Inhibition of neutrophil apoptosis after coronary bypass operation with cardiopulmonary bypass

BACKGROUND: Granulocyte apoptosis is a key control process in the clearance of neutrophils from inflammatory sites, and its rate is modulated both in vitro and in vivo by a number of inflammatory mediators. In this study, we investigated the influence of cardiopulmonary bypass (CPB) on neutrophil apoptosis. METHODS: Twenty patients undergoing coronary operation with CPB were studied. Patients undergoing off-pump (OP) coronary bypass and healthy subjects served respectively as stressed and normal groups. Interleukin-6 (IL-6), IL-8, and tumor necrosis factor-alpha were assessed on plasma collected preoperatively, at the end of CPB, and after intervals of 4, 8, 12, and 24 hours. Neutrophil apoptosis was detected by light microscopy as well as by the annexin-V assay on postoperative samples. The polymorphonuclear leukocyte (PMN) apoptotic receptors, Fas and FasL, were studied together with the activity of caspase 3 in postoperative neutrophils. RESULTS: Spontaneous apoptosis was significantly delayed in PMNs from CPB patients when compared with either the stressed or control patients. Neutrophils were activated, as indicated by increased surface expression of CD11b. Western blot analysis showed a normal expression of the apoptotic receptors Fas and FasL. Caspase 3 activity was found to be significantly reduced in neutrophils from CPB patients after 18 and 24 hours of culture. When control neutrophils were cultured in the presence of postoperative plasma from OP and CPB patients, apoptosis was significantly delayed. Depleting surgical plasma of IL-6 and IL-8 completely abolished this antiapoptotic effect. CONCLUSIONS: Inflammatory mediators during CPB prolong the functional lifespan of neutrophils through modulation of apoptosis, and potentiate the inflammatory response observed after coronary bypass operation.     

Dietary restriction impairs neutrophil exudation by reducing CD11b/CD18 expression and chemokine production

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.     

Inhibition of neutrophil apoptosis after elective surgery.

BACKGROUND: Neutrophils play a crucial role in host defense against infections, but their inappropriate infiltration and activation within tissues can cause host tissue damage through release of reactive oxygen metabolites, metalloproteinases, and proinflammatory cytokines. The termination of a neutrophil-mediated inflammatory response is effected through programmed cell death or apoptosis. Delayed neutrophil apoptosis is associated with proinflammatory diseases, such as the systemic inflammatory response syndrome. Surgery induces a profound inflammatory response; therefore, neutrophil apoptosis of patients undergoing elective surgery was investigated. METHODS: Nonseptic patients undergoing elective orthopedic surgery while under epidural anesthesia had neutrophils and platelet-poor isolated from whole venous blood harvested at 4 time points: pre-epidural, 45 minutes postepidural but before surgical intervention, 1 hour postsurgical incision, and 24 hours postsurgery. Neutrophil apoptosis was quantified at 1, 12, and 24 hours in culture by immunofluorescence flow cytometry of annexin V and propidium iodide staining and confirmed by TUNEL (terminal deoxynucleotidyl transferase nick end labeling) assay for DNA strand breaks. Serum cytokines were quantified by specific enzyme-linked immunosorbent assay. RESULTS: Spontaneous neutrophil apoptosis after elective surgery was significantly (P < .001) inhibited with an effect evident within an hour of surgical incision and persisting at 24 hours postsurgery. The addition of patients' 24 hour postoperative plasma to healthy neutrophils markedly (P < .01) reduced neutrophil apoptosis, whereas plasma taken an hour after surgical incision was ineffective. Interleukin (IL)-6 was notably increased (1395 +/- 196 pg/mL, P < .01) 24 hours postsurgery and at this postoperative concentration inhibited (P < .01) apoptosis of normal neutrophils. Levels of other inflammatory mediators (IL-1 beta, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, soluble Fas, soluble Fas ligand) were unaltered. The anti-inflammatory cytokine IL-10 was only slightly increased 24 hours postsurgery (8.32 +/- 2.99 pg/mL); however, the addition of recombinant human IL-10 (10 ng/mL) counteracted (P < .05) inhibition of neutrophil apoptosis induced by IL-6 and post-surgery plasma. CONCLUSIONS: These results identify marked inhibition of neutrophil apoptosis after elective surgery and suggest that the inhibition of neutrophil apoptosis in the postoperative period is, at least in part, a result of soluble circulating factors. The marked imbalance favoring proinflammatory over anti-inflammatory cytokine release in the immediate postoperative period mediates the overwhelmingly antiapoptotic net capacity of postsurgery plasma.     

