Postinjury neutrophil priming and activation: an early vulnerable window

BACKGROUND: Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. METHODS: PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. RESULTS: In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. CONCLUSIONS: These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.     

Substantiation of MPEL of acoustic exposure in accordance with its informational value]

Our previous studies have proved that a great number of polymorphonuclear neutrophils (PMN) adhered to endothelial lining and induced endothelial cell (EC) injury. This study was attempted to investigate the change in adhesive force between PMN and EC in response to burn patients' serum within 24 hours and effects of antibody against CD11/CD18 on PMN-EC adhesive force. Burn serum was isolated from 4 burn patients (III degrees 20%-50% TBSA) within 48 hours after burn injury. PMNs were isolated from 8 volunteers. Adhesive force between a PMN and a HUVEC was calculated by means of micropipette technique at 1 h, 3 h, 6 h, 12 h, 24 h after PMN or HUVEC incubated with burn serum or normal serum. Five individual PMN-HUVEC pairs were measured for each sample. All data were analyzed statistically using the t test. RESULTS: 1. The adhesive force between a single PMN-HUVEC pair increased sharply after PMN incubated with burn serum, which reached the maximun level at 1 h after incubation and remained at this level in the following 23 hours. However, the adhesive force was reduced by 78.5%, 72.5%, 75.2% at 1 h, 6 h, 24 h, respectively after the PMNs were pretreated with CD11/CD18 mAb; 2. The adhesive force between a single burn serum stimulated-HUVEC and PMN pair increased gradually, reached peak at 12 h and remained at this level in the following 12 hours. CONCLUSIONS: Burn serum could induce PMN-EC adhesion, with related to PMN aggregation in internal organs and EC damage after burn injury. Antibodies against CD11/CD18 could partially block PMN-EC adhesion and might reduce EC injury induced by activated PMNs.     

Effects of trauma and sepsis on soluble L-selectin and cell surface expression of L-selectin and CD11b

OBJECTIVES: To examine (1) the effects of trauma on changes in neutrophil L-selectin and CD11b expression and on the levels of soluble L-selectin and (2) whether these alterations are different on leukocyte subpopulations in those patients who develop multiple organ dysfunction syndrome. MATERIALS AND METHODS: Twenty patients with Injury Severity Score (ISS) > or = 16 and 15 patients with ISS score < 16 were studied. Arterial blood were collected serially after injury. The staining of leukocyte surface adhesion molecules was performed with antibodies against L-selectin and CD11b. Positive cell count and mean fluorescence intensity were determined by flow cytometry. Soluble L-selectin was measured using enzyme-linked immunosorbent assay. RESULTS: In patients with ISS > or = 16, neutrophil L-selectin expression showed an immediate increase, reaching peak levels between 3 to 4 hours after injury (p < 0.05 vs. patients with ISS < 16), followed by a gradual decrease. Plasma levels of soluble L-selectin reached peak levels at 6 hours after injury. However, in patients with ISS < 16, minimal changes in L-selectin expression and soluble L-selectin were observed. Neutrophil CD11b expression showed an immediate increase for the first 3 hours followed by a gradual increase up to 24 hours after injury. In patients who developed multiple organ dysfunction syndrome, CD11b both on neutrophils and lymphocytes remained elevated for 120 hours. CONCLUSIONS: These findings suggest that acute neutrophil activation is an early event after trauma and may be implicated as "a vulnerable window" for leukocyte-mediated end organ injury.     

