Conventional mechanical ventilation is associated with bronchoalveolar lavage-induced activation of polymorphonuclear leukocytes: a possible mechanism to explain the systemic consequences of ventilator-induced lung injury in patients with ARDS

BACKGROUND: Protective ventilatory strategies have resulted in a decreased mortality rate in acute respiratory distress syndrome, but the underlying mechanisms remain unclear. The authors hypothesized that (1) mechanical ventilation modulates activation of polymorphonuclear leukocytes (PMNs), (2) the consequent release of proteinases is correlated with a systemic inflammatory response and with multiple organ dysfunction, and (3) these deleterious effects can be minimized by a protective ventilatory strategy. METHODS: Human PMNs were incubated with bronchoalveolar lavage fluid obtained from patients at entry or 36 h after randomization to ventilation with either a conventional (control) or a lung-protective strategy. PMN oxidant production and surface expression of adhesion molecules and granule markers, including CD18, CD63, and L-selectin, were measured by flow cytometry. Extracellular elastase activity was quantified using a fluorescent substrate. RESULTS: Bronchoalveolar lavage obtained from both groups of patients at entry showed similar effects on PMN oxidant production and expression of surface markers. At 36 h, exposure of PMNs to bronchoalveolar lavage fluid from the control group resulted in increased PMN activation as manifested by a significant increase in oxidant production, CD18, and CD63 surface expression, and shedding of L-selectin. By contrast, these variables were unchanged at 36 h in the lung-protective group. There was a significant correlation between the changes of the variables and changes in interleukin-6 level and the number of failing organs. CONCLUSIONS: Polymorphonuclear leukocytes can be activated by mechanical ventilation, and the consequent release of elastase was correlated with the degree of systemic inflammatory response and multiple organ failure. This result may possibly explain the decreased mortality in acute respiratory distress syndrome patients treated with a lung-protective strategy.     

Changes of pulmonary intercellular adhesion molecule-1 and CD11b／CD18 in peripheral polymorphonuclear neutrophils and their significance at the early stage of burns

Objective: To investigate the role of intercellular adhesion molecule-1 (ICAM-1) in the accumulation of polymorphonuclear neutrophils (PMN) in the lungs at the early stage of burns. Methods: Myeloperoxidase content in lung tissues and bronchoalveolar lavage fluid (BALF) were detected.ICAM-1 and its mRNA expression in lung tissues were determined bycal method and in situ hybridization. CD11b/CD18 expression on the peripheral PMNs was measured by flowcytometry. Results : The levels of myeloperoxidase in lung tissues and BALF after burn injury were markedly higher than those of control. Expression of ICAM-1 and its mRNA in the lung tissues and CD11b/CD18 on peripheral PMNs surface was significantly increased at 2, 6, 12, 24 h after burns. Conclusions. PMNs accumulation in the lungs is related to increased ICAM-1 expression on pulmonary microvascular endothelial cells and CD11b/CD18 expression on PMN at the early stage of burn injury.     

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.     

Neutrophil Adhesion Molecule Expression Is Comparable in Perinatal Rabbits and Humans

Background: Human newborns, particularly those born before full term, are more susceptible to bacterial infections as a result of impaired host defense mechanisms. Compared with adults, circulating leukocytes from human newborns (preterm and full-term gestations) and newborn rabbits (full-term gestation) have low resting levels of CD62L (L-selectin) and do not significantly increase surface expression of CD18 after inflammatory stimulation. To determine the potential utility of preterm rabbits in investigations of perinatal human conditions, the authors compared the surface expression of the beta2 -integrin CD18 and CD62L (L-selectin) on polymorphonuclear leukocytes (PMNs) from perinatal rabbits and perinatal humans, both under resting conditions and after in vitro activation with inflammatory stimulants.

Methods: After erythrocyte lysis of whole-blood samples, leukocytes from 7-day-old, full-term (31-day gestation), and preterm (24-day gestation) rabbits, as well as full-term (37-42 week gestation) and preterm (27-36 week gestation) human newborns were prepared and stimulated in vitro at 37 degrees C with either C5a or phorbol myristate acetate. After fluorescence labeling of CD18 and CD62L with monoclonal antibodies, PMN adhesion molecule expression was assessed by flow cytometry.