Role of the neutrophil in the development of systemic inflammatory response syndrome and sepsis following abdominal aortic surgery.

INTRODUCTION: There is evidence to suggest that the polymorphonuclear neutrophil (PMN) plays a critical early step in the development of the ischaemia-reperfusion syndrome, the systemic inflammatory response syndrome (SIRS) and sepsis. The PMN receptor CD16 plays an important role in phagocytosis, cell-mediated cytotoxicity and the release of free radicals and proteolytic enzymes. The aim of this study was to determine whether there is any relationship between PMN CD16 expression, phagocytosis and the development of sepsis. METHODS: Fifty patients who underwent elective infrarenal abdominal aortic aneurysm repair were studied. Venous blood was taken before operation, throughout surgery and for 7 days after operation. CD16 expression was measured, unstimulated and following further stimulation, by means of flow cytometry. Phagocytosis was determined using flow cytometry. RESULTS: Some 36 patients had an uncomplicated recovery; 14 developed SIRS or sepsis. There was no difference between the two groups with respect to nutritional, co-morbid or technical factors. In the group that developed septic complications after operation, the level of PMN CD16 expression was significantly higher before surgery (mean channel fluorescence (MCF) 30.2 versus 10.4; P < 0.05, Mann-Whitney U test) and throughout the postoperative period. Surgery produced no change in CD16 expression. After operation, stimulation of PMNs in the septic group resulted in a fall in CD16 expression (40.8 versus 20.4 MCF; P < 0.05, Mann-Whitney U test); surgery produced no change in the level of expression in the uncomplicated group. CONCLUSION: This study provides evidence of phenotypic and functional differences in neutrophil behaviour in patients who develop sepsis following aneurysm surgery.     

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.     

The effects of glucocorticoid therapy on inflammatory responses to coronary artery bypass graft surgery

HYPOTHESIS: Delayed or reduced polymorphonuclear leukocyte (PMN) apoptosis may contribute to prolongation of systemic inflammation after cardiopulmonary bypass. BACKGROUND/OBJECTIVE: Preoperative administration of glucocorticoids has been used ostensibly to attenuate the systemic inflammation associated with cardiopulmonary bypass. Therefore, this study evaluated, in patients undergoing cardiopulmonary bypass, the efficacy of glucocorticoids in restoring peripheral blood PMN apoptosis and modulating PMN surface receptors (CD95, tumor necrosis factor receptor [TNFR]) known to be involved in proapoptotic or antiapoptotic signal transduction. DESIGN: Randomized control study. SETTING: Medical school and affiliated tertiary care hospital. PATIENTS: Thirteen patients undergoing coronary artery bypass grafting. INTERVENTION: Patients were randomly assigned to the control group (n = 7) or to receive 1 g of methylprednisolone sodium succinate on anesthetic induction (n = 6). MAIN OUTCOME MEASURES: Blood samples were drawn before induction, 20 minutes after sternotomy and bypass, immediately postoperatively, and on postoperative day 1. Isolated PMNs were incubated for 6 hours with or without the CD95 agonist CH 11. Polymorphonuclear leukocyte apoptosis was measured using propidium iodide-RNAase staining and flow cytometry. Levels of PMN cell-associated receptors (TNFR and CD95), cytokines (TNF-alpha, interleukin 6 [IL-6], IL-8, and IL-10), and soluble receptors (sTNFR1 and sTNFR2) were measured. RESULTS: In all 13 patients, spontaneous and Fas-mediated PMN apoptosis decreased more than 80% from baseline (P<.001) by postoperative day 1. Polymorphonuclear leukocyte CD95 increased (P<.003) by postoperative day 1 compared with baseline, whereas PMN TNFR was unchanged. Methylprednisolone administration did not modulate PMN apoptosis or immunocyte receptor expression; however, such treatment did decrease postoperative IL-6 secretion (P<.001) and increase postoperative IL-10 secretion (P<.001). CONCLUSIONS: The complications of major surgery include persistent inflammation, which can lead to multisystem organ failure. Polymorphonuclear leukocyte resistance to apoptosis may contribute to this process. A single preoperative dose of glucocorticoids did not effect PMN apoptosis or receptor phenotype.     