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Polymorphonuclear leukocyte (PMN) superoxide (.O2-) production has been implicated in the pathogenesis of cardiopulmonary bypass (CPB)-related end organ injury. PMN "priming" has been described as an event which enhances the release of .O2- following a second, activating insult. We hypothesized that PMN priming occurs during CBP and is temporally related to the plasma level of complement (C3a), interleukin (IL)-6, and IL-8. PMNs were isolated from 10 CPB patients pre-bypass (preCPB), 5 min after protamine administration (PROT), and at 6 and 24 h post-CPB. PMN .O2- production was measured by a cytochrome c reduction assay in the presence or absence of either phorbol 12-myristate-13-acetate (PMA, 0.4 microgram/ml) or N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM) and also after priming with 2000 nM platelet-activating factor (PAF) followed by activation with either PMA or FMLP. Plasma levels of C3a, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. PMA-activated PMN .O2- production was significantly elevated at 6 h post-CPB compared to pre-CPB levels (11.04 +/- 0.9 vs 7.62 +/- 0.57, P = 0.009), indicating that CPB is associated with in vivo PMN priming. When PMNs were primed in vitro with PAF and then activated with PMA or FMLP, .O2- release at 6 h post-CPB was also significantly greater than pre-CPB levels (16.04 +/- 0.74 vs 12.2 +/- 0.92, P = 0.038; and 17.33 +/- 1.38 vs 13.33 +/- 1.35, P < 0.05), indicating that CPB acts synergistically with PAF to prime PMNs. Levels of C3a rose significantly over pre-CPB levels at PROT (P = 0.001), and IL-6 and IL-8 rose over pre-CPB levels at 6 h post-CPB (P = 0.01 and P = 0.006, respectively). These findings demonstrate that CPB not only directly primes PMNs, but also potentiates priming of PMNs by PAF. This "primed" PMN state, which coincided with the increased plasma levels of inflammatory mediators, may suggest a mechanism of predisposition to organ dysfunction following CPB.     

Stimulation of inflammatory markers after blunt trauma

BACKGROUND: Inflammatory mediators are released after trauma and may be related to the pathogenesis of sepsis. A prospective combined study of the pattern of release of an inflammatory mediator, interleukin (IL) 6, leucocyte activation (polymorphonuclear leucocyte (PMN) CD11b receptor expression and plasma elastase-alpha1 proteinase inhibitor complex (E-alpha1PI)) and soluble endothelial adhesion molecule expression (soluble E-selectin (sE-selectin) and soluble intracellular adhesion molecule 1 (sICAM-1)) was performed in patients suffering blunt trauma without adult respiratory distress syndrome (ARDS) or multiple organ failure syndrome (MOFS). METHODS: Thirty-one patients with a mean Injury Severity Score (ISS) of 14 (range 9-57) were studied. Venous blood samples were collected within 6 h of injury and then at 1, 3, 5 and 7 days. Leucocyte CD11b expression was quantified by flow cytometry. Serum IL-6, plasma E-alpha1PI, sE-selectin and sICAM-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Serum IL-6, CD11b expression and E-alpha1PI levels were significantly raised above control values (P < 0.0001) on admission, slowly returning towards control values over the study period (median IL-6, 140 pg/ml versus undetectable; CD11b, 14.8 versus 6.4 mean channel fluorescence units; E-alpha1 PI, 208 versus 52 microg/l). The sICAM-1 level rose to a median of 539 ng/ml at 5 days (control 243 ng/ml). The median sE-selectin level also progressively increased to a maximum level of 80 ng/ml at 5 days (control 49 ng/ml). Eleven patients developed postoperative sepsis. Significant differences in CD11b expression were seen at days 3, 5 and 7 and in E-alpha1 PI at 6 h, 24 h and 3 days in patients who subsequently developed sepsis (P < 0.05). Severe injury (ISS 16 or greater) was associated with significantly greater responses in these measurements. CONCLUSION: These data show that markers of inflammation are specifically stimulated by trauma even when ARDS and MOFS do not occur. The CD11b receptor on PMNs may be useful in screening patients destined to develop post-traumatic sepsis.     

Role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis

OBJECTIVE: To evaluate the role of interleukin 8 (IL-8) in the regulation of neutrophil (PMN) apoptosis in normal plasma and plasma from patients with early, fulminant acute respiratory distress syndrome (ARDS). DESIGN: Experimental study using cultured human PMNs. SETTING: University hospital, level I trauma center. PARTICIPANTS: Plasma was obtained from 6 patients with early, fulminant posttraumatic ARDS (mean Injury Severity Score, 26). All samples were drawn within 24 hours after injury. Plasma was also taken from 13 healthy control subjects. These controls were also used as sources of PMNs. MAIN OUTCOME MEASURES: Effect of early, fulminant ARDS and normal plasma on spontaneous apoptosis, CD16, and CD11-b expression in PMNs in vitro; levels of IL-8 in plasma; correlation of extracellular IL-8 concentration with rate of PMN apoptosis; and effect of IL-8 blockade on PMN apoptosis, CD16, and CD11-b expression in ARDS and normal plasma. RESULTS: Plasma from patients with early, fulminant ARDS inhibited spontaneous PMN apoptosis at 24 hours (35%+/-5% vs 54%+/-5%; P=.01). Neither CD16 nor CD1l-b differed significantly between the 2 groups. The mean plasma level of IL-8 in patients with early, fulminant ARDS was 359+/-161 pg/mL vs 3.0+/-0.4 pg/mL in healthy controls (P<.05). Interleukin 8 inhibited apoptosis in plasma-free medium at low doses (1-50 pg/mL) but had no significant effect at higher doses (100-5000 pg/mL) (P<.05). Interleukin 8 blockade with monoclonal antibody suppressed apoptosis in normal plasma (28%+/-5% with monoclonal antibody vs 51%+/-5% without monoclonal antibody; P=.008) but not in plasma from patients with early, fulminant ARDS (29%+/-5% with monoclonal antibody vs 34%+/-6% without monoclonal antibody; P=.67). It had no effect on CD16 or CD11-b expression in either plasma. CONCLUSIONS: Plasma from patients with early, fulminant ARDS contains soluble factors that inhibit PMN apoptosis in vitro. Low levels of IL-8 inhibit PMN apoptosis in normal plasma. Although plasma levels of IL-8 are markedly elevated in early, fulminant ARDS, IL-8 is not directly responsible for the antiapoptotic effect of plasma from patients with early, fulminant ARDS.     