Results: Constitutive CD18 expression was not significantly different between perinatal and adult humans but was reduced in all perinatal rabbits compared with adults. Inflammatory stimulation caused significant increases in CD18 expression in adult human PMNs but not in full-term and preterm newborns. Changes in CD18 expression in adult and preterm rabbits after stimulation, although in the same direction as humans, were more variable. In both species, constitutive CD62L expression on PMNs from all perinates was significantly lower than in adults. However, CD62L was shed to similar degrees after inflammatory stimulation in all groups.     

Posthemorrhagic shock mesenteric lymph primes circulating neutrophils and provokes lung injury.

Mesenteric lymph has recently been invoked as an avenue for gut-derived factors that may result in distant organ injury following hemorrhagic shock. We demonstrate that posthemorrhagic shock mesenteric lymph primes neutrophils (PMNs) and causes lung injury. Methods. Mesenteric lymph was collected from Sprague-Dawley rats from their mesenteric lymph duct prior to, during, and following hemorrhagic shock (MAP 40 for 90 min). The rats were then resuscitated with shed blood plus lactated Ringers (2X shed blood) over 3 h. Lung leak was assessed by transudation of Evan's blue dye into the alveolus as measured by bronchoalveolar lavage. Isolated human PMNs were incubated with 1 and 10% lymph; priming was measured by the fMLP (1 microM)-stimulated production of superoxide and surface expression of CD11b determined by flow cytometry. Results. Mesenteric lymph flow increased significantly during resuscitation: preshock 144.4 microl/h, shock 44.5 microl/h, resuscitation 566.6 microl/h. Furthermore, diversion of this lymph abrogated lung injury as compared to rats without lymph diversion. Finally, mesenteric lymph from postshock animals primed PMNs for superoxide production (nearly three times control cells) as well as increased surface expression of CD11b (2-fold over control). Conclusion. Mesenteric lymph primes PMNs and causes lung injury following hemorrhagic shock. Mesenteric lymph provides a conduit for proinflammatory mediators that may participate in the pathogenesis of MOF.     

Granulocyte colony-stimulating factor and neutrophil-related changes in local host defense during recovery from shock and intra-abdominal sepsis.

BACKGROUND: We have reported that treatment with exogenous granulocyte colony-stimulating factor (G-CSF) improves abscess localization and reduces mortality without aggravating neutrophil (PMN)-mediated reperfusion injury in a model of septic abdominal trauma. The purpose of this study was to determine actions of G-CSF on PMN function in the peritoneum. METHODS: Anesthetized swine were pretreated with broad-spectrum antibiotics and underwent cecal ligation and incision and 35% hemorrhage (trauma). After 1 hour they were resuscitated with shed blood, crystalloid, and either G-CSF (n = 10) or saline solution vehicle (n = 9). The animals were observed for 72 hours. RESULTS: After trauma, saline solution treatment increased PMN infiltration into the peritoneum within 2 hours (P = .035), increased peritoneal PMN elastase production (i.e., cytotoxicity) by 24 hours (P = .004), and decreased adherence of peritoneal PMNs to an artificial substrate from 4 to 72 hrs (P = .043). The mean autopsy score was 7.0 +/- 0.5. With G-CSF treatment peritoneal neutrophilia was enhanced (maximum 48 hours, P = .002) and PMN cytotoxicity was augmented and delayed (maximum 48 hours, P = .004). Despite these changes, adherence of peritoneal PMNs was not significantly changed and there was no evidence for PMN-mediated damage in the lung as judged by bronchoalveolar lavage protein, bronchoalveolar lavage PMNs, lung tissue myeloperoxidase, or histologic changes. The mean autopsy score was improved to 4.1 +/- 0.3 (P < .001). CONCLUSIONS: G-CSF in resuscitation fluids improved localization of an intra-abdominal septic focus by increased production of circulating PMNs, increased PMN extravasation into the peritoneal cavity, and increased PMN cytotoxicity at the abdominal septic focus, without exaggerating PMN-dependent reperfusion injury in the lung. Therefore these data further support the idea that G-CSF in resuscitation fluids might reduce septic complications in the multiply injured trauma patient.     