Hypothermia augments polymorphonuclear leukocyte degranulation and interleukin-8 production from human umbilical vein endothelial cells and increases lipopolysaccharide-induced polymorphonuclear leukocyte-endothelial cell interaction when followed by normothermia

OBJECTIVE: To determine if hypothermia followed by normothermia (rewarmed to 37 degrees C) changes the inflammatory response of polymorphonuclear leukocytes (PMNs) and human umbilical vein endothelial cells (HUVEC). DESIGN: Prospective, controlled, in vitro study. SETTING: University laboratory. PARTICIPANTS: PMNs from 4 healthy volunteers and HUVEC. MEASUREMENTS AND MAIN RESULTS: PMNs and HUVEC were incubated for 3 hours at 20 degrees C, 30 degrees C, or 37 degrees C followed by 37 degrees C again. PMN degranulation, cytokine production from HUVEC, and PMN-HUVEC interaction were compared among the 3 experimental temperatures. Interleukin (IL)-8-induced PMN degranulation measured by myeloperoxidase concentrations was significantly higher in the 20 degrees C and 30 degrees C groups than the 37 degrees C groups. Bacterial lipopolysaccharide (LPS)-induced IL-8 production from HUVEC, measured by enzyme-linked immunosorbent assay, was significantly higher in the 20 degrees C group than the other 2 groups; however, tumor necrosis factor-alpha was not detectable in any of the groups. LPS-induced cell injury measured by cellular (51)Cr release was significantly higher in the 20 degrees C and 30 degrees C groups than in the 37 degrees C groups. This injury was significantly inhibited by IL-8 antibody. CONCLUSION: Hypothermic (20 degrees C and 30 degrees C) incubation followed by rewarming augmented IL-8-induced PMN degranulation and LPS-induced IL-8 production from HUVEC and LPS-induced PMN-endothelial interaction. IL-8 plays an important role in this increased injury. This increased inflammatory response may support the positive outcomes of normothermic CPB.     

严重烧伤早期大鼠中性粒细胞、巨噬细胞凋亡实验研究

目的探讨严重烧伤后中性粒细胞(PMN)凋亡及烧伤血清刺激后PMN、巨噬细胞(Mφ)凋亡,以及巨噬细胞吞噬凋亡PMN的变化.方法选用Ⅲ度30%TBSA烧伤大鼠模型,收集伤后0、6、12、24hPMN,分离伤后24h烧伤血清,应用流式细胞技术检测PMN凋亡百分率;同时收集大鼠腹腔巨噬细胞,检测烧伤血清对Mφ凋亡变化的影响及烧伤后24h大鼠腹腔Mφ对凋亡PMN吞噬能力.结果伤后大鼠PMN凋亡百分率明显降低,伤后6、12、24h各时相点无显著差异.烧伤大鼠血清刺激24h后,PMN凋亡百分率为(15.4±1.2)%,较正常大鼠血清刺激后(50.6±3.9)%显著降低;而Mφ凋亡百分率为(23.2±2.1)%,较正常大鼠血清刺激后(¨.5±1.6)%增多;严重烧伤后大鼠腹腔Mφ对凋亡PMN吞噬能力由(35.0±2.6)%降至(21.8±2.1)%.结论严重烧伤后及在烧伤血清刺激下大鼠PMN凋亡延迟,Mφ吞噬凋亡PMN能力降低,可能凋亡的PMN被Mφ吞噬不完全,最终坏死,释放毒性内容物,是引发SIRS的重要因素之一.     

Neutrophils are primed for cytotoxicity and resist apoptosis in injured patients at risk for multiple organ failure.

BACKGROUND: Postinjury multiple organ failure (MOF) is the result of a dysregulated systemic inflammatory response in which primed neutrophils (PMNs) are sequestered in tissues, vulnerable to activation through secondary insults. Apoptosis is critical to the normal clearance of these sequestered PMNs. Conversely, dysfunctional apoptosis prolongs the PMN functional life span, potentially exacerbating PMN-mediated tissue injury and the development of MOF. We hypothesized that severe trauma, in addition to priming PMNs, provokes dysfunctional PMN apoptosis. METHODS: Neutrophils were harvested daily from 12 severely injured patients at high risk for MOF, cultured for 24 hours, and assessed for apoptosis with use of acridine orange-ethidium bromide staining and fluorescence microscopy. Priming for elastase release was measured in freshly isolated patient PMNs. Plasma from patients was assessed for its ability to delay apoptosis of normal PMNs. RESULTS: Four patients (33%) had MOF. Neutrophil apoptosis was profoundly delayed in severely injured patients throughout the 5-day study period. Priming for elastase release was augmented concomitantly. Patients' plasma delayed apoptosis of normal PMNs. CONCLUSION: In patients at high risk for postinjury MOF, PMNs are not only primed for cytotoxicity but also resist apoptosis. The dysfunctional apoptosis is attributed, at least in part, to a plasma-borne mediator. The net effect may facilitate hyperinflammatory organ injury.     