Role of endothelin-1 in astrocyte responses after acute brain damage

The mucosal injury of active ulcerative colitis (UC) could involve enhanced migration and activation of neutrophils (PMNs). Because, in vitro, PMNs from patients with UC appear normal and are not therefore a likely cause for the enhancements, we hypothesized an abnormal colonic milieu. We previously found that factors in the UC colonic milieu markedly increase production of reactive oxygen species (ROS) by control PMNs. We now hypothesize that these factors also regulate PMN surface integrins, that regulation of UC PMNs is different than that of control PMNs, and that the integrin regulation is consistent with the ROS regulation. Using rectal dialysis, we sampled the colonic milieu in patients with active UC, in patients with inactive UC, and in control subjects. We monitored a key PMN adhesion molecule, CD11b. When control PMNs were tested, active UC rectal dialysate was almost as effective (+115%) as N-formyl-methionyl-leucyl-phenylalanine (+132%) in up-regulating CD11b. When inactive UC PMNs were tested, baseline CD11b was 50% higher than that for control PMNs. In contrast, rectal dialysates failed to up-regulate CD11b of inactive UC PMNs and in fact down-regulated CD11b. Preincubating control PMNs with UC rectal dialysates converted their CD11b response to PMN activators from up-regulation to down-regulation, mimicking inactive UC PMNs. Changes in intracellular calcium levels paralleled these changes in CD11b. Rectal dialysate-induced changes in both CD11b and calcium paralleled our previous findings on rectal dialysate-induced changes in ROS production. Thus the net overall effect of factors in the colonic milieu is a consistent and predictable regulation of PMN function--proinflammatory in UC, anti-inflammatory in control subjects. These factors may be a critical part of the pathophysiology of UC.     

Sequential systemic platelet-activating factor and interleukin 8 primes neutrophils in patients with trauma at risk of multiple organ failure

Plasma from 33 patients at risk of multiple organ failure (MOF) after major trauma was tested for a priming effect on neutrophils, and for the presence of platelet-activating factor (PAF) activity and interleukin (IL) 8. Plasma sampled at 3, 6, 12 and 24 h after injury significantly primed normal neutrophils to release mean(s.e.m.) 1.26(0.19), 1.33(0.26), 1.04(0.14) and 0.86(0.13) nmol superoxide per min per 1.3 x 10(6) neutrophils respectively (P < 0.05). Priming at 3 h after injury was inhibited by mean(s.e.m.) 63.8(7.0) per cent by the PAF antagonist, WEB 2170 (P < 0.01). Mean(s.e.m.) plasma IL-8 was raised at 6 and 12 h after injury to 785(183) and 836(175) pg/ml (P < 0.01). At 12 h after injury the plasma IL-8 level correlated directly with the number of units of red blood cells transfused (r = 0.64, P < 0.01), and was significantly higher in the group of six patients who developed MOF (P < 0.05). These data suggest that after trauma the mediators PAF and IL-8 appear sequentially in the circulation, are potential mechanisms of circulating neutrophil priming, and that IL-8 may also be an early biochemical marker predicting the onset of MOF.     

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.     