25% Albumin modulates adhesive interactions between neutrophils and the endothelium following shock/resuscitation

BACKGROUND: Polymorphonuclear neutrophil (PMN) sequestration in the lung is a hallmark of acute respiratory distress syndrome (ARDS). We have shown that 25% Albumin (A25) resuscitation attenuates lung injury after hemorrhagic shock and lipopolysaccharide (LPS) exposure by reducing lung leukosequestration. We hypothesize that this protective property is mediated by alteration of neutrophil-endothelial cell adhesive interactions. MATERIALS AND METHODS: A 2-hit rodent model of shock resuscitation was used. CD11b and L-selectin were measured using flow cytometry in rat and human neutrophils ex vivo. Intercellular adhesion molecule-1 (ICAM-1) levels were measured by Northern blot and immunohistochemistry. RESULTS: Resuscitation with A25 attenuated the increase in PMN CD11b expression in Ringer's lactate (RL) resuscitated animals at end resuscitation and at 4-hour post-LPS. While PMN L-selectin levels remained stable in RL treated animals, A25 resuscitation resulted in a significant decrease in surface L-selectin expression at 4-hour post-LPS. ICAM-1 lung endothelial cell mRNA, was increased in RL resuscitated animals, however reduced with A25 use by 51%. The LPS induced ICAM-1 endothelial cell protein expression was also prevented with A25 resuscitation. Antioxidant property of albumin was shown to play a critical role in altering CD11b expression. CONCLUSIONS: The A25 exerts its lung-protective activity at various levels including altering the interaction between neutrophils and endothelial cells via suppressed expression of adhesion molecules. These findings suggest a novel role for A25 as an anti-inflammatory agent in PMN-mediated diseases such as ARDS.     

Effects of lung preservation solutions on PMN activation in vitro

Polymorphonuclear leukocyte (PMN) activation and PMN-endothelial cell interactions may cause graft failure due to ischemia-reperfusion injury after lung transplantation. We investigated the effects of Euro-Collins solution (EC), low-potassium dextran solution (LPD), and EC plus pentoxifylline (EC-PTXF) on adhesion molecule (CD11b/CD18 and L-selectin) expression, chemotaxis, and oxidative burst of PMN. PMN from healthy human volunteers were incubated with EC, LPD, and EC-PTXF, and, in controls, without preservation solution. LPD exerted a suppressive effect on PMN chemotaxis as compared to EC (P < 0.05), but had no attenuating effect on the increase of CD11b/CD18, the shedding of L-selectin, and intracellular oxidant generation. EC-PTXF attenuated the expression of CD11b/CD18 and the oxidative burst as compared to EC alone (P < 0.05). These effects of LPD and PTXF on PMN function may contribute to successful organ preservation in transplantation. Received 22 June 98 Received after revision: 30 December 1998 Accepted: 19 January 99     

Superoxide production by neutrophils in a model of adult respiratory distress syndrome

Neutrophils (polymorphonuclear leukocytes [PMNs]) are thought to contribute to the pathophysiology of adult respiratory distress syndrome (ARDS) by the release of toxic oxygen metabolites. This study investigated superoxide production by circulating and bronchoalveolar lavage (BAL) PMNs in a rat model of ARDS induced by chronic Escherichia coli (lipopolysaccharide) endotoxemia. Superoxide production was stimulated by fmet-leu-phe, opsonized zymosan, and phorbol myristate acetate. Circulating and BAL PMNs from lipopolysaccharide-infused rats compared with PMNs from control rats are primed for nonselective increased superoxide production. The BAL PMNs are not only partially primed to release superoxide on adherence, they concomitantly have a depressed superoxide response to a phagocytic (opsonized zymosan) stimulus. These PMN responses may partially explain both the pulmonary injury and the increased susceptibility to pulmonary infection seen in patients with ARDS.     