Role of polymorphonuclear neutrophils and macrophages in the prevention of postoperative infections

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.     

Alteration of chemoattractant receptor expression regulates human neutrophil chemotaxis in vivo

OBJECTIVE: To elucidate the mechanisms that regulate human neutrophil delivery in vivo, as well as the mechanisms that lead to observed reduction in polymorphonuclear (PMN) delivery to remote sites in septic patients. METHODS: Alterations in human PMN chemoattractant receptor expression and chemotactic function in vivo were evaluated in two distinct experiments: exudate PMNs (PMNs that have undergone transmigration to skin window blisters in controls) and septic PMNs (circulating PMNs from septic patients in the intensive care unit) were both separately compared with control circulating PMNs. RESULTS: Exudate PMNs displayed increased C5a receptors and C5a chemotaxis, and reduced interleukin-8 receptors (both IL-8 RA and IL-8 RB) and IL-8 chemotaxis. Septic PMNs displayed reduced C5a and IL-8 receptors and decreased C5a chemotaxis but no change in IL-8 chemotaxis. IL-8 but not C5a receptor gene expression decreased in parallel to receptor alteration. CONCLUSIONS: These results suggest that change in PMN chemoattractant receptor expression serves to regulate PMN chemotaxis in vivo; that exudate PMN chemotaxis depends more on C5a than IL-8; and that diminished chemoattractant receptors and chemotaxis in septic PMNs may explain decreased PMN delivery in these patients.     

Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism.

BACKGROUND: For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. METHODS: PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. RESULTS: There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 mumol/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. CONCLUSION: Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism.     

Cardiopulmonary bypass renders patients at risk for multiple organ failure via early neutrophil priming and late neutrophil disability.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response syndrome (SIRS) and these patients are recognized to be at increased risk for delayed infectious complications. We have documented that circulating neutrophils (PMNs) from patients manifesting SIRS have evidence of early postinjury priming for cytotoxicity. Consequently, we hypothesized that CPB would result in early postoperative PMN hyperresponsiveness (priming). MATERIALS AND METHODS: Six patients (mean age 50 +/- 2.9 years) who underwent CPB for CABG had sequential blood samples obtained perioperatively. PMNs were isolated and superoxide anion (O(-)(2)) generation (nmol O(-)(2)/3.75 x 10(5) PMNs/min) was measured by reduction of cytochrome c after exposure to fMLP, C5a, or PMA; elastase release (% total PMN elastase content) was measured by cleavage of AAPV-pNA after exposure to fMLP or C5a. RESULTS: PMNs were activated for increased elastase release 6 h after initiation of CPB. Significant PMN priming for O(-)(2) production was discovered at 3, 6, and 12 h following CPB and for elastase release at 3 and 6 h after CPB. At 2 to 3 days after CPB, O(-)(2) generation was significantly less than that of the preoperative control. Neutrophil primability with PAF was detected at 6 h after CPB. A similar defect in PAF-primable O(-)(2) production was seen 2 and 3 days post-CPB. Direct PMN interrogation with the receptor-independent activator PMA revealed loss of integrity of the NADPH oxidase at 2 and 3 days following CPB. CONCLUSIONS: A vulnerable window exists between 3 and 12 h after CPB when PMNs are primed for enhanced cytotoxicity via O(-)(2) production and elastase release. Paradoxically, PMN oxidase integrity becomes deficient 48 h post-CPB, while protease degranulation remains intact. These events render the bypass patient at risk for multiple organ failure via both early PMN-mediated tissue injury and delayed infectious complications.     