Neutrophil transfusions: kinetics and functions of neutrophils mobilized with granulocyte-colony-stimulating factor and dexamethasone

BACKGROUND: The collection of adequate numbers of neutrophils (polymorphonuclear leukocytes, PMNs) from normal donors has long hampered the development of neutrophil transfusion therapy. The stimulation of donors with granulocyte-colony-stimulating factor (G-CSF) plus dexamethasone is a promising way of improving PMN collections. STUDY DESIGN AND METHODS: Sixteen normal subjects received G-CSF (600 micrograms subcutaneously) and dexamethasone (8 mg by mouth) 12 hours before leukapheresis. Measurements included PMN morphology, immunophenotype analysis, chemiluminescence, bactericidal activity, in vivo kinetics, and adverse effects. RESULTS: A mean of 77.4 +/- 6.4 x 10(9) PMNs was collected with each leukapheresis; 14 percent were bands. PMNs had increased surface expression of CD11b, CD18, CD14, CD32, and CD64. Bactericidal capacity against Staphylococcus aureus was normal. Inducible respiratory burst was maintained, although the responses to some agonists were diminished. Returned leukapheresis cells labeled with 3H-diisopropylfluorophosphate had a modestly decreased percentage of recovery and circulated with a prolonged half-life. Migration of these cells to skin chambers was approximately equal to that of the subjects' own blood PMNs. Adverse effects included transient bone pain, headache, hunger, and insomnia. CONCLUSIONS: Precollection treatment of leukapheresis donors with G-CSF plus dexamethasone is an effective way to enhance the collection of PMNs with normal or near-normal functional properties for PMN transfusion therapy.     

Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury

BACKGROUND: The interactions between polymorphonuclear neutrophils (PMNs) and sinusoidal endothelial cells (SECs) have been known to be involved in the pathogenesis of acute liver injury. It has been also reported that tumor necrosis factor-alpha (TNF-alpha) up-regulates ICAM-1 expression on SECs and that interleukin-8 (IL-8) provokes rapid activation of CD11/CD18 on PMNs. These findings expand into the relationship between the expression of leukocyte adhesion molecules (ICAM-1, CD11a/CD18 and CD11b/CD18) in liver tissues and plasma TNF and IL-8 levels after lipopolysaccharide (LPS)-induced liver injury in rats. METHODS: Male Wistar rats weighing 200-250 g were treated with 2 mg LPS/kg intravenously in a 0.2- to 0.25-ml volume. Liver and blood samples were obtained at 1, 3, 8, and 12 h after LPS exposure. Plasma TNF and IL-8 levels were measured using bioassay and specific enzyme-linked immunosorbent assay, respectively. Liver samples were fixed and studied by immunohistochemistry using specific monoclonal antibodies against ICAM-1, CD11a, and CD11b. RESULTS: The TNF level showed a peak at 1 h (23.3 +/- 11.4 IU/ml), and the IL-8 level showed a peak at 3 h (343.1 +/- 110.5 ng/ml) after LPS exposure. An increase in the number of PMNs in the liver was observed as early as 1 h and continued until 12 h after LPS exposure. PMNs adhered to degenerated SECs and hepatocytes. ICAM-1 on SECs was diffusely and strongly expressed at 8 h, and PMNs adhered to SECs expressed both CD11a and CD11b. ICAM-1 was also observed on hepatocytes. CONCLUSION: These data suggest that PMN-SEC and PMN-hepatocyte interactions via leukocyte adhesion molecules, related to inflammatory cytokines such as TNF and IL-8, exist and play an important role in the pathogenesis of acute liver injury.     

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.     

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming

Because of uncertainties regarding the comparability of granulocyte-macrophage and granulocyte colony-stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl-leucyl-phenylalanine (fMLP) was not influenced by G-CSF (0.1-100 ng/ml), whereas GM-CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM-CSF and G-CSF in combination was not different from that of PMNs treated with GM-CSF alone. GM-CSF (10 ng/ml) increased the rate of O2- production by 79%, caused a fivefold increase in fMLP-induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G-CSF (50 ng/ml) only slightly increased O2- production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM-CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G-CSF on mature PMNs is negligible compared with that of GM-CSF. Our results are in conflict with previous reports of much more pronounced G-CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM-CSF. We therefore suggest that the primary role of G-CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in vivo.     