Neutrophil CD18 expression and blockade after traumatic shock and endotoxin challenge.

OBJECTIVE: The expression of the leukocyte CD18 adhesion complex on polymorphonuclear leukocytes (PMNs) was measured, and the physiologic effects of blockade of the complex were studied after trauma and sepsis. SUMMARY BACKGROUND DATA: Margination of PMNs occurs early during inflammation and depends, in part, on expression of the CD18 adhesion complex. Blockade of this adherence complex can reduce PMN-mediated damage. This study tests the hypothesis that PMN activation after resuscitated trauma produces an occult endothelial injury that increases the vulnerability to a delayed inflammatory stimulus. METHODS: Anesthetized (fentanyl) mongrel pigs were sham injured or fluid resuscitated from soft tissue injury +35% hemorrhage. Systemic blood was collected at 24-hour intervals from awake animals. The CD18 density on circulating PMNs was determined with flow cytometry using mean channel fluorescence (MCF). The CD18 receptors were blocked with monoclonal antibodies either immediately before trauma or immediately before an endotoxin (lipopolysaccharide [LPS]) challenge that was administered to all groups 3 days after the shock episode. Bronchoscopy was performed before trauma, pre-LPS, and post-LPS, and protein content was measured in bronchoalveolar lavage (BAL). RESULTS: Mean channel fluorescence was reduced on PMNs for 48 hours in animals with trauma versus animals with sham injuries. Anti-CD18 therapy produced higher circulating PMN counts compared with nontreated sham or shock groups. The incremental rise of BAL protein after shock was prevented with anti-CD18; the increment after LPS was attenuated. Anti-CD18 was administered before trauma and reduced the fluids necessary to maintain cardiac filling pressures after LPS. CONCLUSIONS: These data suggest that PMNs are activated after resuscitation from traumatic shock and that these cells produce an endothelial injury that may increase the vulnerability to a septic challenge. The broad implication is that temporarily blocking PMN adhesiveness at the time of trauma might salvage some host tissue and reduce the incidence of septic complications in the post-trauma period.     

Hemodialysis-induced degranulation of polymorphonuclear cells: no correlation between membrane markers and degranulation products

BACKGROUND/AIMS: Degranulation of polymorphonuclear leukocytes (PMN) during hemodialysis (HD) is usually assessed by measuring degranulation products. However, this process might also be estimated by the assessment of cell surface markers. In this study, the relationship between the expression of PMN degranulation markers (CD63 and CD66b) and the release of degranulation products [myeloperoxidase (MPO) and lactoferrin (LF)] was investigated during clinical HD in order to evaluate cell surface markers as a useful index of PMN degranulation. METHODS: The expression of CD63 and CD66b on PMN and the release of MPO and LF were investigated in 10 chronic HD patients, during both heparin (HDhep) and trisodium citrate anticoagulation (HDcit), in a randomized order. Samples were drawn from both the efferent and afferent lines of the dialyzer at 0, 7.5, and 180 min. RESULTS: During HDhep at first passage, a major increase in MPO (from 158 +/- 32 to 448 +/- 177 microg/l, p = 0.001) and LF (from 134 +/- 52 to 260 +/- 120 microg/l, p = 0.01) was found across the dialyzer, whereas marked changes were not observed during HDcit. The expression of CD63 and CD66b increased across the dialyzer during both anticoagulation modalities, but was only significant in the case of HDhep (CD63: mean fluorescence intensity from 247 +/- 61 to 331 +/- 118, p < 0.01; CD66b: mean fluorescence intensity from 340 +/- 76 to 434 +/- 103, p = 0.01). During HDhep a correlation was noted between the degranulation products and markers of both azurophilic and specific granules (MPO and CD63: r = 0.35; p < 0.01; LF and CD66b: r = 0.39, p < 0.01). Significant differences in the expression of CD63 and CD66b between HDhep and HDcit were not observed. When analyzing the combined data for both HDhep and HDcit, no correlation was observed between degranulation products and markers. CONCLUSION: Our data suggest that the measurements of cell surface markers may not be a reliable indicator of the degree of HD-induced PMN degranulation.     