Inhibition of alveolar neutrophil immigration in endotoxemia is macrophage inflammatory protein 2 independent

BACKGROUND: Altered transendothelial migration and delayed apoptosis of neutrophils (PMN) have been implicated as contributing to infection in patients with gram-negative sepsis. Macrophage inflammatory protein 2 (MIP-2) signals PMN immigration and may alter other PMN functions. We tested the hypothesis that sequential endotoxin challenge in vivo alters PMN apoptosis and chemotactic responses. MATERIALS AND METHODS: Endotoxemia was induced in male Wistar rats (250 g) via intraperitoneal (IP) administration of LPS (4 mg/kg). After 18 h, intratracheal (IT) injection of LPS (400 microg/kg) was performed. Control animals received saline injections. Four hours after IT-LPS, circulating and bronchoalveolar lavage (BAL) PMN were isolated. PMN yields were calculated, and apoptosis was quantified after 18 h in culture by annexin V-fluorescein isothiocyanate FACS analysis. BAL MIP-2 concentrations were determined by ELISA. PMN chemotaxis to MIP-2 and IL-8 was determined using a fluorescent in vitro migration assay. RESULTS: Endotoxemia (IP-LPS) significantly decreases BAL PMN yield in response to an in vivo IT-LPS challenge. IT-LPS inhibits BAL PMN apoptosis to the same extent as sequential IP/IT-LPS. Alveolar MIP-2 concentrations are similar in the two groups. In vitro migration to IL-8 and MIP-2 was inhibited in PMN from endotoxemic versus control animals. CONCLUSIONS: These data demonstrate that endotoxemia inhibits PMN migration despite similar MIP-2 concentrations in the alveolus. Sequential insults do not affect the inhibition of apoptosis. In vitro, PMN from endotoxemic animals display impaired chemotaxis to MIP-2 and interleukin-8. This may result in an inadequate host defense that contributes to increased ICU-acquired pneumonia in septic patients.     

Early trauma polymorphonuclear neutrophil responses to chemokines are associated with development of sepsis, pneumonia, and organ failure

OBJECTIVES: The modulation of polymorphonuclear neutrophil (PMN) function by injury is unpredictable, and can predispose either to hyperimmune states (adult respiratory distress syndrome [ARDS], multiple organ failure) or to immune dysfunction, infection, and sepsis. Such outcomes have been related to excess production of the CXC chemokine interleukin (IL)-8, but PMN responses to IL-8 are mediated by both the relatively stable and IL-8 specific CXC receptor 1 (CXCR1) and the labile, promiscuous CXCR2. We hypothesized that progression to septic and multiple organ failure outcomes could be related to early differences in PMN CXC receptor status. METHODS: PMNs were isolated 12 +/- 3 hours after injury from 15 major trauma patients (Injury Severity Score of 34 +/- 2, 11 men and 4 women, age 36 +/- 4 years) who survived at least 7 days. Volunteer normal PMNs (n = 6 donors) were studied for comparison. Cells were stimulated either with the CXCR2 specific agent growth-related oncogene-alpha, or with IL-8, which stimulates CXCR1 and CXRR2. Receptor response was assessed as the mobilization of cell calcium. The development of ARDS, sepsis, and pneumonia was assessed according to standardized criteria. Day 1 receptor activity in the clinical groups was then compared by analysis of variance with Tukey's or t tests as appropriate. RESULTS: In patients that were otherwise comparable, CXCR2 responses were markedly diminished in the PMNs of patients who went on to sepsis and pneumonia, but were elevated in PMNs from the patients who went on to ARDS. CXCR1 responses were modestly lower in trauma patients than volunteers, but showed no significant variations among the various clinical outcome groups. CONCLUSION: The activity of PMN CXCR2 receptors soon after injury may be reflected in the later clinical sequelae of PMN activity. High CXCR2 activity may correlate with PMN hyperfunction and outcomes such as ARDS, whereas the loss of CXCR2 function in inflammatory environments may impair PMN functions in a manner that predisposes to pneumonia or sepsis. Early responses of PMN CXC receptors to injury may influence the clinical course of trauma patients.     

Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis

BACKGROUND: Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response. METHODS: PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Proteinase-3 expression (active 63.04 +/- 5.6% of total number of cells, remission 51.47 +/- 7.9% of total number of cells, control 17.7 +/- 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 +/- 0.8 nmol/L x 10(6) cells, remission 5.15 +/- 0.4 nmol/L/10(6) cells, control 3.63 +/- 0.3 nmol/L/10(6) cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 +/- 3.9% apoptosis, controls 53.2 +/- 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 +/- 6.27, anti-PR3 93.98 +/- 4.90, N IgG 44.89 +/- 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 +/- 6.8 pg/mL, anti-MPO 42.01 +/- 12.3 pg/mL, N IgG 8.04 +/- 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 +/- 0.93 ng/mL, anti-MPO 8.45 +/- 1.46 ng/mL, N IgG 0.96 +/- 0.15 ng/mL, P < 0.01). CONCLUSION: In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.     