Prothymosin alpha 1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients

Immunoregulatory effects of thymic peptides on functions of polymorphonuclear leukocytes (PMNs) are poorly investigated. We studied the effects of prothymosin alpha 1 (Pro alpha 1) on PMNs from patients with colorectal tumors, breast tumors and melanoma (total n = 37) in comparison with healthy donors (n = 18), with respect to chemotaxis, cytotoxicity against HCT-116 colon tumor cells, oxidative response (chemiluminescence reaction) as well as expression of surface marker molecules. We found that Pro alpha 1 was equally effective in stimulating the chemotactic activity of PMNs from tumor patients and healthy donors (43% increase). PMNs from tumor patients, especially with breast tumor, showed a significant enhancement of cytotoxicity against the tumor target cells in comparison with healthy donors. With respect to the PMNs cytotoxicity, only about 50% of the colorectal tumor patients and healthy donors responded to Pro alpha 1 and FMLP. As to the oxidative response of PMNs, elevated levels were found only among colorectal tumor patients. Pro alpha 1 significantly increased the oxidative response in breast and colorectal tumor patients by 55% and 25%, respectively. Pro alpha 1 decreased the expression of CD16 on PMNs of healthy donors, but not that of CD11a, CD11b, CD11c, CD13, CD14, CD15 and CD32. Therefore, we suggest, that Pro alpha 1 may improve some PMN functions of tumor patients, associated with the proposed role in host-tumor interaction.     

The pattern of inflammation in rat sepsis due to enterotoxin-producing Staphylococcus aureus: a comparison with ischemia-reperfusion injury

Sepsis and trauma have similarities in their immunopathologic profiles. Both conditions can result in multi-system organ failure which is sometimes associated with cytokine generation and inflammatory cell activation. Furthermore, decreases in peripheral blood monocyte expression of HLA-DR have been noted in both human sepsis and trauma. However, the magnitude, onset, and time course of such stimuli are often difficult to ascertain in human studies. Thus, to study a more detailed in vivo immunologic profile in these conditions, rat models were employed. Our aim was to describe and analyze cytokine and peripheral blood immunophenotype patterns in bacterially induced rat sepsis and to compare this to rat ischemia-reperfusion injury. Sprague-Dawley rats underwent either bacterial injection with enterotoxin producing Staphylococcus aureus or hind limb ischemia/ reperfusion. Two bacterial doses which were either lethal or sublethal at 24-48 hours were utilized. Peripheral blood neutrophils and B-lymphocytes were studied for expression of beta-integrins (CD11b and CD11b/c) and I-A, respectively, using flow cytometry. Corresponding plasma levels of TNF alpha and interferon gamma were measured by ELISA. At 24 hr, a lethal bacterial lethal bacterial dose injection resulted in significantly higher levels of neutrophil CD11b/c expression (p < 0.005) compared with ischemia-reperfusion treatment. B-cell I-A expression was also higher in lethal sepsis. Gamma interferon levels were significantly higher in lethal sepsis compared with ischemia-reperfusion (p = 0.005). Studies over time showed that CD11b expression and interferon gamma were both more marked at 6 hr than at 24 hr in lethal sepsis. This pattern was not observed in sublethal sepsis or in ischemia-reperfusion. CD11b/c expression on the other hand remained elevated at comparable levels at 6 and 24 hr in lethal sepsis. B-cell I-A expression in ischemia-reperfusion and sublethal sepsis decreased at 24 hr compared with baseline. Lethal sepsis in rats injected with enterotoxin producing staphylococcus results in phasic alterations in neutrophil CD11b and plasma interferon levels prior to death. In analogy to the findings of monocyte decreases in DR expression observed in human trauma and sepsis, rat B-cell I-A expression showed decreases in sublethal sepsis as well as in ischemia-reperfusion injury. However, this was not observed in lethal sepsis. These findings have implications in understanding the immunologic/inflammatory changes observed in human sepsis and trauma.     

Azurophilic granules of human neutrophils contain CD14

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.     

