMN viability at 24 hours (data are given as mean +/- SEM) 52% +/- 3% control vs 60% +/- 3% ARDS, P<.05). Binding of the GM-CSF receptor with a neutralizing mAb significantly reduced PMN viability in ARDS plasma, but not in normal plasma (60% +/- 3% ARDS vs 53% +/- 3% ARDS + mAb, P<.05). Ligation of GM-CSF with mAb had no significant effect on PMN viability in either plasma. Only 1% of PMNs expressed detectable levels of the GM-CSF receptor when incubated for 24 hours in either ARDS or normal plasma. The GM-CSF levels were undetectable (>7 pg/mL) in both ARDS and normal plasma and in culture supernatants taken after 24 hours of incubation in both plasma types. Levels of GM-CSF ranging from 0 to 50000 pg/mL had no effect on PMN Ao in plasma-free medium. CONCLUSIONS: The antiapoptotic effect of ARDS plasma appears to be mediated by the GM-CSF receptor. This effect occurs at both low levels of plasma GM-CSF and surface expression of its PMN receptor. Ligation of GM-CSF had no effect of PMN Ao, suggesting that Ao is triggered by Fc portion-mediated receptor cross-linking. These results provide the theoretical basis for alphaGM-CSF receptor mAb therapy as a novel modality of treatment for ARDS.

Prognostic usefulness of scoring systems in critically ill patients with severe acute pancreatitis.

OBJECTIVE: To compare prognostic scoring systems in a retrospective series of patients with severe acute pancreatitis admitted to a surgical intensive care unit (ICU). METHOD: Between January 1992 and December 1996, the charts of all patients with a discharge code of acute pancreatitis were reviewed. There were 273 charts reviewed. Of these, 12 were admitted to the surgical ICU with a diagnosis of severe acute pancreatitis. A preliminary analysis of the data considers descriptive summary statistics, such as the mean and the range. The Spearman's rank-correlation test was computed to assess concordance between the following: a) length of stay and Ranson criteria; b) length of stay and Acute Physiology and Chronic Health Evaluation (APACHE) III score; and c) length of stay and modified Glasgow Coma score. Also, an unpaired t-test was used to obtain concordance between the following: a) death and Ranson; b) death and APACHE III; and c) death and modified Glasgow Coma score. RESULTS: The prognostic score for APACHE III, Ranson criteria, and modified Glasgow Coma score were compared with the patients' length of stay. Patients who had >5 Ranson criteria, modified Glasgow Coma scores of >4, and APACHE III scores of >30 at 96 hrs (mean 71+/-16 [SD]; p < .0) subsequently died. These two patients were excluded from the Spearman's rank-correlation tests. The mean length of stay in our sample was 61.8 (range, 7-201) days. The mean Ransom criteria was 4.3 (range, 1-9). The mean 96-hr APACHE III score was 33.3 (range, 0-83). The Spearman's rank-correlation between length of stay and Ranson criteria was 0.68, with a corresponding p value of .03. Similar results were observed for the length of stay and APACHE III at 96 hrs (correlation, 0.77; p = .0098) and the length of stay and the modified (correlation, 0.78; p = .007). These data reveal that the magnitude of correlation between the length of stay and the 96-hr APACHE III and modified Imrie is larger than that between length of stay and Ranson criteria. CONCLUSIONS: Once a patient is admitted to the surgical ICU, several predictors of mortality or complications that will require long hospitalization times are evident. In this sample of patients, APACHE III scores >30 at 96 hrs, 5 or more Ranson criteria, and a modified Imrie (Glasgow) score of >3 predicted those who died or had multiple complications. Those patients with combined 48-hr and 96-hr APACHE III scores of >60 either died or had hospitalizations of >60 days. These patients had major pancreatic complications that included pancreatic necrosis, pancreatic abscess, pseudocyst, hemorrhagic pancreatitis, and pancreatic ascites.

Regulation of IL-8RA (CXCR1) expression in polymorphonuclear leukocytes by hypoxia/reoxygenation.