Fracture hematoma is a potent proinflammatory mediator of neutrophil function

BACKGROUND: Patients with multiple skeletal injuries are susceptible to acute respiratory distress syndrome and multiple organ failure, which result from hyperactivation of the immune system. This study was designed to evaluate in vitro the proinflammatory properties of fracture hematoma (FH). METHODS: FH was isolated from patients undergoing emergent open reduction and internal fixation for isolated closed fractures. Neutrophils (PMNs), isolated from healthy volunteers, were exposed to the FH supernatant and activation was examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis, apoptosis, and transmigration across an endothelial barrier were also assessed. RESULTS: FH increased PMN respiratory burst (control, 100; FH-treated, 186) and phagocytosis (control, 100; FH-treated, 172) but had no effect on adhesion receptor expression. Transendothelial migration of PMNs was unaffected, although FH was toxic to endothelial cells. In contrast, apoptosis of FH-treated PMNs was delayed (control, 46; FH-treated, 8). CONCLUSION: These effects, although beneficial at the site of injury in the context of antibactericidal function, may cause PMN-mediated tissue injury systemically.     

Alterations in immunocyte tumor necrosis factor receptor and apoptosis in patients with congestive heart failure

OBJECTIVE: To examine systemic immune cell proinflammatory receptor expression and apoptosis in patients with congestive heart failure (CHF). SUMMARY BACKGROUND DATA: Prior studies have demonstrated that CHF is associated with a chronic myocardial inflammatory state, including increased plasma proinflammatory cytokine and soluble receptor expression. By contrast, it has also been shown that tumor necrosis factor (TNF) receptor protein expression is decreased in the failing myocardium. However, no studies to date have examined systemic immune cell proinflammatory receptor expression or function as disease markers in patients with heart failure. METHODS: Twenty-nine patients were studied prospectively over an 8-month period at a single institution. One group (n = 16) had a history of clinical symptoms of CHF and moderate to severe left ventricular dysfunction. The second group (n = 13) consisted of patients who had coronary artery disease without symptoms of CHF and documented preservation of left ventricular function. Blood samples were analyzed for polymorphonuclear cell (PMN) and monocyte TNF and CD95 membrane-associated receptor expression, spontaneous and CD95 (Fas)-mediated PMN apoptosis, and plasma cytokine and soluble TNF receptor levels. Isolated PMNs were incubated for 6 hours with or without CH 11, a CD95 agonist. Propidium iodide/RNAase staining and flow cytometry was used to assess apoptosis, defined as PMNs expressing hypodiploid DNA (<2 n DNA). Membrane-associated TNF receptor and CD95 were also measured by flow cytometry. Plasma levels of TNF, interleukin (IL)-6, IL-10, and soluble TNF receptors 1 and 2 were quantified using enzyme-linked immunosorbent assay. RESULTS: Compared to patients without CHF, circulating PMN and monocyte TNF receptor levels were significantly decreased in patients with CHF. By contrast, PMN and monocyte CD95 expression was not significantly changed in patients with CHF versus those without CHF. Patients with CHF had a 60% decrease in spontaneous PMN apoptosis compared to patients without CHF, whereas no significant difference in CD95-mediated apoptosis was observed between the two groups. Pearson-product movement correlation of monocyte TNF receptor expression and spontaneous PMN apoptosis rates versus patients' ejection fraction was performed and was statistically significant. Plasma levels of soluble TNF receptor 2 (p75) were elevated in CHF patients versus patients without CHF, while there was no significant difference in soluble TNF receptor 1 (p55), TNF, IL-6, and IL-10 between the two groups. CONCLUSIONS: These data demonstrate a systemic alteration in immune cell phenotype and apoptosis in patients with CHF. These findings provide support for the concept that inflammatory mediators either contribute to myocardial dysfunction or are elaborated systemically by left ventricular compromise. This present study suggests that immune cell TNF receptor expression and diminished PMN apoptosis may serve as biologic markers of myocardial failure.     

