Polymorphonuclear cell-mediated vascular injury in anergic surgical patients

We examined the responses of primed polymorphonuclear neutrophils (PMNs) adhered to vascular endothelium, which can lead to endothelial cell damage as a mechanism of the capillary leak syndrome, the main cause of death in anergic patients. We tested PMNs from (1) preoperative reactive patients, (2) preoperative anergic patients, (3) anergic patients in the surgical intensive care unit, and (4) healthy controls for in vitro adherence and cytotoxicity on cultured human vein endothelial cells. Adherence of PMNs was 12.9% +/- 3.9% in preoperative anergic patients and 13.1% +/- 3.2% in anergic patients in the surgical intensive care unit compared with 9.0% +/- 2.1% in preoperative reactive patients (P < .05). Cytotoxicity was 6.0% +/- 2.8% in preoperative reactive patients, 13.7% +/- 4.1% in preoperative anergic patients, and 14.3% +/- 4.6% in anergic patients in the surgical intensive care unit. The PMNs from preoperative anergic patients were more cytotoxic against human vein endothelial cells when stimulated by Staphylococcus epidermidis or formyl-methionyleucylphenylalanine. We conclude that PMNs from anergic surgical patients adhere more to endothelial cells and can produce increased cytotoxicity that may lead to detrimental results.     

Endothelial cell activation by leukocyte microparticles

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.     

Kinetics of leukocyte-induced changes in endothelial barrier function

1. Extravasation of polymorphonuclear leukocytes (PMN) and associated plasma leakage are key events in the inflammatory process. The kinetics of PMN-induced changes in endothelial barrier function were studied by means of confluent monolayers of bovine aorta or human umbilical vein endothelial cells (EC), cultured on permeable membranes and mounted in a two-compartment diffusion chamber. The model permitted continuous measurement of transendothelial electrical resistance (TEER), and analysis of protein efflux and PMN migration across the EC monolayer. 2. Transendothelial chemotactic stimulation (fMLP or LTB4) of PMN resting on EC in the upper compartment induced a prompt decline in TEER, followed by an increase in protein flux and transmigration of PMN. Adding the chemoattractant together with PMN in the upper compartment provoked adhesion of PMN, fall in TEER and increase in protein permeability, but no transmigration of PMN, whereas inhibition of PMN adhesion to EC by pretreatment with anti-CD18 mAb prevented all responses to chemotactic stimulation. 3. Chemoattractant-induced adhesion of PMN to the EC monolayer induced a rapid rise in EC cytosolic free Ca2+, similar to that obtained by direct stimulation of EC with histamine, indicating an active response of EC to PMN activation and adhesion. 4. In summary, continuous recording of transendothelial electrical resistance in the in vitro model described permits rapid and sensitive analysis of leukocyte activation-induced effects on EC barrier function. The kinetics and specificity of the EC and PMN responses to chemoattractant stimulation suggest that activated PMN, via adhesion-dependent events, have a direct effect on EC junctional integrity independent of whether transmigration occurs or not.     

In vitro correlates of the L. casei animal model of Kawasaki disease

The induction of coronary arteritis in mice by Lactobacillus casei cell wall (CW) is thought to represent an animal model of Kawasaki disease. Treatment of vascular endothelial cells (EC) in vitro with supernatants from CW stimulated human mononuclear cells (MNC) enhanced adherence of human polymorphonuclear leukocyte (PMN) to human EC, and EC expression of intercellular adhesion molecule-1 (ICAM-1) but not HLA-DR. Supernatants contained high concentrations of tumor necrosis factor-alpha (TNF-alpha) and PMN adherence correlated directly with the concentration of TNF-alpha. Intravenous human gamma globulin (IVGG) preparations did not block the effect of cytokine containing MNC supernates upon EC, ICAM-1 expression by EC, or PMN adherence to prestimulated EC. However, both EC ICAM-1 expression and enhanced PMN adherence to EC by CW induced MNC supernatants were blocked by anti-TNF-alpha treatment. The initial coronary inflammatory reaction in the mouse model appears to involve PMN adherence to vascular EC that have been activated by TNF-alpha released by MNC after stimulation with CW.     

