Zymosan-activated plasma causes prolonged decreases in PMN superoxide release in sheep

It is well established that activation of neutrophils within the pulmonary circulation produces acute lung injury in which adherence of neutrophils to endothelial cells is an obligatory step in the mechanism of injury. The effects of in vivo activation of neutrophils on the in vitro responses of these cells to stimulation have not been determined, although such information may be important in understanding how different etiological factors may interact to produce infection or acute respiratory failure. By using an assay to sequentially measure superoxide anion (O2-) release from adherent neutrophils stimulated with phorbol myristate acetate (PMA), we measured the in vitro activation response of peripheral blood neutrophils isolated before and 24 h after infusion of zymosan-activated plasma (ZAP; or untreated plasma as a control), air bubbles, or PMA in awake, instrumented sheep. Each of the three inflammatory agents produced an increase in lung microvascular permeability characteristic of acute lung injury; control plasma did not. For the in vivo ZAP experiments, stimulated O2- release in vitro by using PMA was approximately 50% lower (P less than 0.05) for neutrophils isolated 24 h after the in vivo infusion (4.3 +/- 0.8 nmol/500,000 cells) than before (8.1 +/- 0.2 nmol/500,000 cells). For the air emboli or PMA in vivo experiments, there were no changes in neutrophil activation responses in vitro. Similarly, infusion of control plasma did not result in reduced neutrophil O2- release. These results show that alterations in the inflammatory potential of neutrophils may occur in vivo and that such alterations appear to be dependent on the mechanism and agent by which lung injury is produced.     

Zymosan activated plasma infusion in sheep: Effects of ethanol administration

Zymosan activated plasma infusion induces pulmonary sequestration of neutrophils and the release of TXA₂ into the pulmonary vascular bed causing profound and transient pulmonary hypertension.Since ethanol (ETOH) inhibits several inflammatory functions of neutrophils, including adherence and aggregation, we examined the ability of anesthetic doses of ETOH to alter the hemodynamic and cellular response to the infusion of zymosan activated plasma (ZAP) in vivo. Twenty five ml of autologous ZAP was intravenously infused into five control and seven (ETOH-treated sheep during mechanical ventillation. In control sheep the mean pulmonary artery pressure (PAP) transiently increased from 14.7±1.4 mm Hg (mean±SEM) to a pead of 38+8 mm Hg by three minutes after beginning the infusion of ZAP. Blood leukocyte concentration transiently decreased 19% below the baseline value due to pulmonary sequestration of polymorphonuclear leukocytes (PMN). Plasma TXB₂ levels measured by radioimmunoassay (RIA) increased from 0.2 to 5.4 ng/ml six minutes after the initiation of ZAP infusion.In five sheep, intravenous infusion of 200 ml of 96% ETOH yielded very high plasma concentrations (882±101 mg%) and completely inhibited both the rise of PAP and the increase of plasma TXB₂ levels after ZAP infusion. However, blood leukocytes transiently decreased 58% below the baseline value. Lower plasma levels of ETOH (200 and 400 mg%) did not prevent either the increase of PAP or the elevation of plasma TXB₂ after ZAP infusion.     

P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.

In postcapillary venules, marginating neutrophils (PMNs) are often seen rolling along the vessel wall prior to stopping and emigrating. There is substantial evidence in vitro and in vivo that the adhesion receptors E- and L-selectin participate in this phenomenon on cytokine-stimulated endothelium, and recent evidence has shown that a closely related adhesion receptor, P-selectin, is capable of mediating neutrophil rolling on an artificial membrane. Here we demonstrate and characterize PMN rolling on monolayers of human umbilical vein endothelial cells (HUVECs) stimulated with histamine to induce surface expression of P-selectin. Peak association of PMNs with the HUVECs occurs 10 min after histamine stimulation, and at a postcapillary venular wall shear stress of 2.0 dyn/cm2 the rolling velocity is 14 microns/s. Approximately 95% of the PMNs roll on the endothelial cells, 5% adhere firmly, and none migrate beneath the endothelial monolayer. Monoclonal antibody (MAb) G1, which binds P-selectin and blocks its adhesive function, completely prevents association of the PMNs with histamine-stimulated HUVEC, whereas the nonblocking anti-P-selectin MAb S12 does not. Treatment of PMNs with the anti-L-selectin MAb DREG56 reduces PMN adherence by approximately 50%. Anti-CD54 MAb R6.5 and anti-CD18 MAb R15.7 have little effect on the number of PMNs rolling on the HUVECs but completely prevent PMNs from stopping and significantly increase rolling velocity. Nonblocking control MAbs for R6.5 (CL203) and R15.7 (CL18/1D1) lack these effects. Rolling adhesion of PMNs on histamine-stimulated HUVECs therefore appears to be completely dependent on endothelial cell P-selectin, with a minor adhesion-stabilizing contribution from intercellular adhesion molecule 1 and beta 2 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1