Store-operated calcium channel inhibition attenuates neutrophil function and postshock acute lung injury

BACKGROUND: A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calcium entry (SOCE) channels rather than the voltage operated (L-type) channels that regulate cardiovascular tone. We hypothesized that SOCE inhibition might suppress postshock PMN activation, lessening lung injury without compromising cardiovascular performance. METHODS: Human PMNs were treated in vitro with N-propargyl-nitrendipine (MRS1845 [MRS]) a dihydropyridine Ca2+ channel blocker with relative specificity for SOCE channels. Calcium flux was measured by fura fluorescence. Chemotaxis was studied in modified Boyden chambers. Respiratory burst was studied by dihydrorhodamine fluorescence. Exploratory studies were then performed where rats were subjected to trauma and hemorrhagic shock (T/HS) (laparotomy, then hemorrhage to a mean arterial pressure of 30-40 mm Hg for 90 minutes) after pretreatment with MRS or vehicle given intraperitoneally at laparotomy. In vivo PMN CD11b expression was then assayed by flow cytometry and lung injury was assessed as percentage Evans blue dye leak 3 hours after resuscitation. The shed blood volume required to achieve standardized hypotension was measured. RESULTS: In vitro, MRS suppressed human PMN SOCE without affecting calcium store release; it suppressed chemotaxis (60 +/- 6 vs. 150 +/- 15 x 10(3) PMNs/well, p = 0.002) and suppressed respiratory burst (62 +/- 11% vs. 100%, p < 0.05) at IC50 concentrations similar to those needed to suppress SOCE. In subsequent in vivo rat studies, MRS decreased postshock PMN CD11b expression from 397 +/- 93 to 268 +/- 39 MFU mean flourescent units (p < 0.05) and decreased lung Evans blue dye permeability from 8.1 +/- 1.9% to 3.4 +/- 0.1% (p < 0.05). MRS had no noticeable effect on the relationship between blood pressure and blood loss, with shed blood volume remaining almost identical (26 +/- 2 mL/kg vs. 27 +/- 3 mL/kg, p = not significant). CONCLUSION: Modulation of PMN Ca2+ entry by means of selective SOCE channel inhibition attenuates PMN inflammatory responses in vitro. In vivo, SOCE channel blockade attenuates trauma and hemorrhagic shock-induced PMN priming and lung injury without gross evidence of hemodynamic side effects. The relative specificity of SOCE channel blockade for "nonexcitable" cells such as PMNs may make it a valuable form of chemoprophylaxis for the inflammatory consequences of hemorrhagic shock in trauma patients.     

Plasma from aged stored red blood cells delays neutrophil apoptosis and primes for cytotoxicity: abrogation by poststorage washing but not prestorage leukoreduction

BACKGROUND: Blood transfusion-particularly that of older stored red blood cells (RBCs)--is an independent risk factor for postinjury multiple organ failure. Immunomodulatory effects of RBC transfusion include neutrophil (PMN) priming for cytotoxicity, an effect exacerbated by longer RBC storage times. We have found that delayed PMN apoptosis in trauma patients is provoked by transfusion, independent of injury severity. We hypothesized that aged stored RBCs delay PMN apoptosis, but that prestorage leukodepletion or poststorage washing could abrogate the effect. METHODS: Healthy volunteers each donated 1 unit of blood. One half was leukodepleted, and RBC units were processed in the usual fashion and stored at 4 degrees C. Aliquots were removed on days 1, 14, 21, and 42 and the plasma fraction isolated. Selected aliquots were washed with normal saline before plasma isolation. PMNs harvested from healthy controls were incubated (5% CO2, 37 degrees C) with unmodified, leukoreduced, or washed RBC plasma (20% plasma/80% RPMI 1640), and apoptosis assessed by morphology after 24 hours. Apoptotic index (apoptotic PMNs/total PMNs) was compared. PMN priming for superoxide release was also assessed after plasma exposure. RESULTS: PMN apoptosis was delayed by RBCs stored for 21 or 42 days. Prestorage leukodepletion did not alter the effect. However, washing 42-day-old RBCs abrogated the effect. PMN priming for superoxide was provoked by stored packed RBCs in an identical pattern to delayed apoptosis. CONCLUSION: Plasma from stored RBCs-even if leukoreduced-delays apoptosis and primes PMNs. The effect becomes evident at 21 days and worsens through product outdate (42 days), but may be prevented by poststorage washing. Inflammatory agents contaminating stored blood likely mediate the effect. Modification of transfusion practices (e.g., giving fresher or washed RBCs or blood substitutes) may attenuate adverse immunomodulatory effects of transfusion in trauma patients.