Sevoflurane and isoflurane protect the reperfused guinea pig heart by reducing postischemic adhesion of polymorphonuclear neutrophils.

BACKGROUND: Polymorphonuclear neutrophils (PMNs) contribute to reperfusion injury. Because volatile anesthetics can reduce PMN adhesion in the reperfused, nonworking heart, the authors analyzed whether this action of volatile anesthetics affects cardiac performance after ischemia and reperfusion and further clarified the underlying mechanism. METHODS: Isolated guinea pig hearts perfused with crystalloid buffer and performing pressure-volume work were used. Hearts were subjected to 15 min global ischemia and 20 min reperfusion. In the intervention groups an intracoronary bolus of 3 x 10(6) PMNs was applied in the second min of reperfusion, either in the absence or presence of 0.5 or 1 minimum alveolar concentration sevoflurane or isoflurane. The number of sequestered PMNs was calculated from the difference between coronary input and output (coronary effluent) of PMNs. Performance of external heart work, determined pre- and postischemically, served as criterion for recovery of myocardial function. Additionally, the expression of the integrin CD11b on the cell surface of PMN was measured before and after coronary passage. RESULTS: Injection of PMN in the reperfusion phase, but not under nonischemic conditions, reduced recovery of external heart work significantly (from 55+/-7% to 19+/-11%). Addition of sevoflurane or isoflurane in concentrations of 0.5 and 1 minimum alveolar concentration to the perfusate reduced postischemic PMN adhesion from 36+/-8% to basal values (20+/-7%) and prevented decline of cardiac function. CD11b expression on PMNs increased significantly during postischemic coronary passage under control conditions. Again, both anesthetics in both concentrations inhibited that activation. CONCLUSIONS: Volatile anesthetics reduce PMN adhesion in the reperfused coronary system and thereby preserve cardiac function. Reduced expression of the adhesion molecule CD11b on PMNs in the presence of sevoflurane or isoflurane is, at least in part, responsible for the cardioprotective effect.     

Dynamics of leukocyte receptors after severe burns: an exploratory study

Background

Patients with burns are susceptible to organ failure, and there is indirect evidence that leukocytes may contribute to this process. They may change the expression of cell-surface receptors after certain stimuli, for example, the burn. We therefore aimed to assess the changes induced by the burn in the expression of leukocyte cell-surface receptors CD11b, CD14, CD16, and CD62L on the surface of PMNs and monocytes. We also wanted to examine the dynamics of this activation during the first week after the burn, and to relate it to the size of the injury.

Methods

Ten patients with burns of >15% (TBSA) were included in the study. Blood samples were collected on arrival and every consecutive morning during the first week. Healthy volunteers acted as controls.

Results

PMN CD11b expression was increased. The extent of PMN CD11b expression correlated negatively to the size of the full thickness burn. Monocyte CD14 expression increased initially but there was no relation to the size of the burn. PMN CD16 expression decreased initially during the first days and the decrease was related to burn size. CD62L did not vary depending on the burn in either PMN or monocytes during the first week after the burn.

Conclusion

This study showed that specific receptors on the surface of leukocytes (PMN CD11b, monocyte CD14 and PMN CD16) are affected by the burn. Expression of PMN CD11b and CD16 are related to burn size. Burn-induced effects on the expression of PMN receptors, such as PMN CD11b and CD16, may contribute to burn-induced infection susceptibility.     

Mesenteric lymph is responsible for post-hemorrhagic shock systemic neutrophil priming.