L-selectin (CD62L) blockade does not impair peritoneal neutrophil emigration or subcutaneous host defense to bacteria in rabbits

Neutrophil (PMN) recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. We explored the role of L-selectin (CD62L) in leukocyte emigration following instillation of bacteria into the peritoneum or s.c. skin in rabbits. Pretreatment with blocking mAb against L-selectin (LAM1.3) reduced peritoneal PMN emigration 4 h after i.p. inoculation with 10(10) CFU of Escherichia coli by only 17% compared with animals receiving a nonblocking L-selectin mAb (LAM1.14). Peritoneal PMNs from saline-treated rabbits demonstrated a complete absence of L-selectin, whereas those from LAM1.3-treated animals retained 43% of their baseline L-selectin expression. This suggests that L-selectin shedding is not a requisite event for PMN emigration under these conditions. In rabbits given s.c. inoculations with either Staphylococcus aureus or E coli, pretreatment with mAb LAM1.3 did not significantly impair PMN emigration at 24 h, nor increase the incidence, size, or associated mortality of resulting abscesses at 7 days compared with animals receiving nonblocking mAb LAM1.14. We conclude that: 1) mAb blockade of L-selectin in vivo only modestly affects acute, E. coli-induced peritoneal PMN emigration; and 2) L-selectin blockade does not increase infectious sequelae associated with s.c. bacterial inoculation. These findings of only mildly reduced PMN emigration into the peritoneum and no alteration in s.c. host defense differ from those reported with L-selectin blockade under other, nonbacterial inflammatory conditions, and suggest that redundant selectin-mediated mechanisms (P- and E-selectin) are sufficient for normal PMN emigration in response to bacterial stimulation.     

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.     

The role of neutrophils in peritoneal adhesion formation

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.     

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.     

Effects of antirheumatic drugs on adhesiveness of endothelial cells and neutrophils

Because disease-modifying antirheumatic drugs might exert part of their effects on adhesion of polymorphonuclear neutrophils (PMN) to endothelial cells, this being the first step for PMN migration to inflammatory lesions, we evaluated such drug effects in vitro. Gold sodium thiomalate (GSTM) impaired the ability of interleukin 1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) to express E-selectin and to bind PMN but had no effect on the expression of intercellular adhesion molecule 1 (ICAM-1) or on hyperadhesivity of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN. Auranofin (AF) interacted with HUVEC and PMN adhesiveness but in opposite directions: this drug hampered IL-1beta-induced HUVEC hyperadhesiveness and expression of E-selectin and intercellular adhesion molecule 1, but augmented PMN adherence and CD18 expression. The net effect of auranofin was a reduction of cytokine-driven adhesiveness and enhancement of formylpeptide-induced adhesion. Salazopyrin did not affect HUVEC or PMN adhesiveness or E-selectin and intercellular adhesion molecule 1 expression. Thus, the gold-containing drugs modulated HUVEC and PMN adhesiveness by different mechanisms but ones involving surface adhesion molecules.     

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes.     

Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration

Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.     

Differential antibiotic-induced endotoxin release and interleukin-6 production by human umbilical vein endothelial cells (HUVECs): amplification of the response by coincubation of HUVECs and blood cells

The activation of human vascular endothelial cells (ECs), as assessed by interleukin (IL)-6 production, was investigated in response to antibiotic-induced lipopolysaccharide release from Escherichia coli. Antibiotic-induced killing of E. coli resulted in the activation of human umbilical vein endothelial cells (HUVECs). Imipenem and meropenem induced faster killing of E. coli than ceftriaxone at 2 and 6 h. However, imipenem-induced bacterial killing resulted in significantly less IL-6 release compared with meropenem or ceftriaxone. When HUVECs were coincubated with diluted (4%) human blood, bacterial killing-induced total IL-6 release was significantly higher than that produced by HUVECs or by 4% blood cells alone. These observations suggest that antibiotic-induced lipopolysaccharide release activates both endothelial cells and monocytes and that the effect is significantly amplified when endothelial cells and monocytes are coincubated. Imipenem, which has a greater affinity for penicillin-binding protein (PBP) 2, induced less IL-6 release from blood and endothelial cells than did PBP 3-specific ceftriaxone and meropenem.     