The CD11/18 (LFA-1, Mac-1) molecules participate in neutrophil adhesion to cultured endothelium in vitro and are critical for effective neutrophil localization into inflamed tissues in vivo. More recently, the MEL-14 Ag, which was first defined as a lymphocyte homing receptor, has also been implicated in inflammatory neutrophil extravasation. Here we compare the regulation and function of these adhesion molecules on neutrophils during the in vivo inflammatory response. The MEL-14 Ag is expressed at high levels on bone marrow and peripheral blood neutrophils, but is lost on neutrophils isolated from the thioglycollate-inflamed peritoneal cavity. In contrast, Mac-1 is up-regulated on inflammatory neutrophils and little change is seen in the level of LFA-1 expression. In vitro activation of bone marrow neutrophils with PMA or leukotriene B4 results in a dose dependent increase in Mac-1 and decrease in MEL-14 Ag expression within 1 h after treatment, thus reflecting what is found during inflammation in vivo. Neutrophils activated in vitro or in vivo (MEL-14Low, Mac-1Hi) do not home to inflammatory sites in vivo, correlating with the loss of the MEL-14 Ag and the increased Mac-1 expression. Anti-LFA-1, anti-Mac-1, or MEL-14 antibody given i.v. suppress neutrophil accumulation within the inflamed peritoneum (38%, 30%, and 37% of medium control, respectively) without affecting the levels of circulating neutrophils. However, when FITC-labeled cells are precoated with the mAb and injected i.v., only MEL-14 inhibits extravasation into the inflamed peritoneum (25% of medium control). Finally, in ex vivo adhesion assays of neutrophil binding to high endothelial venules in inflamed-lymph node frozen sections MEL-14 inhibits greater than 90%. anti-LFA-1 20 to 30% and anti-Mac-1 less than 10% of the binding of bone marrow neutrophils to inflamed-lymph node high endothelial venules. These results confirm that both the MEL-14 antigen and Mac-1/LFA-1 are important in neutrophil localization to inflamed sites in vivo, but suggest that their roles in endothelial cell interactions are distinct.     

High mannose type N-linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin β2 subunit. Similarly, IL-1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICAM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley-Liss, Inc.     

Thrombin-induced adherence of neutrophils to cultured endothelial monolayers: increased endothelial adhesiveness

We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.     

Phenotypic changes in neutrophils related to anti-inflammatory therapy

Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.     

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.     

Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.     

Adherence of human neutrophils changes Ca signaling during activation with opsonized particles

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) upon activation of human neutrophils by opsonized particles (serum-treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo-1 loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo-1 loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura-2 loaded neutrophils adherent to serum-coated coverslips. Our study shows that the opsonized particle-induced change in [Ca²⁺]_i in neutrophils is altered during adherence of the cells to a serum-coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotaxis.     

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.     

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil phagocytosis and bactericidal activity

Human leukocyte inhibitory factor (LIF) is a lymphokine initially defined by its ability to inhibit the random migration of neutrophils. We have recently demonstrated that LIF also potentiates a number of f-met-leu-phe-mediated functions as well as enhancing one Fc receptor-mediated function (antibody-dependent cellular cytotoxicity). In this paper, we have extended our studies involving the effects of LIF on the neutrophil, specifically its effect on phagocytosis and bactericidal activity. We demonstrate that LIF (2 U/ml) potentiates phagocytosis of opsonized heat-killed Staphylococcus aureus (up to 57.2%) and sheep erythrocytes (124.4%) as well as unopsonized latex particles (59.9%). Phagocytosis of opsonized sheep erythrocytes was inhibited by an anti-neutrophil Fc receptor antibody with control PMN but not using the LIF-treated PMN. LIF (1/2 to 1 U) also potentiates the killing of S. aureus by up to 51.6%. Higher concentrations of LIF (greater than or equal to 4 U) inhibits killing. These effects were shown not to be associated with an increase in Fc receptor availability. It is therefore possible that potentiation of these neutrophil activities by LIF may occur either as a result of increased receptor turnover or, more likely, secondary to an increase in nonspecific neutrophil adherence. These studies further support the concept that LIF may have an important role in vivo in inflammation and immunity.     