Interleukin-8 (IL-8) is an important mediator of neutrophil (PMN) function and the type A IL-8 receptor (IL-8RA) mediates these pro-inflammatory signals. Hypoxia or hypoxia/reoxygenation (H/R) affects the production of IL-8, but no data is available regarding its effect on IL-8RA expression. The purpose of this study was to determine the effects of hypoxia and/or H/R on the expression of IL-8RA in PMN. We demonstrated that IL-8RA mRNA levels were similar under normoxic and hypoxic conditions but H/R resulted in a significant reduction in mRNA expression between 30 and 60 min. IL-8RA protein also decreased with reoxygenation of whole blood, which was altered by the addition of specific antioxidants. Therefore, H/R appears to attenuate the effect of IL-8 by down-regulating IL-8RA in PMN. These data show that changes in oxygen tension within the wound site not only affect the expression of inflammatory cytokines, but also control their actions by regulating their receptors.

Analysis of the effect of conversion from open to closed surgical intensive care unit.

OBJECTIVE: To compare the effect on clinical outcome of changing a surgical intensive care unit from an open to a closed unit. DESIGN: The study was carried out at a surgical intensive care unit in a large tertiary care hospital, which was changed on January 1, 1996, from an open unit, where private attending physicians contributed and controlled the care of their patients, to a closed unit, where patients' medical care was provided only by the surgical critical care team (ABS or ABA board-certified intensivists). A retrospective review was undertaken over 6 consecutive months in each system, encompassing 274 patients (125 in the open-unit period, 149 in the closed-unit period). Morbidity and mortality were compared between the two periods, along with length-of-stay (LOS) and number of consults obtained. A set of independent variables was also evaluated, including age, gender, APACHE III scores, the presence of preexisting medical conditions, the use of invasive monitoring (Swan-Ganz catheters, central and arterial lines), and the use of antibiotics, low-dose dopamine (LDD) for renal protection, vasopressors, TPN, and enteral feeding. RESULTS: Mortality (14.4% vs. 6.04%, p = 0.012) and the overall complication rate (55.84% vs. 44.14%, p = 0.002) were higher in the open-unit group versus the closed-unit group, respectively. The number of consults obtained was decreased (0.6 vs. 0.4 per patient, p = 0.036), and the rate of occurrence of renal failure was higher in the open-unit group (12.8% vs. 2.67%, p = 0.001). The mean age of the patients was similar in both groups (66.48 years vs. 66.40, p = 0.96). APACHE III scores were slightly higher in the open-unit group but did not reach statistical significance (39.02 vs. 36.16, p = 0.222). There were more men in the first group (63.2% vs. 51.3%). The use of Swan-Ganz catheters or central and arterial lines were identical, as was the use of antibiotics, TPN, and enteral feedings. The use of LDD was higher in the first group, but the LOS was identical. CONCLUSIONS: Conversion of a tertiary care surgical intensive care unit from an open to closed environment reduced dopamine usage and overall complication and mortality rates. These results support the concept that, when possible, patients in surgical intensive care units should be managed by board-certified intensivists in a closed environment.

The role of neutrophil-derived oxidants as second messengers in interleukin 1beta-stimulated cells.

The role of the inflammatory cytokine interleukin 1beta (IL-1beta) as potent agonist of the PMN respiratory burst signal transduction cascade has been described. We hypothesized that this phenomenon is self-limiting and that polymorphonuclear leukocyte (PMN)-derived reactive oxygen intermediates (ROI) might provide feedback regulation on the IL-1beta surface receptor (IL-1betaR)-G-protein-effector enzyme transducing tripartite complex that ultimately leads to NADPH oxidase activation. Therefore, we separately assessed either baseline or IL-1beta-induced activation of each member of the IL-1betaR-G-protein-phospholipase D (PLD) or IL-1betaR-G-protein-phospholipase C (PLC) signaling systems in the presence or absence of one of several specific ROI scavengers/antioxidants. Purified human PMN were lipopolysaccharide primed, adhered for 2 h, and stimulated with 100 ng/mL IL-1beta with or without 1% v/v dimethyl sulfoxide, 10 mM NaN3, 30 mM L-alanine, 200 U catalase, or 300 U superoxide dismutase (SOD). To validate the use of these antioxidants, the production of O2-, H2O2, hypochlorous acid, or myeloperoxidase (MPO) in the employed experimental model was confirmed in a separate set of experiments. The expression of IL-1betaR type I or II was assessed by binding with corresponding 125I-labeled monoclonal antibodies and corrected for nonspecific binding. PLD activation was assessed by measuring phosphatidyl ethanol formation in the presence of ethanol. PLC activation was determined by quantitative measurement of diacylglycerol. The level of Galpha stimulatory and inhibitory subunits was assessed by Western blotting. IL-1betaR type I expression was significantly up-regulated in the presence of catalase and SOD. PLD activation was increased by dimethyl sulfoxide and NaN3, and PLC activation was up-regulated by NaN3, L-alanine, SOD, and catalase. After 5 min of stimulation with IL-1beta, Gialpha expression was significantly down-regulated by NaN3 and SOD, whereas SOD had an up-regulating effect on the expression of Gs alpha. Increasing concentrations of externally added authentic MPO progressively down-regulated both PLD and PLC activity. Thus, PMN-derived ROI, in addition to their role as antibacterial/fungal agents, serve as second messengers in IL-1beta signal transduction, with MPO having the most ubiquitous role as a modulator of PMN second messenger pathways.