Bacterial lipopolysaccharide induces endothelial cells to synthesize a degranulating factor for neutrophils

Enzymes and other factors secreted by degranulating neutrophils (polymorphonuclear leukocytes, PMNs) mediate endothelial injury, thrombosis, and vascular remodeling. In bacteremia and sepsis syndrome and their consequent complications (including acute respiratory distress syndrome and systemic ischemia-reperfusion resulting from septic shock), neutrophil degranulation is an important mechanism of injury. In related studies, we found that human endothelial cells regulate neutrophil degranulation and that inflammatory cytokines induce synthesis of degranulating factors by human endothelial cells. Here we show that lipopolysaccharides (LPS) from gram-negative bacteria were the most potent agonists for release of degranulating activity by endothelial cells when compared to several cytokines and stimulatory factors. LPS also induced the release of degranulating signals for PMNs from a human endothelial cell line, EA.hy 926. Interleukin 8 (IL-8) is synthesized by endothelial and EA.hy 926 cells in response to LPS and induces neutrophil degranulation. However, complementary strategies using receptor desensitization, translation of messenger RNA by Xenopus laevis oocytes, and purification and analysis of factors from conditioned supernatants demonstrated that degranulating factors distinct from IL8 are generated in response to LPS. The characteristics of a partially purified degranulating factor isolated from conditioned supernatants distinguished it from known chemokines and other factors that induce PMN degranulation and are generated by endothelial cells in response to LPS. Thus, cultured human endothelial cells and endothelial cell lines synthesize several unique signaling molecules that can trigger neutrophil granular secretion. If produced in vivo in response to LPS or other pathologic agonists, these degranulating signals may activate PMNs in combination or in sequence, initiating or propagating vascular damage.     

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.     

Laminar shear stress-induced GRO mRNA and protein expression in endothelial cells

BACKGROUND: The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS: The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS: The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.     

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.     

Vascular cell adhesion molecule-1 is involved in mediating hypoxia-induced sickle red blood cell adherence to endothelium: potential role in sickle cell disease

We investigated the effects of hypoxia on red blood cell (RBC)-endothelial cell (EC) adherence and the potential mechanism(s) involved in mediating this effect. We report that hypoxia significantly increased sickle RBC adherence to aortic EC when compared with the normoxia controls. However, hypoxia had no effect on the adherence of normal RBCs. In additional studies, we found that the least dense sickle RBCs containing CD36+ and VLA-4+ reticulocytes were involved in hypoxia-induced adherence. We next evaluated the effects of hypoxia on the expression of EC surface receptors involved in RBC adherence to macrovascular ECs, including vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and the vitronectin receptor (VnR). Hypoxia upregulated the expression of both VCAM-1 and ICAM-1, whereas no effect on VnR was noted. Potential involvement of VCAM-1 and ICAM-1 in mediating hypoxia-induced sickle RBC-EC adhesion was next investigated using monoclonal antibodies against these receptors. Whereas anti-VCAM-1 had no effect on basal adherence, it inhibited hypoxia-induced sickle RBC adherence in a concentration-dependent manner, with 50% to 75% inhibition noted at 10 to 60 micrograms/mL antibody (n = 6, P < .05 to P < .01). Anti-ICAM-1 (10 to 60 micrograms/mL, n = 8) had no effect on either basal or hypoxia-induced adherence. As noted in the bovine aortic ECs, hypoxia stimulated the adherence of sickle RBCs to human retinal capillary ECs, and this response appeared to be mediated via mechanisms similar to those observed with macro-endothelium, ie, via the adhesive receptor combination VCAM-1-VLA-4. Our studies show that hypoxia enhances sickle RBC adhesion to both macrovascular and human microvascular ECs via the adhesive receptor VCAM-1. Our findings are of interest because hypoxia is an integral part of the pathophysiology of the vaso-occlusive phenomenon in sickle cell anemia.     