BACKGROUND: Hemorrhagic shock-induced splanchnic hypoperfusion has been implicated as a priming event in the two event model of multiple organ failure (MOF). We have previously shown that early postinjury neutrophil (PMN) priming identifies the injured patient at risk for MOF. Recent in vitro studies have demonstrated that postshock mesenteric lymph primes isolated human neutrophils. We hypothesize that lymphatic diversion before hemorrhagic shock abrogates systemic PMN priming and subsequent lung injury. METHODS: Sprague-Dawley rats (n >or= 5 per group) underwent hemorrhagic shock (MAP 40 mm Hg x 30 min) and resuscitation (shed blood + 2x crystalloid) with and without mesenteric lymphatic duct diversion. Sham animals underwent anesthesia and laparotomy. Whole blood was taken 2 hours after resuscitation, heparinized, and incubated for 5 min at 37 degrees C. Surface expression of CD11b (a marker for PMN priming) was determined by flow-cytometry compared with isotype controls. In addition, lung myeloperoxidase (MPO) was measured for PMN sequestration, and Evans blue lung leak was assessed in the bronchoalveolar lavage fluid in sham, and shock +/- lymph diversion animals. RESULTS: Hemorrhagic shock resulted in increased surface expression of PMN CD11b relative to sham (23.8 +/- 6.7 vs. 9.9 +/- 0.6). Mesenteric lymphatic diversion before hemorrhagic shock abrogated this effect (8.0 +/- 2.6). Lung PMN accumulation, as assessed by MPO, was greater in the lungs of nondiverted (113 +/- 14 MPO/mg lung) versus sham (55 +/- 4 MPO/mg lung, p < 0.05); lymph diversion reduced lung PMNs to control levels (71 +/- 6.5 MPO/mg lung, p < 0.05). Evans blue lung leak was 1.6 times sham in the hemorrhagic shock group; this was returned to sham levels after lymph diversion (p < 0.05). CONCLUSION: Post-hemorrhagic shock mesenteric lymph primes circulating PMNs, promotes lung PMN accumulation, and provokes acute lung injury. Lymphatic diversion abrogates these pathologic events. These observations further implicate the central role of mesenteric lymph in hemorrhagic shock-induced lung injury. Characterizing the PMN priming agents could provide insight into the pathogenesis of postinjury MOF and ultimately new therapeutic strategies.     

Effects of spermidine and p-cresol on polymorphonuclear cell apoptosis and function

Polymorphonuclear leukocytes (PMNs) from chronic kidney disease (CKD) patients display accelerated apoptosis and dysfunction, which may predispose CKD patients to infections. In this study, we investigated the effect of spermidine and p-cresol on apoptosis and function on PMN from healthy subjects. We measured the effect of spermidine and p-cresol on apoptosis, ROS production unstimulated and stimulated (S. aureus and PMA) and expression of CD95, caspase 3, and CD11b on PMN. After incubation with p-cresol and spermidine, we did not observe any changes in apoptosis, viability or expression of caspase 3 and CD95 in PMN from healthy subjects. PMN incubated for 10 minutes with spermidine demonstrated a significant reduction in spontaneous, S. aureus and PMA-stimulated ROS production. p-cresol induced a decrease in PMA-stimulated ROS production. Spermidine and p-cresol also induced a decrease in the expression of CD11b on PMN. Spermidine and p-cresol decreased the expression of CD11b and oxidative burst of PMN from healthy subjects and had no effect on PMN apoptosis and viability.     

Sevoflurane and Isoflurane Protect the Reperfused Guinea Pig Heart by Reducing Postischemic Adhesion of Polymorphonuclear Neutrophils

Background: Polymorphonuclear neutrophils (PMNs) contribute to reperfusion injury. Because volatile anesthetics can reduce PMN adhesion in the reperfused, nonworking heart, the authors analyzed whether this action of volatile anesthetics affects cardiac performance after ischemia and reperfusion and further clarified the underlying mechanism.

Methods: Isolated guinea pig hearts perfused with crystalloid buffer and performing pressure-volume work were used. Hearts were subjected to 15 min global ischemia and 20 min reperfusion. In the intervention groups an intracoronary bolus of 3 x 106 PMNs was applied in the second min of reperfusion, either in the absence or presence of 0.5 or 1 minimum alveolar concentration sevoflurane or isoflurane. The number of sequestered PMNs was calculated from the difference between coronary input and output (coronary effluent) of PMNs. Performance of external heart work, determined pre- and postischemically, served as criterion for recovery of myocardial function. Additionally, the expression of the integrin CD11b on the cell surface of PMN was measured before and after coronary passage.