Chemiluminescence response and adherence of neutrophils to cultured endothelial cells–influence of immunoglobulin G

The effect of different concentrations (0.87, 4.35, 8.7, 17.5, 25 and 35 mg/mL) of intravenous immunoglobulin G (Endobulin®) on neutrophil–endothelial cell interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury the influence of immunoglobulin G on adherence has been investigated. A second aim of the present study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 8.7 mg immunoglobulin/mL test during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 8.7 mg immunoglobulin/mL test (p < 0.025). The present results indicate that this preparation of immunoglobulin G might exert a protective effect on neutrophil–endothelial cell interaction by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Therefore, the current investigation points to a probable protective effect of immunoglobulin G in oxidative diseases, such as the adult respiratory distress syndrome.     

In vitro effect of ascorbic acid on neutrophil–endothelial cell interaction

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil–endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).     

Neutrophil-endothelial interactions: Modulation of neutrophil activation responses by endothelial cells

There is controversy concerning whether intravascular activation of neutrophils during acute inflammation injures contiguous endothelial cells in vivo. Several physiologic defense mechanisms tend to limit such injury. In this paper we have examined evidence for one of these putative protective mechanisms: endothelial cell modulation of the activation responses of neutrophils during adherence and diapedesis. In vitro, endothelial cells co-incubated with neutrophils inhibit the release of superoxide anion when stimulated by receptor-mediated activators. The possible mechanisms include contact-linked down-regulation of neutrophil activation, the release from endothelial cells of soluble mediators which attenuate neutrophil activation responses, and the presence of free radical scavengers in endothelial cells which are active at the interface between endothelial cells and adherent neutrophils. There may be a broad spectrum of mechanisms by which intercellular interactions protect the lining cells of the vascular lumen from 'inadvertent' destruction by phagocytes which become activated while in an intravascular location.     

Distinct adhesive properties of granulocytes and monocytes to endothelial cells under static and stirred conditions

Adherence of neutrophils and monocytes to endothelium is an important event in the sequence of leukocyte responses to inflammatory disease. Double-color flow microfluorimetry analysis was used to determine neutrophil and monocyte adherence to suspended endothelial cells under stirred conditions. The static adherence to endothelial cell monolayers was simultaneously determined. In both assays, neutrophils behaved in a similar way. Basal adherence of neutrophils was very low. Activation by PMA or by the chemoattractants platelet-activating factor and FMLP induced rapid binding, primarily mediated by CR3. Nonactivated neutrophils showed CD18-dependent (lymphocyte function-associated Ag-1 and CR3) and CD18-independent adherence to endothelial cells when the endothelial cells were prestimulated with rIL-1 beta. In contrast to neutrophils, nonactivated monocytes adhered avidly to resting endothelial cells. This adherence was partly CD18 dependent and partly CD18 independent. Whereas monocyte adherence under stirred conditions strongly increased upon activation by PMA, a significant decrease in adherence was found under static conditions, which was prevented by cytochalasin B. This decrease was limited to a distinct CD18-independent binding mechanism, and absent under stirred conditions. We conclude that monocytes adhere with (at least) three binding mechanisms to endothelial cells, a CD18-dependent and two CD18-independent mechanisms.     

Differential effector responses by circulating/blood and tissue/peritoneal neutrophils following burn combined with Enterococcus faecalis infection

Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions.     

Complement activated granulocytes can cause autologous tissue destruction in man

Activation of polymorphonuclear granulocytes (PMNs) by C5a is thought to be important in the pathogenesis of multiple organ failure during sepsis and after trauma. In our experiment exposure of human PMNs to autologous zymosan activated plasma (ZAP) leads to a rapid increase in chemiluminescence. Heating the ZAP at 56( degrees )C for 30 min did not alter the changes, while untreated plasma induced only baseline activity. The respiratory burst could be completely abolished by decomplementation and preincubation with rabbit antihuman C5a antibodies. Observation of human omentum using electron microscopy showed intravascular aggregation of PMNs, with capillary thrombosis and diapedesis of the cells through endothelial junctions 90 s after exposure to ZAP. PMNs caused disruption of connections between the mesothelial cells. After 4 min the mesothelium was completely destroyed, and connective tissue and fat cells exposed. Native plasma and minimum essential medium did not induce any morphological changes. These data support the concept that C5a activated PMNs can cause endothelial and mesothelial damage in man. Even though a causal relationship between anaphylatoxins and organ failure cannot be proved by these experiments C5a seems to be an important mediator in the pathogenesis of changes induced by severe sepsis and trauma in man.     

IL-4 increases surfactant and regulates metabolism in vivo

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.