A randomized study of combined zidovudine-lamivudine versus didanosine monotherapy in children with symptomatic therapy-naive HIV-1 infection. The Pediatric AIDS Clinical Trials Group Protocol 300 Study Team

Platelet-derived adherence-inhibiting factor (AIF) has been demonstrated to regulate the neutrophil binding to type IV collagen. In this study, we have examined the effect of AIF on neutrophil adherence to confluently cultured human umbilical vein endothelial cells (EC). AIF inhibited neutrophil adherence to thrombin- or tumor necrosis factor alpha (TNF-alpha)-stimulated EC by 75 or 43%, respectively, but hardly affected neutrophil adherence to resting EC. It is interesting to note that the inhibitory activity of AIF was reduced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of neutrophils. Pretreatment of neutrophils or EC with AIF inhibited neutrophil adherence to thrombin- or TNF-alpha-stimulated EC, suggesting that neutrophils and EC have AIF-binding proteins. Using AIF-Sepharose affinity chromatography, AIF-binding proteins containing L-selectin were isolated from 125I-labeled resting neutrophils. However, L-selectin was markedly decreased in the AIF-binding fraction from fMLP-stimulated neutrophils. With the use of AIF-affinity chromatography, P- and E-selectins were obtained in the AIF-binding fractions from resting, thrombin-, and TNF-alpha-stimulated EC. It is important to note that P- and E-selectin were greatly increased in the AIF-binding fractions from thrombin- and TNF-alpha-stimulated EC, respectively. Furthermore, AIF was able to bind to L-selectin-IgG chimeric protein and inhibit the binding of chimeric protein to thrombin or TNF-alpha-stimulated EC. In addition, AIF inhibited the binding of anti-P- or anti-E-selectin monoclonal antibody to the lysates of thrombin- or TNF-alpha-stimulated EC. Together these observations indicate that AIF could recognize L-, P-, and E-selectins, and modulate neutrophil adherence to EC through interactions with selectin molecules.     

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.     

Antibodies to proteinase-3 mediate expression of intercellular adhesion molecule-1 (ICAM-1, CD 54)

We investigated the role of intercellular adhesion molecule-1 (ICAM-1) in the adhesion of polymorphonuclear neutrophils (PMN) to classic antineutrophil cytoplasmic antibody (C-ANCA)-treated endothelial cells, independently of cytokines. Human umbilical vein endothelial cells (HUVEC) grown to confluence in cytokine-free conditions were stimulated with C-ANCA sera and affinity-purified anti-proteinase 3 antibodies (PR3) from Wegener's granulomatosis (WG) patients. Non-activated PMN were added to treated HUVEC and adhesion was measured. In parallel experiments, treated HUVEC were fixed and ICAM-1 and E-selectin were assayed by cyto-ELISA; in other experiments anti-ELAM-1 and anti-ICAM-1 antibodies were assessed. In this in vitro model, adhesion of non-activated PMN to anti-PR3-stimulated HUVEC was enhanced. Adhesion was greater with anti-PR3 antibodies than with control and normal immunoglobulins, and correlated with the level of anti-PR3 antibodies. Neutralization of anti-PR3 antibodies by neutrophil azurophilic granule proteins abolished adhesion. This adhesion increased at the fourth hour after simulation, peaked at the twelfth hour and then decreased. This phenomenon occurred mainly through endothelial expression of ICAM-1 (the main counter-receptor for integrins, involved in firm PMN adhesion and migration) and E-selectin on HUVEC membranes. Anti-adhesion molecule antibodies inhibited this adhesion. This work supports the hypothesis of a direct effect of C-ANCA in endothelial stimulation, namely, on endothelium-PMN adhesion, and strengthens the major role of ICAM-1, directly involved in firm sticking of PMN to HUVEC, besides E-selectin. C-ANCA upregulate endothelial adhesiveness and thus participate in inflammatory reactions by providing endothelial adhesive structures for neutrophils. This might be one of the first steps leading to clinical expression of the disease. These results provide new insights into the pathogenesis of C-ANCA-related diseases.