Results: Injection of PMN in the reperfusion phase, but not under nonischemic conditions, reduced recovery of external heart work significantly (from 55 +/- 7% to 19 +/- 11%). Addition of sevoflurane or isoflurane in concentrations of 0.5 and 1 minimum alveolar concentration to the perfusate reduced postischemic PMN adhesion from 36 +/- 8% to basal values (20 +/- 7%) and prevented decline of cardiac function. CD11b expression on PMNs increased significantly during postischemic coronary passage under control conditions. Again, both anesthetics in both concentrations inhibited that activation.     

Neutrophil phagocytosis during endotoxin-induced lung injury

Depressed neutrophil (PMN) phagocytosis in patients with ARDS may contribute to the known increased incidence of pulmonary sepsis. To evaluate changes in phagocytosis, circulating PMNs from normal rats were compared to circulating and alveolar PMNs (obtained by bronchoalveolar lavage, BAL) from rats after 72 hr of endotoxin infusion (LPS-Rx)-induced acute lung injury. Since phagocytosis correlates with adherence, PMN adherence to coverslips and to a standard nylon wool column was also measured. PMN adherence to nylon wool was 65% for control, 77% for circulating LPS-Rx, and 20% for BAL PMNs. As a measure of phagocytosis the PMNs were incubated for 30 min with opsonized fluorescent (FITC) tagged yeast. Total PMN with yeast were 95.4 +/- 2.1% for control; 96.4 +/- 1.8% for circulating LPS-Rx; and 78.7 +/- 7.8% (P less than 0.05 compared to control) for BAL PMNs. Total numbers of yeast particles per 100 PMN are 270 +/- 64 for control, 300 +/- 42 for circulating LPS-Rx, and 170 +/- 45 (P less than 0.05 compared to control) for BAL PMN. Conclusions: (1) Intraalveolar (BAL) PMNs have decreased adherence; (2) nonadherent PMNs have decreased uptake of yeast; (3) BAL PMNs, overall, have a significantly decreased uptake of yeast; (4) this depression in BAL PMN phagocytosis may partially explain the known decreased rate of bacterial clearance in injured lungs and the increased risk of pulmonary sepsis with adult respiratory distress syndrome.     

Cytokine-modulated inhibition of neutrophil apoptosis at local site augments exudative neutrophil functions and reflects inflammatory response after surgery

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

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.     

Decreased leukocyte adhesion with anti-CD18 monoclonal antibodies is mediated by receptor internalization.

BACKGROUND. Adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells is mediated partially by CD11/CD18 integrins. The purpose of this study was to define (1) the response of PMNs to anti-CD18 monoclonal antibody binding, and (2) the mechanism responsible for anti-CD18 monoclonal antibody-mediated decreases in PMN adhesion to endothelial cells. METHODS. Canine PMN O2- production, myeloperoxidase, and lysozyme release in response to the anti-CD18 monoclonal antibody IB4 were measured by standard assays. To examine endocytosis of CD18 receptors, PMNs incubated with IB4 and a fluorescein isothiocyanate secondary antibody were analyzed by flow cytometry. RESULTS. Treatment of PMNs with IB4 did not stimulate O2- production or degranulation but decreased adhesion of 51Cr-labeled PMNs to ex vivo canine aorta. Incubation of PMNs at 25 degrees C resulted in a decrease in fluorescence intensity that was not affected by NaN3 or vanadate but was blocked by NaF, 4 degrees C, and bafilomycin, which prevents endosomal acidification. Treatment with an antifluorescein antibody decreased the fluorescence intensity in NaF and 4 degrees C, but not in bafilomycin-treated neutrophils. CONCLUSIONS. IB4 decreases PMN-endothelial cell adhesion but does not stimulate neutrophil oxidative metabolism or degranulation. These data suggest that reduced adhesion may be the result of internalization of the CD18/IB4 complex. Anti-CD18 monoclonal antibodies may be useful in preventing PMN adhesion without the potentially deleterious effects of cell activation.