Intestinal permeability correlates with severity of injury in trauma patients

BACKGROUND: Increased intestinal permeability (IP) and the release of toxic intraluminal materials have been implicated in the systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) observed in patients after severe trauma. Previous studies of intestinal permeability have failed to demonstrate a correlation between early measurements of IP and indicators of injury severity. This study examines the relationship between standard measures of injury severity and the early (day 1) and delayed (day 4) changes in IP. Associations between IP and the development of SIRS, MOF, and infectious complications were also studied. METHODS: The metabolically inactive markers lactulose (L) and mannitol (M) were used to measure IP in 29 consecutive patients who sustained injuries that required admission to the surgical intensive care unit and in 10 healthy control subjects. Measurements were made within 24 hours of admission and on hospital day 4. Severity of injury was assessed by A Severity Characterization of Trauma (ASCOT), Trauma and Injury Severity Score (TRISS), Injury Severity Score (ISS), Revised Trauma Score (RTS), and Acute Physiology and Chronic Health Evaluation (APACHE) II score. Postinjury infections and parameters of SIRS and MOF were recorded. RESULTS: The IP of healthy volunteers (L/M, 0.025 +/- 0.008) was within the normal range (L/M < or = 0.03), whereas the average IP in injured patients was increased both within 24 hours (L/M, 0.139 +/- 0.172) and on the fourth hospital day (L/M, 0.346 +/- 0.699). No significant correlation between severity of injury and increased IP was seen within 24 hours of injury. A significant correlation was seen on hospital day 4, however, with all severity indices measured (ASCOT: r = 0.93, R2 = 0.87, p < 0.001; TRISS: r = 0.93, R2 = 0.87, p < 0.001; ISS: r = 0.84, R2 = 0.70, p < 0.001; RTS: r = 0.68, R2 = 0.47, p = 0.002; APACHE II score: r = 0.51, R2 = 0.26, p = 0.04). Patients with markedly increased IP (L/M > or = 0.100) experienced a significant increase in the development of SIRS (83 vs. 44%; p = 0.03) and subsequent infectious complications (58 vs. 13%; p = 0.01) and showed close correlation with the multiple organ dysfunction scores (r = 0.87, R2 = 0.76, p < 0.001). CONCLUSION: These observations demonstrate that the increased IP observed after trauma correlates with severity of injury only after 72 to 96 hours and not within the initial 24 hours of injury. A large increase in IP is associated with the development of SIRS, multiple organ dysfunction, and an increased incidence of infectious complications.     

Hypoxia reoxygenation-induced injury of cultured pulmonary microvessel endothelial cells

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.     

Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects

OBJECTIVE: To evaluate neutrophil functions in the elderly. METHODS: We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects. RESULTS: No difference was found in PMN migration in vivo (62.9 +/- 21.3 x 10(6) and 65.5 +/- 9.1 x 10(6) PMN/cm2/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6 +/- 2.7 and 9.3 +/- 3.3 nMOLES O2-/10(6) PMN respectively, p < 0.0001) and by exudate PMNs (13.6 +/- 4.3 and 19.4 +/- 6 nMOLES O2-/10(6) PMNs respectively, p < 0.005). CONCLUSION: Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Accelerated healing of gingival incisions by the helium-neon diode laser: a preliminary study

Platelet-activating factor (PAF) concordantly primes neutrophils (PMNs) for superoxide generation and elastase release. beta-Adrenergic stimulation of PMNs enhances cAMP-dependent protein kinase A (PKA) activity and has been shown to inhibit PAF-mediated NADPH-oxidase activity. PMN superoxide generation is thought to play a predominate microbicidal role, whereas elastase is known to mediate untoward PMN-endothelial interactions. We hypothesized that beta-adrenergic neutrophil stimulation has disparate effects on PAF-mediated PMN superoxide generation versus elastase release. Human PMNs were isolated using a standard Ficoll/Hypaque gradient. PMNs were then primed with PAF (200 nM) and activated with fMLP (1 microM). Subsets of PMNs were pretreated for 5 min with a beta agonist (10(-4) M isoprotereno) or an adenylate cyclase agonist (10(-5) M forskolin). Superoxide generation was determined by superoxide dismutase inhibitive cytochrome c reduction. Elastase activity was measured by the cleavage of n-methoxylsuccinyl-A-A-P-V-p-nitroanilide. Pretreatment with isoproterenol and forskolin yielded superoxide generation of 3.2 +/- 0.6 and 3.1 +/- 1.2 nmole/2.5 x 10(5) PMN/min compared to 9.0 +/- 0.6 nmole/2.5 x 10(5) PMN/min for PAF/fMLP alone, whereas isoproterenol and forskolin did not significantly affect PAF-mediated neutrophil elastase release, 22.4 +/- 5.3 and 24.0 +/- 3.6%, respectively, compared to 39.4 +/- 9.1% for PAF/fMLP alone. Disparate PMN signal transduction for superoxide generation versus elastase release may explain the SICU clinical paradox, in which patients are both susceptible to infection and vulnerable to PMN-mediated multiple organ failure.