Interleukin-8-induced suppression of polymorphonuclear leukocyte apoptosis is mediated by suppressing CD95 (Fas/Apo-1) Fas-1 interactions.

BACKGROUND: Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS: PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS: After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS: IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.

Studies on polymorphonuclear leukocyte bactericidal function III: the role of extracellular matrix proteins.

We investigated the effect of the extracellular matrix proteins fibronectin (Fn) and laminin (Ln) on polymorphonuclear leukocytes (PMN) bactericidal activity. Adherence of PMN to increasing concentrations of Ln significantly increased the killing of Escherichia coli after 240 min of adherence, while fibronectin significantly increased PMN staphlacidal activity after 240 min of adherence. The addition of IL-1beta and IL-8 but not TNF-alpha increased PMN bactericidal activity against E. coli when PMN were adhered to Ln, while TNF-alpha and IL-8 increased PMN bactericidal activity against Staphylococcus aureus when PMN were adhered to Ln. TNF-alpha increased PMN killing of E. coli when PMN were adhered to Fn, while only IL-1beta increased the killing of S. aureus when PMN were adhered to FN. Anti-VLA-3 (alpha3/beta1) monoclonal antibodies (mAbs) inhibited the effect of Ln on PMN bactericidal activity, while anti-VLA-5 (alpha5/beta1) mAbs inhibited the effect of Fn on PMN bactericidal activity. Progressive cross-linkage of these two receptors led to a dose-dependent reduction in PMN bactericidal activity for both pathogens when PMN were adhered to Ln or Fn, respectively. These results demonstrate that extracellular matrix proteins +/- exogenously added cytokines have the capacity to regulate PMN bactericidal activity. The signals sent by these matrix proteins to increase PMN bactericidal activity are transduced primarily via separate alpha subunits of the beta1 integrin complex. Stimulation of these receptors might lead to potential upregulation of PMN bactericidal activity which would be potentially advantageous in vivo at sites of infection.

Integrin regulation of polymorphonuclear leukocyte apoxis during hypoxia is primarily dependent on very late activation antigens 3 and 5.

BACKGROUND: Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response. The regulation of polymorphonuclear leukocyte (PMN) apoptosis during hypoxia has not been previously characterized, and we hypothesized that integrin signaling by matrix proteins (laminin) would regulate PMN apoptosis. METHODS: PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during normoxia or hypoxia. Apoptosis was determined both by cellular histologic evaluation and the TUNEL assays (Tdt). Phagocytosis in apoptotic PMNs was determined with two-color flow cytometric analyses with rhodamine-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (563 nm). Western blot analyses were performed on nine apoptotic regulatory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cross-linkage. RESULTS: Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/- 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin). Apoptosis-positive PMNs exhibited significantly greater phagocytosis than apoptosis-negative PMNs +/- laminin. Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia. Cross-linkage of very late activation antigen-3 (alpha 3/beta 1) resulted in the phosphorylation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds. CONCLUSIONS: (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis. (2) The reduction of apoptosis is mediated primarily by very late activation antigen-3, which leads to a subsequent increase in the intracellular expression of p53 and increased bacterial phagocytosis.